Chapter 7: Immune System in Organisms
In the winter of 1891, a child lay dying of diphtheria in a Berlin hospital. Diphtheria was then one of the most feared diseases of childhood: a grey membrane grew across the throat, and the toxin released by the bacteria attacked the heart and nerves. Doctors could do almost nothing except watch. That night the child was given an injection of serum — the clear, cell-free liquid part of blood — drawn from an animal that had earlier been exposed to small, non-lethal doses of diphtheria toxin. The child recovered.
The idea behind that injection came from Emil von Behring, working in Berlin with his colleague Shibasaburo Kitasato. They had shown that an animal which survived a controlled exposure to a toxin developed something in its blood that could neutralise that toxin — and, remarkably, that this protective something could be transferred to a second animal, which was then protected without ever having met the disease. They called it antitoxin. We now call such molecules antibodies. For this work von Behring received the first Nobel Prize in Physiology or Medicine in 1901.
What that Berlin experiment revealed is the central puzzle of this chapter: the body can learn, remember, and even lend out its protection.
Diagram
Fig. 7.1 – Opening illustration: a syringe of clear serum being transferred from an immunised animal to a sick child, with a magnified inset showing Y-shaped antibody molecules in the serum locking onto toxin molecules
By the end of this chapter you will be able to answer every one of these questions yourself.
7.1 Immunity and Its Types
Every day, without noticing it, you swallow, breathe in and touch enormous numbers of bacteria, viruses, fungal spores and foreign proteins. Most of the time nothing happens. The reason nothing happens is that the body carries a defence system capable of recognising what belongs to it and what does not, and of removing the intruder before it can multiply and cause disease.
This ability of an organism to resist infection and to neutralise foreign substances is called immunity. The word comes from a Latin term meaning “exempt” — an immune person is, in effect, exempt from a disease that others around them are catching.
Immunity is not one single mechanism. It is broadly divided into two kinds, depending on whether the protection was present from birth or was built up later during the individual’s own lifetime. The complete classification is set out in FC 7.1, and it is worth keeping this framework in mind, because every later idea in this chapter fits somewhere inside it.
Flowchart
FC 7.1 – Types of immunity: Immunity → branches into (1) Innate (inborn/natural) immunity — present from birth, non-specific; and (2) Acquired immunity — developed during lifetime, specific → Acquired branches into (a) Active — antibodies made by the individual’s own cells, following infection or vaccination, slow to appear but long lasting; and (b) Passive — ready-made antibodies received from outside, immediate but short lived
Innate, Inborn or Natural Immunity
Innate immunity is the protection an individual is born with. It is inherited from the parents, it is present before any pathogen has ever been encountered, and it does not improve with repeated exposure. Because it acts against a wide range of intruders rather than against one particular organism, it is described as non-specific.
Part of this inherited protection operates at the level of the whole species. Human beings do not catch foot-and-mouth disease from cattle, and they do not catch distemper from dogs, however close the contact. The viruses concerned simply cannot attach to and enter human cells in the way they enter the cells of their natural hosts. This kind of built-in resistance costs the body no effort at all — it is a consequence of the cells we inherited.
Acquired Immunity
Acquired immunity is developed during an individual’s own lifetime as a result of gradual exposure to antigens. That exposure may be accidental, as when a person catches an infection, or deliberate, as when a person is vaccinated. Unlike innate immunity, acquired immunity is specific: the protection built up against measles works against measles and against nothing else.
Acquired immunity is of two types, depending on who manufactured the protective molecules.
Active Immunity
In active immunity, the antibodies are produced by the individual’s own cells in response to an antigen that has entered the body. The antigen may arrive as a natural infection, or it may be introduced deliberately as a vaccine.
Because the body has to recognise the antigen, multiply the responding cells and only then begin manufacturing antibodies, active immunity takes days to weeks to become effective. What it loses in speed, it gains in durability. During the response the body also sets aside long-lived memory cells, so that a later encounter with the same antigen produces a much faster and much larger response. Active immunity is therefore long lasting, and in some cases lifelong.
Passive Immunity
In passive immunity, antibodies raised in some other individual — a human donor or an immunised animal — are inoculated directly into the patient. Nothing is manufactured by the recipient’s own body; the protection is borrowed rather than earned.
The advantage is speed. The antibodies are already made, so they begin neutralising the toxin or pathogen almost immediately. The disadvantage follows from the same fact. Antibody molecules are proteins, and like all proteins in the blood they are gradually broken down and removed. No memory cells are formed, because the recipient’s own immune cells were never activated. Passive immunity is therefore short lived, lasting only weeks.
Passive immunity is used exactly where speed matters more than duration: anti-rabies serum after the bite of a suspect animal, tetanus antitoxin after a deep or contaminated wound, antitoxin against food poisoning caused by Salmonella and Clostridium species, and anti-venom after a snakebite. In every one of these situations the toxin is already in the body and there is no time to wait for the patient to build a response. A newborn baby receives natural passive immunity too, in the form of maternal antibodies that crossed the placenta before birth and that continue to arrive in breast milk.
Immunisation
The deliberate use of these principles to protect a population is called immunisation. When immunisation works by giving a vaccine — a preparation of a killed, weakened (attenuated) or fragmented pathogen, or of a purified antigen from it — the body is being tricked into mounting a full active response against an organism that cannot actually cause the disease. The individual ends up with antibodies and memory cells, but skips the illness.
Why Some Immunities Last a Lifetime
Not every infection leaves the same quality of protection behind, and the difference is a useful test of whether the idea of memory has really been understood.
An attack of diphtheria, whooping cough, smallpox or mumps generally produces lifetime immunity. The antigens on these pathogens are stable, so the memory cells stored after the first encounter still recognise the organism decades later. On a second exposure the response is so fast that the person never becomes ill and often never even knows they were exposed. Fig. 7.2 compares the slow, small primary response with the rapid, much larger secondary response that memory makes possible.
Diagram
Fig. 7.2 – Graph of antibody concentration in blood (y-axis) against time in days (x-axis), showing a small, slow primary response after first exposure to antigen and a steep, high, rapid secondary response after a second exposure to the same antigen; mark the lag period on each
By contrast, an attack of the common cold, influenza, pneumonia or bacterial dysentery gives only short-lived immunity. Here the memory cells are formed perfectly well — but the microbes undergo quick mutation. By the following season the surface antigens have changed enough that the stored memory no longer matches what arrives, and the body must start almost from scratch. This is why influenza vaccines are reformulated each year, while a single childhood measles immunisation lasts for life.
7.2 The Human Defence System
Innate and acquired immunity are ways of classifying protection by its origin. The human body can also be looked at in a second way — as a series of defences arranged one behind the other, so that an intruder that gets past one is met by the next. Immunologists usually describe three such lines: two of them non-specific and inherited, the third specific and acquired.
The whole arrangement is summarised in FC 7.2. Notice that a pathogen only ever reaches the third line if the first two have failed.
Flowchart
FC 7.2 – Path of an invading bacterium: bacterium on intact skin → blocked by first line of defence (physical and chemical barriers) → if skin is cut, bacterium enters tissue → met by second line of defence (phagocytic leucocytes and macrophages, inflammation, fever, interferons in viral infection) → if infection persists → third line of defence (specific immunity: lymphocytes and antibodies) → pathogen cleared and memory cells retained
Non-Specific Defence
Non-specific defences act in the same way against every intruder. They do not identify the pathogen, they do not improve with practice, and they are ready from the moment of birth.
First Line of Defence
The first line consists of barriers that stop the pathogen from entering the body at all. These are of two kinds.
Physical barriers shut the door mechanically. The skin, with its tough outer layer of dead keratinised cells, is the largest of these. Wherever the body must remain open to the outside — the airways, the gut, the urinary and reproductive tracts — the surface is lined instead by a mucous membrane. Here mucus traps incoming particles and bacteria, and the beating of cilia sweeps the loaded mucus steadily outwards, away from the lungs. This is one reason why any injury to skin or mucous membrane, from a burn to the damage caused by cigarette smoke, is followed so often by infection: the door has been left open.
Chemical barriers attack whatever lands on those surfaces. Sweat, saliva and tears all contain the enzyme lysozyme, which digests the peptidoglycan of bacterial cell walls and so bursts the cell. The stomach adds a defence of a different kind — hydrochloric acid, which brings the contents to a pH of about 1.5 to 2.0 and destroys most of the bacteria swallowed with food.
Second Line of Defence
If a pathogen breaches the barrier — through a cut, an insect bite or an inhaled droplet — it meets the internal, non-specific defences.
The main cells here are the leucocytes (white blood cells) and the macrophages, large phagocytic cells found in the tissues. Both work by phagocytosis: the cell flows around the bacterium, engulfs it into a vesicle, and then fuses that vesicle with a lysosome so that digestive enzymes destroy the intruder. In your practical work you will make a blood smear and stain it with Leishmann’s stain; the different leucocytes you identify under the microscope are precisely the cells doing this job, and their numbers rise sharply during a bacterial infection.
The inflammatory response follows almost at once. Mast cells and basophils release chemicals such as histamine, which widen the local blood vessels and make their walls more permeable. More blood reaches the site — hence the redness, warmth and swelling around an infected cut — and, more importantly, phagocytic cells and defensive proteins leak out of the vessels into the tissue where the fight is taking place.
Pyrexia, or fever, is the third element. A raised body temperature is not the infection harming the body; it is the body raising the temperature deliberately. Most pathogens grow best at normal body temperature, so a few degrees above it slows their multiplication, while the body’s own metabolic and immune activity speeds up. This is why suppressing every mild fever is not always helpful.
Interferons
Viruses present a special problem for the second line. Once a virus is inside a host cell, phagocytes cannot reach it without destroying the cell itself. The body’s answer is a chemical warning system.
A cell infected by a virus produces and releases small proteins called interferons. These diffuse to neighbouring, still-healthy cells and switch on antiviral defences in them. A virus that later enters such a prepared cell finds that its messenger RNA cannot be translated efficiently at the ribosome, so new viral proteins are not made and the virus cannot multiply.
Two properties of interferons are worth holding on to, because they are frequently tested. Interferons are not virus specific but species specific — the interferon released during an influenza infection will protect a cell against many other viruses as well, but human interferon works in human cells and not in the cells of another species. This is why interferon acts as a broad first response while the specific, antibody-based response is still being organised. Interferon is not a single substance but a group of more than twenty, designated alpha (IFN-α), beta (IFN-β) and gamma (IFN-γ).
Specific Defence
If the non-specific defences cannot clear the infection, the third line takes over. This response is specific: it identifies the particular pathogen by the molecules on its surface and builds a defence tailored to it, carried out by lymphocytes and by the antibodies they cause to be made. Because it is targeted, it is also slower to start — and because it leaves memory cells behind, it is the only line of defence that gets better with experience.
To understand how such precise targeting is possible, we first need to look at what exactly the immune system recognises. That is the subject of the next section.
Diagram
Fig. 7.3 – The three lines of defence in the human body, drawn as three concentric shells around a body outline: outer shell labelled with skin, mucous membrane, cilia, lysozyme in tears and saliva, HCl in stomach; middle shell labelled with phagocytic leucocytes, macrophages, inflammation, fever, interferons; inner shell labelled with lymphocytes and antibodies
7.3 Antigens and Antigenic Determinants
The specific defences of the body are precise because they respond to particular molecules rather than to intruders in general. Any substance that the immune system recognises as foreign and responds to is called an antigen. The word is a contraction of “antibody generator” — a reminder of what these substances provoke the body into making.
An antigen need not be a whole pathogen. The proteins studding the surface of a virus, the polysaccharides of a bacterial capsule, a toxin released by a bacterium, the proteins on the surface of a transfused red blood cell or a transplanted kidney — each of these is an antigen in its own right. This is exactly the principle behind the blood grouping practical you perform in the laboratory: the A and B antigens carried on the surface of red cells are detected by adding antisera containing the corresponding antibodies, and the visible clumping tells you which antigens are present.
What Makes a Substance Antigenic
Not every foreign molecule provokes a response. Two properties matter most.
Size. Most antigens are large molecules, usually proteins or polysaccharides, with a molecular weight greater than about 10,000 daltons (Da). Large size helps in two separate ways. A big molecule offers more surface for immune cells to recognise, and it is also taken up more readily by phagocytosis — an essential first step in starting an immune response, since the antigen must be engulfed and processed before it can be displayed to lymphocytes.
Chemical complexity. A large molecule built from a monotonous repeat of one unit gives the immune system little to recognise. Proteins, with twenty different amino acids arranged in a unique sequence and folded into a specific three-dimensional shape, are chemically the most varied molecules in the body — which is why proteins are the best antigens.
The 10,000 Da figure is a rough guide rather than a rule. Several relatively small proteins are antigenic, including ribonuclease (≈13,700 Da), insulin (≈6,000 Da), glucagon (≈3,800 Da) and angiotensin II (≈1,031 Da), and the last of these is antigenic despite being smaller than many substances that are not.
Certain substances generally fail to act as antigens. Lipids such as lecithin (≈750–800 Da) and cholesterol (≈386 Da) are too small and too simple. Nucleic acids, though large, are usually non-antigenic in isolated form. Simple carbohydrates (usually <1,000 Da) and peptones, which are low molecular weight protein fragments, are likewise poor antigens.
Size alone is therefore not enough, and gelatin proves it. Gelatin is a very large molecule (≈50,000–100,000 Da), yet it is only weakly antigenic, because it is a repetitive protein lacking the structural variety the immune system needs to recognise.
Antigenic Determinants or Epitopes
The immune system does not recognise the whole of a large antigen. It recognises small, specific regions on its surface, called antigenic determinants or epitopes. These are the parts an antibody actually binds to, in much the same way that an enzyme binds its substrate only at the active site rather than over its whole surface.
A single antigen molecule usually carries two or more epitopes, and they need not be identical. This is why large antigens provoke strong responses: each different epitope can stimulate a different set of lymphocytes, so one antigen molecule ends up being attacked by several different antibodies at once, from several directions. Fig. 7.4 shows this — notice that the antibodies binding at the different epitopes are not the same shape as one another.
Diagram
Fig. 7.4 – A single large antigen molecule with three differently shaped epitopes on its surface, each with a differently shaped antibody binding site fitting into it; label antigen, antigenic determinant (epitope), antibody
The number of epitopes is therefore one of the things that determines how strong an immune response will be. It is also why boiling or otherwise denaturing a protein can weaken its antigenicity: unfolding the protein destroys the three-dimensional shape of the epitopes, and an antibody that no longer fits cannot bind.
Haptens — Incomplete Antigens
Some small molecules cannot provoke an immune response on their own, yet clearly do provoke one in the body. Such a molecule is called a hapten.
A hapten is often described as an incomplete antigen. It has the specificity of an antigen — it carries a recognisable epitope — but it lacks the size needed to trigger a response. Attaching it to a large carrier supplies the missing bulk, and the combined molecule then behaves as a full antigen.
The clinical importance of this is considerable. A derivative of penicillin (≈350–400 Da) is far too small to be an antigen, but it can bind covalently to proteins in the blood. The penicillin–protein complex is then treated as foreign, and the allergic reactions that some patients suffer after a penicillin injection are the result. The same mechanism explains contact reactions to poison ivy and poison oak, and to certain dyes and cosmetics: the reactive small molecule enters the skin, couples to skin proteins, and the immune system responds to the pair.
Having established what the immune system recognises, we can now look at the cells that do the recognising.
7.4 Cells Involved in the Immune Response
The specific immune response is not carried out by an organ in the way digestion is carried out by the gut. It is carried out by a population of cells scattered through the blood, the lymph and the lymphoid tissues, working together with the proteins they secrete.
Remarkably, this system begins to be assembled long before the body it will defend has ever met a pathogen. It appears to originate in the foetus itself, about two months after conception.
Origin of Immune Cells
At that stage, primordial cells called stem cells arise in the bone marrow. Stem cells are unspecialised cells that retain the ability to divide and to give rise to specialised cell types. They differentiate along two main lines:
- Erythropoietic cells, which become the erythrocytes — the red blood cells that carry oxygen.
- Lymphopoietic cells, which become the lymphocytes — the cells that form the working core of the immune system.
Only the second line concerns us here. The lymphopoietic cells produced in the marrow are not yet able to do anything useful; they must first be processed, and the route they take during that processing decides what kind of lymphocyte they become. The whole pathway is set out in FC 7.3.
Flowchart
FC 7.3 – Origin of lymphocytes: stem cell in bone marrow → branches into erythropoietic cells → erythrocytes; and lymphopoietic cells → branches into (a) cells passing through the thymus → T-lymphocytes (T-cells) → further differentiate into killer T-cells, helper T-cells, suppressor T-cells and memory T-cells; and (b) cells maturing in the bone marrow (Bursa of Fabricius in birds) → B-lymphocytes (B-cells) → on stimulation give plasma cells and memory B-cells
Where Lymphocytes Mature
Some of the lymphopoietic cells travel to the thymus, a gland lying behind the breastbone. Inside the thymus they are modified into thymus-dependent lymphocytes, better known as T-lymphocytes or T-cells. The thymus is large and highly active in childhood, which is when the body is building most of its immunological experience, and it gradually shrinks in adult life.
Other lymphopoietic cells mature without passing through the thymus and become B-lymphocytes or B-cells. In humans, B-cells mature in the foetal liver before birth and in the bone marrow thereafter. B-cells carry distinctive chemical substances on their surface — receptor molecules — which is what makes them recognisably different from T-cells, since the two look almost identical under an ordinary light microscope.
Types of T-Cells
Mature T-cells are not all alike. Three functional types are important at this level, and the differences between them are worth learning carefully, because questions frequently ask you to match a T-cell to its job.
Killer T-cells (KT-cells) destroy body cells that have been infected. Once an antigen has stimulated them, they contact the infected cell and lyse it — that is, they break it open — so that the pathogen multiplying inside is destroyed along with its shelter.
Helper T-cells (HT-cells) bind to antigens and assist the response made by B-cells. They do not kill anything themselves. Their role is to enable, and it is a role the immune system cannot do without: when helper T-cells are lost, as happens in HIV infection, the rest of the system is left largely unable to act even though its other cells are intact.
Suppressor T-cells (ST-cells) work in the opposite direction. They interfere with the activity of B-cells and hold the response back, preventing it from becoming exaggerated. Just as importantly, they help prevent an immune response against one’s own tissues.
Together, these three types illustrate something that runs through the whole of immunology: a defence system needs brakes as much as it needs weapons. A response that cannot be switched off damages the body it was meant to protect.
B-Cells
B-cells remain in the lymphoid tissue and respond to foreign antigens that reach them. When stimulated, a B-cell divides repeatedly to form a clone — a population of genetically identical cells derived from a single parent cell. Most of these become antibody-secreting cells; some are set aside as memory cells, the long-lived cells introduced in Section 7.1 that make the second response to an antigen so much faster than the first.
Diagram
Fig. 7.5 – Origin and differentiation of immune cells: bone marrow at top with stem cells; arrows to erythrocytes on one side and lymphocytes on the other; lymphocytes branching to thymus → T-cells (killer, helper, suppressor) and to bone marrow → B-cells → plasma cells secreting antibodies and memory cells; label each cell type
Macrophages, met earlier as phagocytes of the second line of defence, also take part here. After engulfing an intruder they display fragments of its antigens on their own surface, where lymphocytes can inspect them. In this way the non-specific and specific defences are not two separate systems but one continuous chain of events.
With the cells identified, we can follow what actually happens when an antigen meets them — and why the response then divides into two distinct pathways.
7.5 Cell-Mediated and Humoral Immunity
When an antigen finally reaches the lymphocytes, the immune response divides into two branches. Which branch dominates depends on which type of lymphocyte is stimulated. A response driven by T-cells is called cell-mediated immunity; a response driven by B-cells is called humoral immunity. Both are specific, both leave memory behind, and in a real infection both are usually running at the same time.
The point at which the pathways separate is shown in FC 7.4. Follow it once before reading further, because the rest of this section simply explains each branch in turn.
Flowchart
FC 7.4 – Divergence of the immune response: antigen enters body → recognised by lymphocytes → branch 1: T-cells stimulated → killer T-cells contact and lyse infected body cells → cell-mediated immunity (acts within tissues); branch 2: B-cells stimulated, assisted by helper T-cells → B-cells divide to form a clone of plasma cells → plasma cells secrete antibodies into blood → antigen–antibody reaction in blood → humoral immunity; both branches → surviving cells retained as memory T-cells and memory B-cells
Cell-Mediated Immunity
Cell-mediated immunity is the part of the immune system stimulated by T-cells. It is placed under the direct control of the lymphocytes themselves: the T-cell does not send out a molecule to do the work, it goes to the target and acts on it.
Because the action happens on or very near the body’s own cells, and because it takes place within the tissues rather than in the circulating fluid, cell-mediated immunity is also called tissue immunity.
The sequence is straightforward. An antigen displayed on the surface of an infected cell stimulates killer T-cells. The killer T-cell makes contact with that infected cell and lyses it, destroying the pathogen along with the cell that was sheltering it. Helper T-cells bind to antigens and assist the response, while suppressor T-cells hold the reaction within limits so that healthy tissue is not attacked.
This branch matters most against pathogens that hide inside cells, where antibodies cannot reach them — viruses, and bacteria such as the tuberculosis bacillus that survive within the very macrophages sent to destroy them. It is also the chief means of resistance against fungi, many pathogenic protozoa and helminthic parasites.
The same mechanism has a consequence that is not defensive at all. Cells of a transplanted organ carry surface proteins that the recipient’s T-cells read as foreign, so the transplant is attacked exactly as an infected cell would be. This is the basis of graft rejection, and it is why the next section but one deals with the proteins that make a tissue recognisably “self”.
Humoral Immunity
Humoral immunity is the branch stimulated by B-cells, with the assistance of helper T-cells. Its name comes from an old word for the body fluids — the “humours” — because the actual antigen–antibody reaction takes place in the blood rather than in the tissues.
The B-lymphocytes are not themselves directly involved in the attack. They remain in the lymphoid tissue and, once stimulated by an antigen, divide repeatedly to form a large clone of cells called plasma cells. Plasma cells are about two to three times the size of the B-cells they came from — an increase in size that reflects what they are for. A plasma cell is essentially a protein factory, and it is packed with the machinery needed to manufacture and export antibodies in enormous quantities.
The antibodies are then released into the blood, where they find and bind the antigen. Their synthesis is under the control of a set of genes called Ir genes, or immune response genes.
Because its weapon is a soluble molecule carried in the circulation, humoral immunity is the body’s main defence against pathogens and toxins that are outside cells — bacteria multiplying in the tissue fluid, and the toxins they release. The diphtheria antitoxin described at the start of this chapter was an antibody of exactly this kind, and vaccination works largely by building this branch of the response in advance.
Memory in Both Branches
Whichever branch responds, some of the activated cells are not used up in the fight. Both T-cells and B-cells survive as memory cells. On a second exposure to the same antigen these cells are already present, already specific and already numerous, so the secondary response is far more rapid and far larger than the first — the difference you saw plotted in Fig. 7.2.
Fig. 7.6 brings the whole of the human immune response together in a single picture, from stem cell to memory cell.
Diagram
Fig. 7.6 – The human immune system: bone marrow stem cells → lymphocytes → left pathway through thymus to T-cells (killer and helper) leading to cell-mediated immunity, showing a killer T-cell lysing an infected body cell; right pathway to B-cells which, on meeting antigen, multiply into a clone of plasma cells secreting Y-shaped antibodies that bind the microorganism, leading to humoral immunity; both pathways feeding into a central box labelled memory cells with an arrow marked “second exposure — rapid secondary response”
Everything so far has treated the antibody as a molecule that simply “binds the antigen”. It is time to look at what that molecule actually is.
7.6 Antibodies or Immunoglobulins
Antibodies are the working molecules of humoral immunity. They are proteins, and they are present in the blood in quantity — about 17 per cent of the total protein in the bloodstream is antibody. That figure alone tells you how much of the body’s protein-making capacity is devoted to defence.
Antibodies belong to the globulin group of blood proteins and show the general properties of that group, which is why they are also called immunoglobulins, abbreviated Ig. The two names refer to the same molecules: “antibody” describes what they do, “immunoglobulin” describes what they are chemically.
Structure of an Antibody Molecule
The basic antibody molecule is built from four polypeptide chains — two identical heavy (H) chains and two identical light (L) chains. Each heavy chain has about 400 amino acids and each light chain about 200, so the heavy chains are roughly twice the length of the light ones.
These four chains are held together by disulphide linkages, the same \(-\mathrm{S}-\mathrm{S}-\) bridges that stabilise tertiary and quaternary structure in other proteins. The bridges join each light chain to a heavy chain, and the two heavy chains to each other. The result is a Y-shaped molecule, with the two light chains lying along the arms of the Y and the heavy chains running through both the arms and the stem. Study Fig. 7.7 carefully, since diagram-labelling questions on this molecule are common.
Diagram
Fig. 7.7 – Schematic representation of an antibody: Y-shaped molecule with two heavy chains and two light chains; label antigen-binding site at the tip of each arm, variable region of heavy chain, variable region of light chain, constant region of heavy chain, constant region of light chain, and the disulphide bridges joining light chain to heavy chain and heavy chain to heavy chain
Variable and Constant Regions
Each chain, heavy or light, has two kinds of region along its length.
The variable region, at the tip of each arm of the Y, differs in amino acid sequence from one antibody to another. The variable regions of a heavy chain and a light chain together fold into a pocket whose shape is unique to that antibody — the antigen-binding site. Because the arms of the Y are identical, one antibody molecule has two identical antigen-binding sites.
The constant region makes up the rest of the molecule and is the same for all antibodies of a given class. It does not decide what the antibody binds; it decides what happens after binding — whether the antibody can be transported across the placenta, whether it can activate complement, whether phagocytes will recognise it.
Having two binding sites also has a practical consequence. A single antibody can hold two antigen particles at once, so antibodies can bridge bacteria or cells into large clumps — the effect you see as visible clumping in the blood grouping practical, where the antibodies in the antiserum link the red cells together.
Classes of Immunoglobulins
Antibodies are grouped into five classes according to the type of heavy chain in their constant region. Each class has a characteristic job, and questions often test the match between class and function.
| Class | Where it is found | Principal role |
|---|---|---|
| IgG | Most abundant in blood and tissue fluid | Main antibody of the secondary response; the only class that crosses the placenta, giving the newborn passive immunity |
| IgM | Blood; a large molecule | First antibody produced on initial exposure to an antigen; a powerful agglutinin |
| IgA | Secretions — saliva, tears, mucus, breast milk | Protects the mucosal surfaces that form the first line of defence |
| IgE | Bound to mast cells and basophils | Mediates allergic reactions |
| IgD | Surface of B-cells | Acts as an antigen receptor on the B-cell surface |
Classification of Antibodies by Function
Antibodies may also be classified by what their binding accomplishes. Five functional types are recognised.
- Antitoxins These develop in response to toxins and neutralise them by binding to the toxin molecule so that it can no longer reach its target in the body. Diphtheria, tetanus and botulinum antitoxins are examples.
- Agglutinins These clump together particulate antigens or bacterial suspensions, converting a cloud of separate particles into a few large clumps that phagocytes can deal with efficiently. Haemagglutinins and bacterial agglutinins belong here.
- Precipitins These act on antigens that are dissolved rather than particulate, forming a large antigen–antibody complex that comes out of solution as a visible precipitate. Antibodies against the polysaccharide C substance of Streptococcus pyogenes, capsular antibodies and antitoxins show this behaviour.
- Lysins These bring about the disintegration of antigenic cells, but only with the help of complement, a group of blood proteins that punches holes in the target cell membrane once antibody has marked it. Bacteriolysins and haemolysins are examples. It must be remembered that an antibody cannot kill the antigen itself — it marks the target and recruits the machinery that does the killing.
- Opsonins These combine with surface components of bacteria and so make them far easier to engulf. A phagocyte grips an antibody-coated bacterium readily, whereas a bacterium with a slippery capsule may escape it altogether. The process is called opsonisation.
Diagram
Fig. 7.8 – Four ways an antibody acts, drawn as four small panels: (a) neutralisation — antibody binding a toxin molecule; (b) agglutination — antibodies bridging several bacteria into a clump; (c) opsonisation — antibody-coated bacterium being engulfed by a phagocyte; (d) lysis — antibody bound to a cell with complement proteins forming a pore in the membrane
7.7 Human Leucocyte Antigens and Organ Transplantation
An immune system that attacks anything foreign faces an obvious problem: it must be able to tell foreign from familiar. Every cell in your body therefore carries a set of surface proteins that act as an identity label, marking the cell as belonging to you. These labels are what killer T-cells inspect when they patrol the tissues, and they are the reason a transplanted organ is treated as an intruder.
The group of genes that codes for these labels is called the major histocompatibility complex, or MHC — the name means, quite literally, the complex of genes concerned with tissue compatibility.
In human beings the MHC proteins are known as human leucocyte antigens, abbreviated HLA, because they were first detected on the surface of leucocytes. They are chemically glycoproteins — proteins carrying attached carbohydrate chains.
The genes coding for these proteins lie on chromosome 6 in humans. In the mouse, the equivalent group of genes is carried on chromosome 17, and much of what is known about the system was first established in mice for exactly this reason — the genes were easy to map and the strains easy to breed.
Diagram
Fig. 7.9 – Left: a human chromosome 6 with the HLA gene region marked. Right: two body cells side by side, one from the patient and one from a donor, each drawn with a set of small surface markers of a different shape; a killer T-cell shown inspecting both, ignoring the patient’s cell and attaching to the donor cell. Label self antigens, non-self antigens, HLA proteins
The Three Categories of MHC Proteins
MHC proteins fall into three main categories, distinguished by where they are found and what they do.
- The first category is found on almost all the nucleated cells of the body. These are the general identity labels. Because they are everywhere, any infected or foreign cell can be checked by a passing T-cell.
- The second category is associated only with certain leucocytes — the macrophages, B-cells and other cells that take part in the cellular immune response. These proteins are used for displaying processed antigen fragments to other lymphocytes, the step by which the non-specific and specific defences are joined, as described in Section 7.4.
- The third category consists of secreted proteins which possess immune functions rather than sitting on a cell surface. Several components of the complement system belong here.
Why HLA Types Differ Between People
The HLA genes are among the most variable genes in the human population: many different versions of each exist, and each person inherits one set from each parent. The combination a person ends up with is therefore almost as individual as a fingerprint.
Two consequences follow. First, an unrelated donor is very unlikely to share a patient’s HLA type. Second, brothers and sisters have a reasonable chance of matching, since both draw their sets from the same two parents, which is why siblings are checked first when a donor is being sought.
Graft Rejection
When an organ is transplanted, the recipient’s T-cells encounter cells carrying HLA proteins they do not recognise. They respond exactly as they would to an infected cell, by the cell-mediated pathway described in Section 7.5. The graft is attacked, its blood supply is damaged and the organ fails. This is graft rejection, and it is the single greatest obstacle to transplantation.
Flowchart
FC 7.5 – Graft rejection: donor organ transplanted into recipient → recipient’s T-cells inspect donor cell surface → donor HLA proteins recognised as non-self → killer T-cells activated and multiply → donor cells attacked and lysed → graft damaged and rejected. Branch: if HLA types closely matched and immunosuppressive drugs given → response weak → graft accepted
The closer the match between donor and recipient HLA types, the weaker the response and the better the chance that the organ will survive. Matching is established before surgery by tissue typing.
Even a good match is rarely perfect, so recipients are also given immunosuppressive drugs, which damp down the immune response and allow the graft to survive. The price is a general weakening of defence: a patient on these drugs is more vulnerable to infection and must be monitored carefully. Here again the immune system shows its double character — the same machinery that protects the body is the machinery that must be restrained for a transplant to work.
7.8 Enzyme Linked Immuno Sorbent Assay (ELISA)
Everything discussed so far has been the body’s own use of the antigen–antibody reaction. That same reaction can be taken out of the body and used as a laboratory tool, and when it is, it becomes one of the most widely used diagnostic techniques in medicine. The best known of these tools is the Enzyme Linked Immuno Sorbent Assay, universally abbreviated to ELISA.
ELISA is used so widely because it combines four qualities that rarely occur together: it is highly sensitive, highly specific, simple to perform and reliable.
The Principle
ELISA rests on the specific binding between an antigen and its antibody — the lock-and-key fit between an epitope and an antigen-binding site described in Section 7.3.
Specificity by itself, however, would only tell you that binding had happened somewhere in a clear liquid, which you cannot see. ELISA solves this by attaching an enzyme to one of the two partners. Wherever binding occurs, the enzyme is brought along with it and held there. Adding the enzyme’s substrate then produces a coloured product at exactly that spot. In effect, the enzyme acts as a signal amplifier: a single bound antibody molecule carries an enzyme that goes on converting substrate molecules, so an invisible binding event is turned into a colour strong enough to see and to measure.
The word sorbent in the name refers to the second essential feature. Either the antigen or the antibody is immobilised — fixed to a solid surface, usually the plastic wells of a microplate. Because one partner is anchored, everything that has not bound to it can simply be washed away.
How the Test Is Performed
The washing steps are the part beginners tend to overlook, and they are the part on which the whole test depends. If unbound enzyme-linked antibody were left in the well, it would generate colour whether or not the target was present, and every sample would appear positive. Each wash removes what has not bound specifically, so that the final colour reflects only genuine antigen–antibody binding.
Flowchart
FC 7.6 – ELISA workflow: coat well with antibody → wash → add patient’s sample; target antigen binds → wash → add enzyme-linked detection antibody → wash → add substrate → enzyme converts substrate to coloured product → measure colour intensity in colorimeter → compare with standard curve to obtain concentration
Diagram
Fig. 7.10 – Section through a single microplate well at four stages, drawn side by side: (a) capture antibody coated on the well surface; (b) antigen from the sample bound to it; (c) enzyme-linked detection antibody bound to the antigen, forming a sandwich; (d) substrate added and coloured product filling the well. Beside it, a whole 96-well microplate showing a row of wells graded from colourless to deep colour
Reading the Result
The intensity of the colour formed is proportional to the amount of antigen or antibody present in the sample, so the test both detects and quantifies. Note carefully that the proportionality may be direct or inverse depending on the format used — in most formats more target gives more colour, but in the competitive format described below, more target in the sample gives less colour.
For a quantitative answer, the colour is read as optical density in a colorimeter or spectrophotometer, the same instrument you use in the laboratory for Bradford’s assay. Standards of known concentration are run alongside the unknown, a standard curve of optical density against concentration is plotted, and the concentration of the unknown is read off that curve.
Types of ELISA
ELISA is not a single procedure but a family of them, differing in how the target is captured and detected.
- Direct ELISA The target molecule is immobilised directly on the solid surface and detected using a labelled primary antibody specific to it. The detection signal comes from the enzyme conjugated directly to that primary antibody. It is the quickest format, with the fewest steps.
- Indirect ELISA The target is immobilised and detected first with an unlabelled primary antibody. A secondary antibody, labelled with an enzyme and directed against the primary antibody, is then added. This two-step process amplifies the signal, because several secondary antibodies can bind to each primary antibody.
- Sandwich ELISA The target is captured between two antibodies — an immobilised capture antibody and a labelled detection antibody. The capture antibody binds one epitope on the target while the detection antibody recognises a different one. This format is particularly useful for antigens carrying multiple epitopes, and it is highly specific, since only a molecule bearing both epitopes gives a signal.
- Competitive ELISA Used to measure the presence or concentration of an antigen. The antigen in the sample competes with a labelled antigen for binding to a limited amount of specific antibody. The more antigen the sample contains, the less labelled antigen can bind, so the signal obtained is inversely proportional to the antigen concentration.
- Multiplex ELISA Allows several different target molecules to be detected and quantified in one sample at the same time. Microbeads or microarray platforms carry different capture antibodies, each labelled with a unique fluorescent dye or identifier, so many targets can be read simultaneously.
Applications of ELISA
- Disease diagnosis Detection of diseases such as HIV infection, cholera and influenza.
- Detection of antibodies Identifying past infection or an immune response, including the response to a vaccine.
- Pregnancy testing Detection of hormones such as hCG.
- Allergy testing Identification of allergens such as milk, peanuts and eggs.
- Food and environmental testing Detection of contaminants, GMOs and toxins.
- Biomedical research Quantification of proteins, hormones and biomarkers.
7.9 Secondary Metabolites in Plants and Their Significance
Everything discussed so far belongs to animals. Plants face the same enemies — insects, grazing animals, fungi, bacteria, viruses — but they meet them under a severe handicap. A plant is stationary. It cannot run from a herbivore, it cannot shake off a fungus, and it has no circulating cells and no antibodies with which to hunt down an intruder.
What plants have instead is chemistry. Over evolutionary time they have developed the ability to manufacture an extraordinary range of compounds that are toxic, repellent or inhibitory to other organisms. These compounds are the secondary metabolites, and they form the backbone of plant defence.
The contrast with primary metabolites is worth fixing clearly, because the two are easily confused.
Secondary metabolites are produced through complex biochemical pathways and are frequently stored in specialised tissues or cellular compartments — in latex canals, in resin ducts, in the vacuole. Storing a toxin in a sealed compartment matters: it keeps the plant from poisoning its own metabolism, and it means the compound is released chiefly when the tissue is torn, which is exactly when a herbivore is biting.
Major Classes of Secondary Metabolites in Plant Defence
Alkaloids
Alkaloids are nitrogen-containing organic compounds, usually bitter in taste and highly biologically active. They are among the most important groups of plant defence chemicals.
Their commonest target is the nervous system of the herbivore, where they interfere with feeding behaviour and survival. Many are toxic outright and can also inhibit the growth of microbes, so a single compound may deter a grazing animal and a bacterium at once. Bitterness itself is part of the defence — an animal that tastes the compound and stops eating has been repelled without the plant needing to poison it.
Quinine, derived from the bark of Cinchona, protects the plant from herbivores and has long been used in medicine against malaria. Nicotine, found in tobacco, acts as a powerful insecticide by affecting nerve transmission in insects.
Phenolic Compounds
Phenolic compounds include a wide variety of substances such as tannins, flavonoids and curcumins. Chemically they are characterised by hydroxyl groups attached to aromatic rings, and they possess strong antioxidant and antimicrobial properties.
Their defensive action works in an unusual way: they bind to proteins. By binding the proteins of plant tissue they make it tougher and much less digestible, so a herbivore that eats the tissue extracts little nourishment from it. They also inhibit microbial growth, reduce herbivore feeding and strengthen plant tissues generally.
Tannins, found in bark, leaves and fruits, reduce palatability and digestion in herbivores — the astringent, mouth-drying sensation of an unripe fruit is tannin at work, discouraging animals from eating the fruit before its seeds are ready. Curcumin, present in turmeric, has antimicrobial and protective roles.
Phytoalexins
The classes described so far are present in the healthy plant, waiting. Phytoalexins work on a different principle altogether.
Phytoalexins are special defensive compounds that are not present in healthy plants in large amounts, but are synthesised rapidly in response to pathogen attack or injury. They are therefore called induced defence molecules: their production is triggered by infection. They accumulate at the site of infection, where they prevent the growth and spread of the pathogen and so limit the damage to the surrounding tissue.
Flowchart
FC 7.7 – Induced defence by phytoalexins: fungal spore lands on leaf and penetrates tissue → plant cells detect the invasion → genes for phytoalexin synthesis are switched on → phytoalexin is synthesised rapidly and accumulates at the site of infection → growth of the pathogen is inhibited → infection contained within a small area
Gossypol, found in cotton plants, protects against fungal infections. Phaseolin, found in beans, inhibits pathogen growth.
Terpenoids
Terpenoids are the largest and most diverse class of secondary metabolites. They are derived from isoprene units and include essential oils, resins and pigments.
Many terpenoids are toxic or repellent to herbivores, and they act variously as toxins, repellents or signalling molecules against herbivores and pathogens. Some, however, serve the plant in the opposite direction: the scent compounds that attract pollinators to a flower are terpenoids too. The same class of chemistry is used both to drive away what harms the plant and to draw in what helps it.
Taxol, obtained from the yew tree, acts as a defence compound in the plant and has strong anti-cancer properties.
Diagram
Fig. 7.11 – A plant defending itself chemically: a caterpillar feeding on a leaf with an arrow to a bitter alkaloid molecule; a fungal infection on a second leaf with an arrow to phytoalexin accumulating at the infection site; roots releasing allelochemicals into the soil with a stunted seedling growing nearby. Label alkaloid, phytoalexin, allelochemical
Allelopathy
Not all of a plant’s chemical warfare is directed at animals and microbes. Some of it is aimed at other plants.
These chemicals may be released through the roots, leached from the leaves, or set free as plant parts decompose on the ground. Once in the soil they can inhibit seed germination, restrict root growth and slow the overall development of competing plants.
The advantage is straightforward. A plant that suppresses its neighbours faces less competition for water, nutrients and light, and so improves its own chances of survival and dominance. Allelopathy is competition conducted chemically rather than by growing faster.
Black walnut releases juglone, which suppresses the growth of nearby plants — gardeners have long known that few vegetables thrive under a walnut tree. Eucalyptus releases chemicals that inhibit undergrowth vegetation, which is one reason the ground beneath a eucalyptus plantation is often strikingly bare.
Significance of Secondary Metabolites
For the plant, the significance of these compounds is survival: protection against predators, resistance to infection, and success in competition, achieved by an organism that cannot move.
For human beings, the significance is at least as great. A large proportion of our medicines are plant secondary metabolites or are derived from them — quinine against malaria and taxol against cancer are two examples already met in this section. In agriculture, the same compounds are used as natural insecticides, and crop varieties are bred for higher levels of their own defensive chemicals so that less pesticide is needed. In biotechnology, cultured plant cells are grown in bioreactors to manufacture valuable secondary metabolites without harvesting the plant at all — a use of plant tissue culture that you will study in Class XII. These compounds also account for a great deal of the flavour, colour and aroma of the food we eat.
7.10 Defence Strategies in Bacteria — Endospores and R Plasmids
A bacterium is a single cell with no tissues, no organs and no specialised defensive cells to call upon. Yet bacteria survive in boiling springs, in desert soil, inside the bodies of animals that are actively trying to destroy them, and in hospital wards saturated with disinfectant. Their defences are correspondingly simple and correspondingly effective.
Chemical Defence and the Capsule
Bacteria compete with one another as well as with the organisms they infect, and much of their chemistry is directed at their neighbours. Many produce hydrolytic enzymes, antibiotics and other antimicrobial compounds that destroy or inhibit competing microorganisms. It is worth remembering that most of the antibiotics used in medicine were originally found in microorganisms, where they had evolved for exactly this purpose — to suppress a rival.
Against the host’s immune system, many bacteria rely on a capsule, a protective outer covering of slimy material outside the cell wall. The capsule works by being difficult to grip. A phagocytic cell such as a macrophage or a neutrophil cannot engulf what it cannot hold, so an encapsulated bacterium escapes phagocytosis and survives far longer in the body.
Endospore Formation
The major defence mechanism of bacteria against a hostile environment is endospore formation. It is used by certain bacteria — the genera Bacillus and Clostridium are the familiar examples — when conditions become unfavourable, as in extreme temperature, dryness or lack of nutrients.
In this process the cytoplasm condenses and forms a thick-walled, highly resistant structure inside the cell. The bacterium is not protecting itself from the outside in, but packing its essential contents — chromosome, ribosomes and a minimum of cytoplasm — into a small, heavily armoured, dehydrated container within itself.
The result is remarkable. Endospores can withstand heat, radiation, chemicals and even antibiotics. The reason antibiotics fail against them is instructive: most antibiotics act on processes such as cell wall synthesis or protein synthesis, and a dormant endospore is carrying out neither. There is nothing for the drug to interrupt.
Dormancy is not death. When favourable conditions return, the endospore germinates and develops back into a normal, active bacterial cell — which may then multiply rapidly in the newly hospitable surroundings.
Flowchart
FC 7.8 – Endospore formation and germination: actively growing bacterial cell → conditions become unfavourable (heat, dryness, lack of nutrients) → chromosome copied and cytoplasm condenses around it → thick resistant spore coat laid down inside the cell → remainder of the parent cell breaks down, releasing the free endospore → dormant endospore survives heat, radiation, chemicals and antibiotics → favourable conditions return → endospore germinates → normal active bacterial cell resumes growth and division
Diagram
Fig. 7.12 – Three bacterial cells drawn side by side: (a) a normal rod-shaped cell with chromosome and flagellum; (b) the same cell containing a dense, thick-walled endospore within it; (c) a cell containing a plasmid drawn as a small separate circle of DNA beside the chromosome. Label cell wall, chromosome, endospore, spore coat, plasmid
This is a matter of real practical importance. Sterilisation in the laboratory is designed around endospores rather than around ordinary bacteria: boiling water will not reliably destroy them, which is why culture media and glassware are autoclaved under steam at pressure — the conditions used in your practical work are chosen precisely because they destroy spores. Medically, the spores of Clostridium tetani survive in soil and dust for years, which is why a deep wound contaminated with soil carries a risk of tetanus and is treated as urgently as it is.
Resistant Plasmids (R Plasmids)
Bacteria also defend themselves through plasmids — the small, circular, extra-chromosomal DNA molecules present in bacteria in addition to the main chromosome. Some plasmids carry resistance genes that enable the bacterium to survive in the presence of antibiotics and toxic substances. Such plasmids are called resistant plasmids or R plasmids.
The genes on an R plasmid typically work by producing an enzyme that destroys or chemically modifies the antibiotic, by altering the target the antibiotic acts on, or by pumping the drug back out of the cell.
What makes R plasmids far more serious than an ordinary mutation is that they can be transferred between bacteria. A plasmid passed from one cell to another carries its resistance with it, so resistance spreads sideways through a bacterial population instead of only downwards from parent to daughter cell — and it can cross between different species of bacteria.
Flowchart
FC 7.9 – Spread of antibiotic resistance: a bacterium acquires an R plasmid carrying a resistance gene → antibiotic is applied → sensitive bacteria are killed while the resistant one survives → the survivor multiplies freely in the space left behind → it also transfers copies of the R plasmid to neighbouring bacteria, including bacteria of other species → a largely resistant population develops → the antibiotic is no longer effective as a treatment
Taken together, the three parts of this chapter describe the same problem solved three ways. Animals defend themselves with specialised cells and specific recognition, plants with chemistry, and bacteria with dormancy and genetic exchange — and in every case the defence has both a cost and a limit.
7.11 Summary
- Immunity is the ability of an organism to resist infection by pathogens or to neutralise foreign substances and their toxins.
- Innate (inborn or natural) immunity is inherited from the parents, is present from birth, acts against all intruders alike and is therefore non-specific. Human resistance to foot-and-mouth disease of cattle and to distemper of dogs is an example.
- Acquired immunity develops during an individual’s own lifetime through exposure to antigens, accidental or deliberate, and is specific to the antigen concerned.
- In active immunity the individual’s own cells make the antibodies, following infection or vaccination. It takes days to weeks to develop, forms memory cells and is long lasting.
- In passive immunity, ready-made antibodies raised in another individual or animal are injected. Protection is immediate but short lived, and no memory cells are formed. Anti-rabies serum, tetanus antitoxin and anti-venom are examples, as are the maternal antibodies a newborn receives.
- Immunisation is the deliberate production of immunity — active, by giving an antigen as a vaccine, or passive, by giving ready-made antibodies.
- Diphtheria, whooping cough, smallpox and mumps generally give lifetime immunity because their antigens are stable. The common cold, influenza, pneumonia and bacterial dysentery give short-lived immunity because these microbes undergo quick mutation, so stored memory no longer matches the antigen that arrives next time.
- The human defence system is arranged in three lines. The first two are non-specific and inherited; the third is specific and acquired.
- The first line of defence consists of physical barriers — skin, and mucous membranes with mucus and cilia — and chemical barriers — lysozyme in sweat, saliva and tears, and hydrochloric acid in the stomach.
- The second line of defence acts inside the body and includes phagocytosis by leucocytes and macrophages, the inflammatory response mediated by mast cells and basophils, pyrexia (fever), and interferons.
- Interferons are antiviral proteins released by virus-infected cells that make neighbouring cells resistant to viral multiplication by blocking translation of viral mRNA. They are not virus specific but species specific, and occur as IFN-α, IFN-β and IFN-γ.
- An antigen is a substance recognised by the immune system as foreign, which triggers a specific immune response. Most antigens are proteins or polysaccharides with a molecular weight above about 10,000 Da.
- Antigenicity depends on size and on chemical complexity together. Lipids, simple carbohydrates and isolated nucleic acids are poor antigens; gelatin is large but only weakly antigenic because it is structurally repetitive.
- An antigenic determinant, or epitope, is the small region of an antigen that an antibody actually binds. One antigen usually carries several epitopes, which is why large antigens provoke strong responses.
- A hapten is a small molecule that is not antigenic alone but becomes antigenic on binding to a larger carrier molecule. A penicillin derivative acting as a hapten on body proteins can cause allergic reactions.
- Immune cells arise from stem cells in the bone marrow. Lymphopoietic cells maturing in the thymus become T-lymphocytes; those maturing in the bone marrow (the Bursa of Fabricius in birds) become B-lymphocytes.
- Killer T-cells lyse infected body cells, helper T-cells assist the response of B-cells, and suppressor T-cells restrain the response and help prevent reaction against one’s own tissues.
- Cell-mediated immunity is driven by T-cells acting within the tissues on or near infected cells, and is therefore also called tissue immunity. It is the chief defence against intracellular pathogens, fungi, protozoa and helminths, and it causes graft rejection.
- Humoral immunity is driven by B-cells assisted by helper T-cells. Stimulated B-cells form a clone of plasma cells, two to three times their own size, which secrete antibodies under the control of Ir genes. The antigen–antibody reaction takes place in the blood.
- Both branches leave memory T-cells and memory B-cells behind, which make the secondary response faster and larger than the primary response.
- Antibodies, or immunoglobulins, form about 17 per cent of blood protein. Each molecule is Y-shaped and built from two heavy chains of about 400 amino acids and two light chains of about 200, joined by disulphide linkages.
- The variable regions at the tips of the arms form the two antigen-binding sites and give the antibody its specificity; the constant region determines what happens after binding. The structure was established by Gerald M. Edelman and Rodney R. Porter.
- The five classes of immunoglobulin are IgG, IgM, IgA, IgE and IgD; IgE mediates allergic reactions and IgG is the only class that crosses the placenta.
- By function, antibodies are classified as antitoxins, agglutinins, precipitins, lysins and opsonins. An antibody cannot kill an antigen by itself — lysins act with complement, and opsonins act by making bacteria easier for phagocytes to engulf.
- The major histocompatibility complex (MHC) codes for cell-surface glycoproteins that mark a cell as self. In humans these are the human leucocyte antigens (HLA), coded by genes on chromosome 6; the equivalent genes lie on chromosome 17 in the mouse.
- MHC proteins fall into three categories: those on almost all nucleated cells, those confined to certain leucocytes taking part in cellular immunity, and secreted proteins with immune functions.
- Because HLA types differ between individuals, a transplanted organ is recognised as non-self and attacked by the recipient’s T-cells, causing graft rejection. Tissue typing is used to find a close match, and immunosuppressive drugs are given to protect the graft.
- ELISA (Enzyme Linked Immuno Sorbent Assay) detects and quantifies an antigen or antibody using an enzyme-linked antibody or antigen that produces a colour reaction. It depends on specific antigen–antibody binding, on immobilisation of one partner on a solid surface, and on washing away everything unbound at each stage.
- Colour intensity is proportional to the amount of target present — directly in most formats, inversely in competitive ELISA — and is read against a standard curve using a colorimeter or spectrophotometer.
- The main formats are direct, indirect, sandwich, competitive and multiplex ELISA. Applications include disease diagnosis, detection of antibodies, pregnancy testing, allergy testing, food and environmental testing, and biomedical research.
- Secondary metabolites are plant compounds not directly required for growth, development or reproduction, which defend the plant against herbivores, microorganisms and environmental stress.
- Alkaloids are bitter, nitrogen-containing compounds that act mainly on the herbivore’s nervous system — quinine from Cinchona and nicotine from tobacco.
- Phenolic compounds such as tannins and curcumin bind proteins, making tissues less digestible, and act as antioxidants and antimicrobials.
- Phytoalexins are induced defence molecules, synthesised rapidly only after pathogen attack or injury and accumulating at the site of infection — gossypol in cotton and phaseolin in beans.
- Terpenoids, derived from isoprene units, are the largest and most diverse class and include essential oils, resins and pigments. Taxol from the yew tree is both a plant defence compound and an anti-cancer drug.
- Allelopathy is the release of allelochemicals into the surroundings to inhibit the germination and growth of competing plants, as with juglone from black walnut and the chemicals released by eucalyptus.
- Bacteria defend themselves by producing hydrolytic enzymes, antibiotics and antimicrobial compounds, and by forming a capsule that prevents phagocytosis by macrophages and neutrophils.
- Endospores are thick-walled, highly resistant dormant structures formed within certain bacterial cells under unfavourable conditions. They withstand heat, radiation, chemicals and antibiotics, and germinate into active cells when conditions improve.
- R plasmids are small circular extra-chromosomal DNA molecules carrying resistance genes. Because they can be transferred between bacteria, including between species, they spread antibiotic resistance sideways through a population as well as to daughter cells.
- Animals, plants and bacteria therefore solve the same problem in three different ways — by specialised cells and specific recognition, by chemistry, and by dormancy and genetic exchange.
Flowchart
FC 7.10 – The chapter at a glance: pathogen approaches host → first line of defence (skin, mucus, lysozyme, HCl) → if breached → second line of defence (phagocytes, inflammation, fever, interferons) → if infection persists → antigen recognised → branches into cell-mediated immunity (T-cells lyse infected cells) and humoral immunity (B-cells → plasma cells → antibodies) → antigen neutralised → memory cells retained for a rapid secondary response. Parallel branches: plant attacked → secondary metabolites (alkaloids, phenolics, phytoalexins, terpenoids) and allelochemicals; bacterium threatened → capsule, endospore formation, R plasmid-mediated resistance
7.12 Key Terms
Immunity the ability of an organism to resist infection by pathogens or to neutralise foreign substances and their toxins.
Innate immunity the non-specific protection inherited from the parents and present from birth, which does not improve with repeated exposure.
Acquired immunity the specific protection developed during an individual’s own lifetime as a result of exposure to antigens.
Active immunity acquired immunity in which the antibodies are produced by the individual’s own cells after infection or vaccination; slow to develop but long lasting.
Passive immunity acquired immunity in which ready-made antibodies raised in another individual or animal are introduced into the body; immediate but short lived.
Memory cells long-lived lymphocytes retained after an immune response, which make the second response to the same antigen faster and larger.
Immunisation the deliberate process of making an individual immune to a disease, either by giving an antigen (active) or by giving ready-made antibodies (passive).
Vaccine a preparation of a killed, weakened or fragmented pathogen, or of a purified antigen from it, given to produce active immunity without causing the disease.
Attenuated weakened, so that an organism can still stimulate an immune response but can no longer cause the disease.
Non-specific defence defence that acts in the same way against every intruder, without identifying it and without forming memory.
Lysozyme an enzyme present in tears, saliva and sweat that destroys bacteria by digesting the peptidoglycan of their cell walls.
Leucocytes the white blood cells, many of which destroy invading organisms by phagocytosis.
Macrophages large phagocytic cells of the tissues that engulf intruders and display fragments of their antigens to lymphocytes.
Phagocytosis the process by which a cell engulfs a particle into a vesicle and digests it with lysosomal enzymes.
Inflammatory response the local reaction mediated by mast cells and basophils, in which blood vessels widen and become more permeable so that defensive cells reach the site of infection.
Pyrexia fever; a deliberate rise in body temperature that slows the multiplication of pathogens and speeds up the body’s own defensive activity.
Interferons antiviral proteins released by virus-infected cells that make neighbouring cells resistant to multiplication of the virus.
Antigen any substance recognised by the immune system as foreign, which triggers a specific immune response.
Antigenic determinant (epitope) the small specific region on the surface of an antigen to which an antibody or lymphocyte receptor binds.
Hapten a small molecule that is not antigenic by itself but becomes antigenic when bound to a larger carrier molecule.
Immune system the complex series of cells, factors and processes that together provide a specific response to antigens.
Stem cells unspecialised cells of the bone marrow that divide and differentiate to give rise to specialised cell types, including all the blood and immune cells.
Lymphocytes the white blood cells responsible for specific immunity, comprising T-cells and B-cells.
T-lymphocytes (T-cells) lymphocytes that mature in the thymus gland and carry out cell-mediated immunity.
B-lymphocytes (B-cells) lymphocytes that mature in the bone marrow and give rise to antibody-secreting plasma cells.
Bursa of Fabricius the organ in birds in which B-lymphocytes mature, and the source of the letter B in their name.
Killer T-cells T-cells that contact and lyse body cells which have become infected.
Helper T-cells T-cells that bind antigen and assist the response made by B-cells.
Suppressor T-cells T-cells that restrain the immune response and help prevent reaction against the body’s own tissues.
Clone a population of genetically identical cells derived from a single parent cell.
Cell-mediated immunity specific immunity in which T-lymphocytes act directly on or near infected cells within the tissues; also called tissue immunity.
Humoral immunity specific immunity in which plasma cells secrete antibodies into the blood and lymph, where the antigen–antibody reaction takes place.
Plasma cells antibody-secreting cells, two to three times the size of a B-cell, formed when a B-cell is stimulated by an antigen.
Ir genes the immune response genes that control the synthesis of antibodies.
Antibody a protein produced by plasma cells that binds specifically to an antigen and helps in its neutralisation or destruction.
Immunoglobulin (Ig) the chemical name for an antibody, reflecting that these molecules belong to the globulin group of blood proteins.
Heavy chain (H chain) the longer of the two kinds of polypeptide chain in an antibody, about 400 amino acids in length.
Light chain (L chain) the shorter of the two kinds of polypeptide chain in an antibody, about 200 amino acids in length.
Disulphide linkage an \(-\mathrm{S}-\mathrm{S}-\) bridge joining polypeptide chains, which holds the four chains of an antibody in their Y shape.
Variable region the part of an antibody chain that differs between antibodies and forms the antigen-binding site.
Constant region the part of an antibody chain that is the same within a class and determines what happens after the antigen is bound.
Antigen-binding site the pocket formed by the variable regions of a heavy and a light chain, which fits a particular epitope.
Antitoxins antibodies that neutralise toxins by binding to them.
Agglutinins antibodies that clump particulate antigens or bacterial suspensions together.
Precipitins antibodies that form a large complex with soluble antigens, bringing them out of solution as a precipitate.
Lysins antibodies that bring about disintegration of antigenic cells with the help of complement.
Complement a group of blood proteins that, once an antibody has marked a target cell, damages its membrane and destroys it.
Opsonins antibodies that coat the surface of bacteria and so make them easier for phagocytes to engulf.
Major histocompatibility complex (MHC) the group of genes coding for cell-surface glycoproteins by which the immune system distinguishes self from non-self.
Human leucocyte antigens (HLA) the MHC proteins of human beings, coded by genes on chromosome 6.
Graft rejection the destruction of a transplanted organ by the recipient’s T-cells, which recognise its HLA proteins as non-self.
Tissue typing the identification and comparison of the HLA proteins of donor and recipient before transplantation.
Immunosuppressive drugs drugs that damp down the immune response so that a transplanted organ is not rejected.
ELISA (Enzyme Linked Immuno Sorbent Assay) an immunological technique that detects and quantifies an antigen or antibody using an enzyme-linked antibody or antigen that produces a colour reaction.
Radioimmunoassay (RIA) an older and highly sensitive immunoassay using a radioactive rather than an enzyme label, now largely replaced by ELISA.
Secondary metabolites plant compounds not directly involved in growth, development or reproduction, which defend the plant against herbivores, microorganisms and environmental stress.
Alkaloids bitter, nitrogen-containing secondary metabolites that act chiefly on the nervous system of herbivores, such as quinine and nicotine.
Phenolic compounds secondary metabolites bearing hydroxyl groups on aromatic rings, such as tannins and curcumin, which bind proteins and act as antioxidants and antimicrobials.
Phytoalexins induced defence compounds synthesised rapidly at the site of infection or injury rather than stored in the healthy plant.
Terpenoids the largest class of secondary metabolites, derived from isoprene units, including essential oils, resins and pigments.
Allelopathy the release of chemicals by a plant into its surroundings to inhibit the germination and growth of competing plants.
Allelochemicals the substances released by a plant during allelopathy, such as juglone from black walnut.
Capsule a slimy protective covering outside the bacterial cell wall that prevents the cell from being engulfed by phagocytes.
Endospore a thick-walled, highly resistant dormant structure formed inside certain bacterial cells under unfavourable conditions.
Plasmid a small, circular, extra-chromosomal DNA molecule present in bacteria in addition to the main chromosome.
R plasmid (resistant plasmid) a plasmid carrying resistance genes that allow a bacterium to survive antibiotics and toxic substances, and which can be transferred to other bacteria.
7.13 Points to Ponder
The immune system must attack anything foreign and yet leave the body’s own tissues untouched. Considering suppressor T-cells and the screening of lymphocytes during maturation, is it fair to say that tolerance of self is as much an achievement of the immune system as the destruction of intruders?
Fever, inflammation and killer T-cells all cause the body some harm while defending it. Where should the line be drawn between a defence that is working and a defence that has become the illness?
Passive immunity gives immediate protection but no memory; active immunity gives lasting protection but arrives too late in an emergency. What does this trade-off suggest about why the body evolved both a fast non-specific system and a slow specific one, rather than a single system?
Influenza vaccines are reformulated every year while the measles vaccine is not. Think about what this says about the arms race between a mutating pathogen and an immune system that can only remember what it has already met.
A person whose HLA type is uncommon in the donor pool may wait years for a transplant while another patient receives one within months. If organs are allocated strictly by tissue match, is the outcome fair, and on what grounds might a different allocation rule be defended?
Some countries assume consent for organ donation after death unless a person has objected during life; others require explicit consent. Consider what each system implies about the relationship between an individual, their body and the community.
ELISA can reveal that a person carries antibodies to a disease long before they feel unwell. Who should have access to such a result — the person, their doctor, their employer, an insurer — and why does the answer differ from one to the next?
A diagnostic test that occasionally reports a positive result for a healthy person and one that occasionally misses a genuine case do different kinds of harm. For a screening test used on a whole population, which error would you rather the test made, and does your answer change with the disease being screened for?
Plants defend themselves with compounds that are toxic by design, and many of those same compounds are our medicines, our spices and our insecticides. What does this suggest about the assumption that a substance is safe simply because it is natural and plant-derived?
Taxol was found in the bark of a slow-growing tree that had to be destroyed to obtain it. When a valuable medicine is locked inside a wild species, what obligations arise towards the species itself, and towards the communities and countries where it grows?
Bacteria did not invent antibiotic resistance in response to medicine — the genes were already there. If human use only decides which bacteria are favoured, how far can any policy on antibiotics be expected to solve the problem rather than slow it down?
Giving antibiotics routinely to healthy farm animals raises yields and lowers food prices, while contributing to resistance that costs lives elsewhere. How should a benefit that is immediate and local be weighed against a harm that is delayed and shared by everyone?
A patient given immunosuppressive drugs to protect a transplanted kidney becomes vulnerable to infections that would not trouble anyone else. Reflect on what this says about the price of every medical intervention that works by switching off a natural process.
Animals defend themselves with specialised cells, plants with chemistry, and bacteria with dormancy and gene transfer. Is it more useful to think of these as three different solutions to one problem, or as three different problems that happen to share a name?
7.14 Objective Questions
- Which of the following is the correct expansion of ELISA as given in the syllabus?
- Enzyme Labelled Immuno Specific Assay
- Enzyme Linked Immuno Sorbent Assay
- Enzyme Linked Immunological Serum Analysis
- Enzyme Labelled Immuno Sorbent Analysis
- Human leucocyte antigens are coded by genes located on chromosome number:
- 6
- 11
- 17
- 21
- The number of antigen-binding sites on one basic antibody molecule is:
- One
- Two
- Four
- Ten
- The specificity of an antibody for a particular antigen is decided by its:
- Constant region of the heavy chain
- Constant region of the light chain
- Variable regions of the heavy and light chains
- Disulphide bridges
- Approximately how many amino acids are present in one heavy chain of an antibody?
- 100
- 200
- 400
- 800
- Which class of immunoglobulin can cross the placenta and protect the newborn?
- IgA
- IgD
- IgG
- IgM
- Antibodies that coat bacteria and make them easier for phagocytes to engulf are called:
- Precipitins
- Opsonins
- Agglutinins
- Antitoxins
- Lysins bring about destruction of antigenic cells only with the help of:
- Lysozyme
- Complement
- Interferon
- Histamine
- Precipitins differ from agglutinins mainly in that precipitins act on antigens that are:
- Particulate
- Soluble
- Radioactive
- Attached to red cells
- In birds, B-lymphocytes mature in the:
- Thymus
- Spleen
- Bursa of Fabricius
- Foetal liver
- Plasma cells differ from the B-cells that produce them in that plasma cells:
- Are about two to three times larger and secrete antibodies
- Mature in the thymus gland
- Lyse infected body cells directly
- Survive as memory cells for life
- The synthesis of antibodies is under the control of:
- Ir genes
- R plasmids
- Oncogenes
- Operator genes
- Cell-mediated immunity is also called tissue immunity because:
- It is produced only in connective tissue
- The reaction takes place within the tissues rather than in the blood
- It requires transplanted tissue to occur
- Antibodies are stored in the tissues
- Interferons are described as species specific but not virus specific. This means interferon produced in a human cell will:
- Act only on the virus that induced it, in any species
- Act on many viruses, but only in human cells
- Act on all viruses in all species
- Act only in the cell that produced it
- In an ELISA, the purpose of the washing step after adding the enzyme-linked antibody is to:
- Increase the amount of colour produced
- Remove unbound enzyme-linked antibody so that colour reflects only specific binding
- Denature the immobilised antigen
- Convert the substrate into a coloured product
- Which format of ELISA gives a signal that is inversely proportional to the amount of antigen in the sample?
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
- In a sandwich ELISA, the target antigen is held between:
- Two identical antibodies binding the same epitope
- A capture antibody and a labelled detection antibody binding different epitopes
- An antibody and a radioactive label
- Two layers of substrate
- Indirect ELISA produces a stronger signal than direct ELISA because:
- The substrate used is more concentrated
- Several labelled secondary antibodies can bind to each primary antibody
- The plate is washed fewer times
- The antigen is present in larger amounts
- Which of the following pairs is correctly matched?
- Quinine – terpenoid
- Taxol – alkaloid
- Gossypol – phytoalexin
- Juglone – phenolic compound of turmeric
- Phytoalexins are described as induced defence compounds because they are:
- Stored in large amounts in the healthy plant
- Synthesised rapidly only after pathogen attack or injury
- Released into the soil to suppress other plants
- Produced only in the roots
- Tannins reduce herbivore feeding chiefly because they:
- Bind proteins and make plant tissue less digestible
- Kill herbivores outright within minutes
- Attract predators of the herbivore
- Prevent seed germination in the herbivore’s gut
- Juglone released by black walnut is an example of:
- An induced defence molecule
- An allelochemical
- A primary metabolite
- A bacterial toxin
- Antibiotics are ineffective against a bacterial endospore mainly because:
- The spore coat is impermeable to all molecules
- The spore is dormant and is not carrying out the processes that antibiotics block
- The spore produces its own antibiotics
- The spore has no DNA
- A bacterial capsule chiefly protects the cell by:
- Resisting high temperature
- Preventing engulfment by phagocytic cells
- Carrying resistance genes
- Allowing the cell to become dormant
- R plasmids are more dangerous than a chromosomal resistance mutation because they:
- Cannot be lost by the cell
- Can be transferred to other bacteria, including other species
- Increase the growth rate of the bacterium
- Produce endospores
For questions B-Q26 to B-Q35, choose the correct option from the following: a. Both Assertion and Reason are true and Reason is the correct explanation of Assertion. b. Both Assertion and Reason are true but Reason is not the correct explanation of Assertion. c. Assertion is true, but Reason is false. d. Both Assertion and Reason are false.
- For the following questions, choose from the options below:
- Assertion (A): Gelatin is only weakly antigenic although it is a very large molecule. Reason (R): Antigenicity depends on chemical complexity as well as on molecular size.
- Assertion (A): An antibody cannot by itself kill the antigen it binds. Reason (R): The constant region of the antibody recruits complement and phagocytes, which carry out the destruction.
- Assertion (A): The thymus is largest and most active during childhood. Reason (R): T-lymphocytes are destroyed in the thymus as soon as they mature.
- Assertion (A): Suppressor T-cells are as necessary to the immune system as killer T-cells. Reason (R): They restrain the response and help prevent reaction against the body’s own tissues.
- Assertion (A): Tissue from one part of a person’s body can be grafted to another part without rejection. Reason (R): The grafted tissue carries the same HLA proteins as the recipient.
- Assertion (A): A patient on immunosuppressive drugs after a transplant must be watched carefully for infection. Reason (R): The drugs damp down the immune response generally, not only the response to the graft.
- Assertion (A): Standards of known concentration are run on the same ELISA plate as the unknown sample. Reason (R): The colour developed depends on incubation conditions and enzyme activity, which vary from run to run.
- Assertion (A): A newborn infant is protected against several diseases during its first few months. Reason (R): Maternal antibodies received across the placenta and in breast milk provide passive immunity.
- Assertion (A): Plants keep phytoalexin production switched off until they are attacked. Reason (R): Manufacturing defensive chemicals costs energy and materials that would otherwise support growth.
- Assertion (A): Culture media are sterilised in an autoclave rather than by boiling. Reason (R): Bacterial endospores can survive boiling but are destroyed by steam under pressure.
- The major site of T-lymphocyte maturation is:
- Bone marrow
- Spleen
- Thymus
- Liver
- Which immunoglobulin is mainly involved in allergic reactions?
- IgA
- IgE
- IgG
- IgM
- The enzyme present in tears that protects against microbes is:
- Pepsin
- Lysozyme
- Trypsin
- Lipase
- Which of the following is a phytoalexin?
- Nicotine
- Gossypol
- Tannin
- Curcumin
- R plasmids in bacteria are associated with:
- Protein synthesis
- Energy production
- Antibiotic resistance
- Cell division
- Passive immunity provides immediate protection because:
- Memory cells are formed
- Antibodies are directly supplied
- Antigens are destroyed
- T-cells are activated
- Antigens are usually large molecules because:
- They diffuse easily
- They resist digestion
- They contain multiple epitopes
- They dissolve in blood
- Fever helps in defence by:
- Killing all body cells
- Increasing metabolic activity
- Reducing immune response
- Destroying antibodies
- Interferons are effective against viruses because they:
- Destroy viral DNA directly
- Prevent viral entry
- Inhibit viral protein synthesis
- Kill infected cells directly
- Haptens become antigenic when they:
- Increase in size
- Bind with carrier molecules
- Enter nucleus
- Undergo mutation
- A patient is injected with anti-rabies serum after a dog bite. This represents:
- Active immunity
- Passive immunity
- Innate immunity
- Cellular immunity
- Which technique would you use to detect antigen concentration in blood?
- PCR
- ELISA
- Chromatography
- Centrifugation
- A transplanted organ is rejected due to mismatch in:
- Antibodies
- Hormones
- MHC proteins
- Enzymes
- A plant produces bitter compounds to avoid herbivory. These are:
- Proteins
- Alkaloids
- Lipids
- Vitamins
- During bacterial infection, which cells mainly perform phagocytosis?
- RBCs
- Platelets
- Macrophages
- Neurons
- Which combination correctly represents humoral immunity?
- T-cells + tissues
- B-cells + antibodies
- Macrophages + enzymes
- Platelets + plasma
- Which of the following best explains short-lived immunity in common cold?
- Weak antibodies
- Rapid mutation of virus
- Lack of T-cells
- Absence of antigens
- Which pair is correctly matched?
- Killer T-cells – Antibody production
- Helper T-cells – Assist B-cells
- Suppressor T-cells – Destroy pathogens
- B-cells – Direct cell lysis
- Which of the following is NOT antigenic by itself?
- Protein
- Polysaccharide
- Hapten
- Large enzyme
- Which structure helps bacteria survive extreme conditions?
- Capsule
- Flagella
- Endospore
- Ribosome
- Which option best justifies vaccination over natural infection?
- Faster disease spread
- Controlled immune response
- No antibody production
- No memory formation
- A molecule of 300 Da is unlikely to act as an antigen because:
- It is too large
- It lacks complexity
- It is toxic
- It is soluble
- Which condition would most likely lead to organ transplant success?
- High antibody levels
- Matching MHC
- Increased fever
- Low RBC count
- Which method is most reliable for early disease detection?
- Microscopy
- ELISA
- Staining
- Culture
- Which factor most increases antigenicity?
- Small size
- Simple structure
- High molecular weight
- Lipid nature
- Which sequence correctly represents the immune response?
- Antibody → Antigen → Response
- Antigen → Immune cells → Antibody
- Immune cells → Antigen → Response
- Response → Antigen → Antibody
- Which combination shows integrated defence in humans?
- Skin + antibodies + T-cells
- Hormones + enzymes + DNA
- Lipids + vitamins + minerals
- RNA + ribosome + nucleus
- Which example shows interaction of biotechnology and immunity?
- Photosynthesis
- ELISA test
- Respiration
- Digestion
- Which situation best shows cooperation between B-cells and T-cells?
- Direct killing of pathogen
- Antibody production with T-cell assistance
- Phagocytosis by macrophages
- Formation of endospores
- Which combination best explains survival strategies across organisms?
- Immunity + mutation + metabolism
- Plant chemicals + bacterial spores + human immunity
- Respiration + digestion + excretion
- Growth + reproduction + movement
- For the following questions, choose from the options below:
- Assertion (A): Active immunity provides long-lasting protection. Reason (R): Memory cells are formed during an active immune response.
- Assertion (A): Passive immunity provides immediate protection. Reason (R): Antibodies are directly transferred into the body.
- Assertion (A): Lipids generally do not act as antigens. Reason (R): Antigens are usually large and complex molecules.
- Assertion (A): ELISA is widely used for disease diagnosis. Reason (R): It detects antigen–antibody interactions using a colour change.
- Assertion (A): Cell-mediated immunity is effective against infected body cells. Reason (R): T-lymphocytes directly destroy infected cells.
- Assertion (A): Haptens alone cannot trigger an immune response. Reason (R): They are small molecules that cannot trigger a response unless attached to a carrier.
- Assertion (A): Endospores help bacteria survive unfavourable conditions. Reason (R): Endospores are highly resistant structures formed within bacterial cells.
- Assertion (A): Interferons are effective against viral infections. Reason (R): They inhibit viral replication inside host cells.
- Assertion (A): MHC compatibility is essential for successful organ transplantation. Reason (R): MHC molecules help the immune system distinguish self from non-self.
Immunity which is inherited from parents is called _______ immunity.
Active immunity involves production of _______ by the body itself.
The first line of defence includes physical barriers like _______ and mucous membrane.
Antibodies are also known as _______.
Interferons were discovered by _______ and Lindenmann.
Passive immunity is _______ lasting because antibodies are not produced by the body.
Fever is an example of the _______ line of defence.
Antigen–antibody interaction in blood is called _______ immunity.
T-lymphocytes mature in the _______ gland.
Interferons prevent viral multiplication by interfering with _______ binding to ribosomes.
Vaccination provides _______ immunity.
Anti-venom injection provides _______ immunity.
Cells that directly kill infected cells are called _______ T-cells.
B-cells differentiate into _______ cells that produce antibodies.
The ELISA technique is used to detect _______ or antibodies in a sample.
Cell-mediated immunity is controlled by _______ lymphocytes.
Antibodies consist of two heavy chains and two _______ chains.
MHC proteins are encoded on chromosome number _______ in humans.
Rapid mutation of microbes results in _______ immunity (short-lived / long-lived).
A stronger immune response is associated with a _______ number of antigenic determinants.
An exaggerated immune response is prevented by _______ T-cells.
Failure of MHC matching can lead to _______ of transplanted organs.
A colour change in ELISA indicates the presence of the _______ molecule.
Alkaloids protect plants mainly by affecting the _______ system of herbivores.
Phytoalexins are produced in response to _______ attack.
Allelopathy helps plants reduce _______ for resources.
Endospores help bacteria survive under _______ conditions.
R plasmids provide resistance against _______ in bacteria.
7.15 Check Your Concepts
Why is innate immunity described as non-specific, while acquired immunity is described as specific? Explain the difference using one example of each.
A doctor treating a patient bitten by a snake gives anti-venom rather than a vaccine against snake venom. Explain the reasoning behind this choice in terms of the time each type of immunity takes to act.
What would happen to a person’s long-term protection if their immune system formed antibodies normally but failed to produce any memory cells?
Human beings do not catch distemper from dogs however close the contact. Explain what this tells you about the nature of innate immunity.
Compare active and passive immunity with respect to the source of antibodies, speed of protection, formation of memory cells and duration of protection.
Why must the influenza vaccine be reformulated almost every year, when a single measles immunisation protects for life? Base your answer on what happens to the antigens of the two viruses.
Arrange the following in the order in which they would act against a bacterium entering through a cut in the skin: antibodies secreted by plasma cells; lysozyme in sweat; inflammatory response and phagocytosis; formation of memory cells.
Explain why an injury to the skin or to a mucous membrane is so often followed by infection, even when the injury itself is minor.
Cilia lining the airways beat continuously in one direction. What would be the consequence for the first line of defence if this beating stopped?
Fever is uncomfortable and is often suppressed with medicine. Give two reasons why a mild fever may actually be helping the body.
A virus is multiplying inside a liver cell. Explain why phagocytosis alone cannot deal with this infection, and name the two defences that can.
Interferon released during an influenza infection also protects the cell against several unrelated viruses, yet human interferon does not work in the cells of a mouse. Explain both observations.
Why is a protein generally a better antigen than a polysaccharide of the same molecular weight?
Explain why boiling a protein solution may reduce its ability to be recognised by an antibody raised against that protein.
A single large antigen molecule is attacked by several different antibodies at the same time. Explain how this is possible.
A patient develops a rash after a penicillin injection, although penicillin is far too small to be an antigen. Explain how the rash arises, and state what would happen if the same penicillin derivative were injected without any carrier protein being available.
Match the cell in Column A with its function in Column B.
| Column A | Column B |
|---|---|
| a. Killer T-cell | i. Secretes antibodies in large quantity |
| b. Helper T-cell | ii. Restrains the immune response |
| c. Suppressor T-cell | iii. Lyses infected body cells |
| d. Plasma cell | iv. Engulfs intruders and displays their antigens |
| e. Macrophage | v. Assists the response made by B-cells |
Both T-cells and B-cells arise from the same stem cells in the bone marrow, yet they behave quite differently. Explain what makes the difference between them.
Lymphocytes are screened during maturation and those reacting against the body’s own molecules are eliminated. Predict what would happen if this screening failed.
Explain why a person who has lost most of their helper T-cells becomes seriously vulnerable to infection, even though their B-cells and killer T-cells are intact.
Killer T-cells destroy the body’s own cells. Justify why this is regarded as a defensive act rather than as damage.
Distinguish between cell-mediated and humoral immunity on the basis of the lymphocyte responsible, the effector agent, the site of action and the kinds of pathogen each deals with best.
Protection against a disease can be transferred from one animal to another using serum alone, with all cells removed. What does this tell you about which branch of immunity is involved, and why would the same transfer fail for cell-mediated immunity?
A plasma cell is two to three times the size of the B-cell it came from. Relate this increase in size to the function the plasma cell performs.
Draw a labelled diagram of an antibody molecule, marking the heavy chains, light chains, disulphide bridges, variable regions, constant regions and antigen-binding sites.
Explain why an antibody molecule needs its constant region, given that only the variable region binds antigen.
A bacterial suspension is mixed with a specific antiserum and forms visible clumps; a dissolved toxin mixed with its antiserum forms a precipitate instead. Name the type of antibody involved in each case and explain the difference in the result.
A patient produces plenty of antibody against a bacterium, but the bacterium continues to multiply. Suggest two possible reasons, one involving complement and one involving phagocytes.
Explain why MHC proteins must be present on almost all nucleated cells rather than only on the cells of the immune system.
Two brothers are being considered as kidney donors for their sister, along with several unrelated volunteers. Explain why the brothers are tested first.
Graft rejection and the destruction of a virus-infected cell are carried out by the same mechanism. Explain how this can be so, and why the outcome is desirable in one case and not in the other.
A student performing an ELISA obtains a strong colour in every well, including the well containing no sample at all. Identify the most likely error in the procedure and explain how it produced this result.
Arrange the following ELISA steps in the correct order: add substrate; coat the well with capture antibody; add enzyme-linked detection antibody; read the colour; add the sample; wash between each stage.
In a competitive ELISA, a patient’s sample gives a very faint colour. Explain what this indicates about the concentration of antigen in the sample, and why the answer would be the opposite for a sandwich ELISA.
Explain the role of the enzyme in an ELISA. Why is the test not simply performed by mixing antigen and antibody and looking for binding?
Distinguish between primary and secondary metabolites of plants, using two examples of each.
Alkaloids are bitter as well as toxic. Explain why bitterness alone is useful to the plant, even before any toxic effect occurs.
Compare the defensive strategies of tannins and phytoalexins, and explain the advantage to the plant of keeping one permanently present and producing the other only on demand.
Terpenoids repel herbivores in the leaves of a plant and attract pollinators to its flowers. Explain how compounds of the same class can serve these opposite purposes.
Little vegetation grows beneath a eucalyptus plantation or a black walnut tree. Explain the phenomenon and name the type of substance responsible.
Explain why an endospore is described as dormant rather than dead, and state what happens to it when favourable conditions return.
A capsule and an endospore both protect a bacterium, but against different threats and in different ways. Compare them on the basis of position, the threat each resists and the metabolic state of the cell.
Explain how antibodies overcome the protection that a bacterial capsule provides, and relate your answer to how a vaccine against an encapsulated bacterium works.
A patient stops a course of antibiotics as soon as they feel better. Explain, in terms of R plasmids and selection, why this behaviour contributes to the spread of resistance.
Resistance can spread through a bacterial population far faster than a favourable mutation normally would. Explain why, using the properties of R plasmids.
Name the organ where B-lymphocytes are believed to mature in humans.
What type of immunity is developed after natural infection?
Which enzyme is present in tears and saliva that acts as a defence?
Name the antibody class involved in allergic reactions.
What is the full form of ELISA?
Why are nucleic acids generally non-antigenic?
Why is passive immunity short-lived?
Why are epitopes essential for antigen recognition?
A person receives ready-made antibodies after a snake bite — identify the type of immunity.
Which immune cells would increase during a bacterial infection?
If a protein has more epitopes, how will the immune response change?
Which test would you use to detect HIV antigens in blood?
Which plant compound would you expect to reduce herbivore feeding — an alkaloid or a carbohydrate?
Differentiate based on function: helper T-cells versus killer T-cells (one point).
How does antigen size influence phagocytosis?
Why do some diseases like the common cold not provide long-term immunity?
Why are haptens unable to trigger an immune response alone?
Would a 500 Da molecule act as an antigen independently? Why?
Which is more effective for long-term protection — vaccination or antiserum? Justify.
Why is MHC compatibility essential in organ transplantation?
Why is ELISA preferred over simple diagnostic methods?
Discuss how secondary metabolites help plants survive without mobility.
How can bacterial plasmids contribute to the spread of antibiotic resistance?
Relate endospore formation with environmental adaptation in bacteria.
How do antibodies and the complement system work together?
Explain how ELISA can be used in both medical diagnosis and food safety.
Define antigen and state any two characteristics that make a substance antigenic.
What are antibodies? Describe their basic structure.
List any three components of the first line of defence in humans.
What are interferons? State their general function.
Explain why large molecules are generally more antigenic than small ones.
Distinguish between innate and acquired immunity (any two points).
Explain the role of helper T-cells in the immune response.
Why are haptens considered incomplete antigens? Explain with reason.
Explain why fever is considered a protective response of the body.
A patient is given tetanus antitoxin after injury. Identify the type of immunity and justify.
During a viral infection, which defence mechanism becomes active first — interferon or antibody? Explain.
How would you use ELISA to detect a specific disease in a patient? (brief steps)
A transplanted organ is rejected by the body. Which immune component is responsible? Explain.
Explain how antibodies help in neutralising toxins in the body.
Analyse the role of antigenic determinants in the specificity of the immune response.
Mention the difference between the functions of B-cells and T-cells in immunity.
Why is vaccination considered better than natural infection for protection? Explain.
Why is gelatin weakly antigenic despite its large size? Explain logically.
Assess the importance of MHC proteins in organ transplantation.
Why are antibiotics ineffective against viral infections, while interferons are effective?
Describe the significance of secondary metabolites in plant defence.
Explain how the different lines of defence work together to protect the human body.
Integrate the roles of T-cells and B-cells in generating an effective immune response.
Describe how ELISA combines the principles of antigen–antibody interaction and enzyme activity.
Explain how plants and microbes use different strategies to survive environmental stress.
Explain how misuse of antibiotics can lead to the development of resistant bacteria through plasmids.
Describe the different types of immunity with suitable examples.
Explain the structure of antibodies and name their different functional types.
Describe the components of the human defence system under the first and second lines of defence.
Describe the different classes of secondary metabolites involved in plant defence with examples.
Explain the mechanism of antigen–antibody interaction and its significance in immunity.
Describe how interferons provide protection against viral infections.
Explain the concept of antigenic determinants and their role in immune specificity.
Describe how humoral immunity is generated and maintained in the body.
Explain how endospore formation helps bacteria survive under adverse conditions.
Explain how ELISA is used in diagnosing infectious diseases, outlining the steps and principle involved.
Describe how vaccination leads to long-term immunity with reference to immune memory.
Explain how antibodies can be used in medical treatment and disease prevention.
Suggest how knowledge of MHC can be applied in successful organ transplantation.
Explain how plant secondary metabolites can be utilised in medicine and agriculture.
Discuss the role of the different types of T-cells (helper, killer, suppressor) in regulating the immune response.
Differentiate between antigens, haptens and non-antigenic substances with suitable examples.
Compare plant defence mechanisms with microbial defence strategies.
Evaluate the importance of vaccination in preventing infectious diseases compared with natural immunity.
Assess the role of ELISA as a diagnostic tool over conventional methods.
Evaluate the significance of antigen size and complexity in determining immunogenicity.
Evaluate the impact of antibiotic resistance in bacteria and the role of R plasmids.
Explain how the different components of the immune system work together to provide a coordinated defence against pathogens.
Integrate the roles of innate and acquired immunity in protecting the human body during an infection.
Describe the relationship between antigen structure, immune recognition and antibody production.
Explain how knowledge of the immune system is applied in biotechnology techniques such as ELISA and vaccine development.
Integrate plant, microbial and human defence strategies to explain survival mechanisms across living organisms.
Amina, a school student, received a tetanus injection after stepping on a rusty nail. The doctor informed her that she was given ready-made antibodies for immediate protection. Identify the type of immunity provided and explain why it is short-lived.
Fatima noticed that after recovering from chickenpox, her grandmother never got the disease again. Identify the type of immunity developed and explain the reason behind its long-lasting effect.
Carlos was curious why proteins act as better antigens compared with lipids. His teacher explained that antigenicity depends on molecular size and complexity. Explain why large molecules are generally more antigenic than small ones.
Omar learns that haptens cannot trigger immune responses on their own but can do so when attached to larger molecules. Explain why haptens are considered incomplete antigens, with an example.
Elena visited a diagnostic lab where ELISA was used to detect a viral infection. She observed a colour change in the test sample. Explain the principle of ELISA and what the colour change indicates.
Arjun noticed that farmers use certain plants to repel insects naturally. These plants produce bitter chemicals. Identify the type of compounds responsible and explain how they protect plants.
Kevin reads that antigenic determinants (epitopes) are essential for immune recognition. Analyse how the number of epitopes influences the strength of the immune response.
Peter argues that vaccination is better than natural infection for disease prevention. Evaluate this statement with suitable justification.
Malti compares ELISA with older diagnostic methods and finds ELISA more reliable. Justify why ELISA is preferred over conventional diagnostic techniques.
Zara is designing a model to explain how different lines of defence work together in the human body. Describe how the first line, the second line and specific immunity act in coordination.
Leila is studying how misuse of antibiotics leads to resistant bacteria. She learns about R plasmids. Explain how R plasmids contribute to antibiotic resistance and its impact on human health.
7.16 Case-Based Questions
- A farm worker comes to a rural health centre with a deep wound in the foot, contaminated with soil. The medical officer gives an injection of tetanus antitoxin at once, and also begins a course of tetanus vaccine to be completed over the following weeks. She explains that the soil itself is the danger, because the organism concerned survives in it for years in a dormant form.
- Identify the type of immunity provided by the antitoxin and the type provided by the vaccine.
- Explain why the medical officer gives both rather than either one alone.
- Name the dormant structure that allows the organism to survive in soil, and state two conditions it can withstand.
- State whether memory cells are formed by the antitoxin injection, and give a reason for your answer.
- During an outbreak of measles in a town, the health department finds that children who were immunised several years earlier remain unaffected, while unimmunised children fall ill. In the same season, many adults in the town catch influenza for the third or fourth year in succession, although influenza vaccine is offered every year.
- Explain why a single measles immunisation given years ago is still protecting those children.
- Explain why immunity to influenza does not last in the same way.
- Which cells account for the difference in outcome between the two diseases?
- Sketch the general shape of the antibody response to a first and to a second exposure to the same antigen, and mark which is faster.
- A technician runs a sandwich ELISA to measure the concentration of a hormone in a patient’s serum. She coats the wells with capture antibody, adds the sample, adds the enzyme-linked detection antibody, adds the substrate, and reads the plate. Every well develops a deep colour, including the blank well containing buffer only.
- Identify the step she has omitted from the procedure.
- Explain how omitting it produced colour in the blank well.
- State the principle on which ELISA depends, and explain the role of the enzyme.
- Describe how she would obtain a numerical concentration once the plate has been correctly run.
- A woman with kidney failure is placed on a transplant waiting list. Her two sisters are tested as possible donors, along with three unrelated volunteers. One sister proves to be a close match and donates a kidney. After the operation, the patient is placed on immunosuppressive drugs and is advised to avoid crowded places for some months.
- Name the group of proteins that is compared during the matching, and state the chromosome on which the genes coding for them lie.
- Explain why sisters are more likely to match than unrelated volunteers.
- Which branch of the immune response would attack the transplanted kidney, and which cells would carry out the attack?
- Explain why the patient must avoid crowded places while on immunosuppressive drugs.
- A boy is brought to a clinic with a spreading rash and swelling that began a few hours after he was given a penicillin injection for a throat infection. The doctor records a drug allergy in his file and notes that penicillin itself is a very small molecule, of about 350 to 400 Da.
- Explain how a molecule of this size can provoke an immune response at all.
- What name is given to such a small molecule, and why is it called an incomplete antigen?
- Name the class of immunoglobulin mainly involved in allergic reactions of this kind.
- Name two other everyday substances that can act in the same way through the skin.
- A patient with tuberculosis is found to have bacteria surviving inside the very macrophages that engulfed them. Antibodies against the organism are present in his blood in good quantity, but the infection continues.
- Explain why circulating antibodies are of limited use against this infection.
- Which branch of specific immunity is chiefly responsible for controlling such an infection?
- Describe how a killer T-cell deals with a body cell harbouring a pathogen.
- Suggest why this branch of immunity is also described as tissue immunity.
- In a cotton field, plants attacked by a fungus are found to accumulate a compound at the site of infection that was almost absent in healthy plants sampled a week earlier. In an adjacent field, an untreated crop suffers heavy insect damage, while a variety bred for higher alkaloid content is barely touched.
- Name the class of secondary metabolite accumulating at the infection site, and name the specific compound found in cotton.
- Explain why this compound was almost absent in the healthy plants.
- Explain how alkaloids protect the second crop from insects.
- Suggest one advantage of breeding crops for higher levels of their own defensive compounds rather than spraying insecticide.
- In a hospital ward, a bacterium that was treatable with a common antibiotic five years ago is now resistant to it, and the same resistance has appeared in a different bacterial species on the same ward. Investigation shows that the resistance gene is carried on a small circular piece of DNA separate from the bacterial chromosome.
- Identify the structure carrying the resistance gene.
- Explain how the resistance came to appear in a second, unrelated species.
- Explain the role played by the use of the antibiotic itself in producing this situation.
- Suggest two measures a hospital could take to slow the spread of such resistance.
- A student prepares nutrient broth for a practical, boils it for ten minutes and leaves it overnight at room temperature. The next morning the broth is cloudy with bacterial growth. A second flask, autoclaved with steam under pressure, remains clear.
- Explain why boiling failed to sterilise the first flask.
- Describe how the surviving structures gave rise to the growth seen the next morning.
- State two conditions, other than heat, that these structures can withstand.
- Explain why the presence of antibiotics in a medium would not, by itself, guarantee sterility.
- A national transplant registry has one donor liver available and three suitable patients waiting. Patient A is the closest tissue match; patient B has waited longest; patient C is the most severely ill and may not survive another month. The registry’s published rules give weight to all three factors.
- Explain, in terms of what you have learnt about HLA, why the degree of tissue match affects how long a transplanted organ is likely to survive.
- Give one argument in favour of allocating the organ strictly by tissue match, and one against it.
- Explain why most countries prohibit the sale of organs.
- Suggest why a registry publishes its allocation criteria openly rather than deciding each case privately.
- Kim, a biology enthusiast, observes that tears and saliva help prevent infections. He also reads that skin acts as a barrier.
- Name the type of defence these belong to.
- Mention any two components involved in this line of defence.
- Name the enzyme present in tears responsible for antimicrobial action.
- Thomson was bitten by a snake and was immediately given anti-venom by doctors.
- Identify the type of immunity provided.
- Explain why this method gives immediate protection.
- State whether memory cells are formed in this case.
- Aisha is analysing organ transplantation cases and learns about MHC compatibility.
- What is MHC?
- Why is MHC matching important in transplantation?
- Predict what happens if MHC is not compatible.
- Nikolas is creating a project on survival strategies across organisms, including plants, microbes and humans.
- Explain one defence mechanism in plants.
- Explain one defence mechanism in bacteria.
- Explain how human immunity differs from these strategies.
- Ayaan is studying the structure of antibodies and observes a Y-shaped molecule with different regions (refer to Fig. 7.7).
- Identify the labelled parts: heavy chain, light chain and antigen-binding site.
- Explain the role of the variable and constant regions in antibody function.
- How do disulphide bonds contribute to antibody structure?
- Why can one antibody bind specifically to a particular antigen?
- Sangita observes a diagram showing two pathways of immune response involving T-cells and B-cells (refer to Fig. 7.6).
- Identify which pathway represents humoral immunity and which represents cell-mediated immunity.
- Name the cells responsible for each pathway.
- Explain how plasma cells are formed and state their function.
- Why is cell-mediated immunity important against infected body cells?
- Elizabeth studies a diagram showing the origin and differentiation of lymphocytes from stem cells (refer to Fig. 7.5).
- Identify the site of origin of lymphocytes.
- Where do T-cells and B-cells mature?
- Differentiate between the roles of T-cells and B-cells.
- What is the significance of the memory cells shown in the diagram?
- Kevin observes a diagram showing different survival strategies of bacteria, including endospore formation and plasmids (refer to Fig. 7.12).
- Identify the structure responsible for survival in extreme conditions.
- Explain the role of R plasmids in bacteria.
- How does the capsule protect bacteria from host defences?
- Why are these mechanisms a concern in medical science?
- Kamini studies a diagram showing plants releasing chemicals to defend against herbivores and pathogens (refer to Fig. 7.11).
- Name two types of secondary metabolites shown.
- Explain how alkaloids protect plants.
- What is allelopathy as seen in the diagram?
- How do phytoalexins differ from pre-existing defence compounds?
7.17 Practice With Data
- A sandwich ELISA is used to measure the concentration of a protein biomarker. Standards of known concentration give the optical density (OD) readings shown below.
| Concentration (ng/mL) | 0 | 5 | 10 | 15 | 20 | 25 |
|---|---|---|---|---|---|---|
| Optical density | 0.06 | 0.22 | 0.39 | 0.55 | 0.72 | 0.88 |
- Plot the standard curve of optical density against concentration.
- A patient’s serum, diluted 1 in 10 before the assay, gives an OD of 0.47. Read the concentration from your curve.
- Calculate the concentration of the biomarker in the patient’s undiluted serum.
- State why the standards were run on the same plate as the patient’s sample.
- A competitive ELISA is used to measure a small hormone. The readings obtained are given below.
| Hormone concentration (ng/mL) | 0 | 2 | 4 | 8 | 16 |
|---|---|---|---|---|---|
| Optical density | 1.20 | 0.94 | 0.71 | 0.44 | 0.21 |
- Describe the relationship between hormone concentration and optical density shown by these data.
- Explain, in terms of competition for a limited amount of antibody, why the relationship takes this form.
- Two samples give OD readings of 0.85 and 0.30. State which contains more hormone.
- State how the shape of this curve would differ if the same hormone were measured by a sandwich ELISA.
- To find the antibody titre of a patient’s serum, the serum is diluted in a series of two-fold steps — 1:2, 1:4, 1:8 and so on — and each dilution is tested for a visible agglutination reaction. Agglutination is seen up to the 1:256 dilution but not at 1:512.
- State the antibody titre of the serum.
- How many two-fold dilution steps were made from the undiluted serum to reach 1:256?
- A second sample from the same patient three weeks later shows agglutination up to 1:1024. Calculate how many times greater this titre is than the first.
- State what a rising titre suggests about the stage of the patient’s infection.
- A rabbit is injected with an antigen on day 0 and again with the same antigen on day 28. The concentration of antibody in its blood, in arbitrary units, is measured as follows.
| Day | 0 | 4 | 7 | 14 | 21 | 28 | 30 | 33 | 38 |
|---|---|---|---|---|---|---|---|---|---|
| Antibody (units) | 0 | 0 | 12 | 40 | 32 | 20 | 190 | 460 | 410 |
- Plot antibody concentration against time.
- State the lag period, in days, before antibody first appeared after the primary injection.
- Calculate how many times greater the peak of the secondary response is than the peak of the primary response.
- Account for both the shorter lag and the higher peak of the secondary response.
- A single plasma cell can secrete about 2000 antibody molecules every second.
- Calculate the number of antibody molecules one plasma cell secretes in 24 hours. Express your answer in standard form.
- A clone of \(5 \times 10^{4}\) such plasma cells is formed from one stimulated B-cell. Calculate the number of antibody molecules the clone secretes in 24 hours.
- Comment on how this figure explains why a plasma cell is two to three times the size of the B-cell it came from.
- A technician has a stock of serum already diluted 1 in 20, and needs 10.0 mL of serum at a final dilution of 1 in 200 for an assay. The relationship \(C_1V_1 = C_2V_2\) applies, where \(C\) is concentration and \(V\) is volume.
- Calculate the volume of the 1 in 20 stock required.
- Calculate the volume of buffer that must be added to it.
- State one reason why serum is diluted before an ELISA rather than being used undiluted.
- A haemocytometer is used to count the white blood cells in a diluted blood sample. The blood is diluted 1 in 20, and 400 cells are counted over a total counted volume of \(4.0 \times 10^{-3}\) mL.
- Calculate the number of white blood cells per mL in the diluted sample.
- Calculate the number of white blood cells per mL in the undiluted blood.
- The normal range is 4000–11000 cells per microlitre (\(\mu\)L). State whether this count lies within the normal range, showing your conversion.
- Suggest what a count well above the normal range might indicate.
- A hospital records the percentage of isolates of one bacterial species that are resistant to a particular antibiotic.
| Year | 2020 | 2021 | 2022 | 2023 | 2024 | 2025 |
|---|---|---|---|---|---|---|
| Resistant isolates (%) | 4 | 7 | 13 | 22 | 34 | 48 |
- Plot these data as a line graph.
- Calculate the percentage point increase between 2020 and 2025.
- Describe the shape of the trend and suggest, in terms of selection and plasmid transfer, why resistance spreads in this way rather than at a steady rate.
- Predict, with a reason, what would happen to the trend if the hospital restricted the use of this antibiotic from 2026 onwards.
- A suspension containing \(1.0 \times 10^{6}\) bacterial endospores per mL is heated, and the number of surviving spores per mL is counted at intervals.
| Time (min) | 0 | 5 | 10 | 15 | 20 |
|---|---|---|---|---|---|
| Survivors per mL at 100 °C | \(1.0 \times 10^{6}\) | \(8.0 \times 10^{5}\) | \(6.5 \times 10^{5}\) | \(5.0 \times 10^{5}\) | \(4.0 \times 10^{5}\) |
| Survivors per mL at 121 °C under pressure | \(1.0 \times 10^{6}\) | \(1.0 \times 10^{4}\) | \(1.0 \times 10^{2}\) | \(1\) | \(0\) |
- Calculate the percentage of spores surviving 20 minutes at 100 °C.
- State the time taken at 121 °C for the spore count to fall to one-hundredth of its starting value.
- Use these data to explain why culture media are autoclaved rather than boiled.
- Explain why a count of surviving spores, rather than of actively growing cells, is the correct measure of whether sterilisation has succeeded.
- An anti-cancer terpenoid is extracted from the bark of a slow-growing tree. About 3.0 kg of dried bark yields 1.0 g of the compound, and one tree yields about 6.0 kg of dried bark. A single patient’s course of treatment requires 2.0 g of the compound.
- Calculate the mass of dried bark needed for one patient’s course.
- Calculate the number of trees needed to treat 500 patients.
- The trees take decades to reach harvestable size and are killed when the bark is stripped. Comment on what your answer to (b) implies for the supply of the drug.
- Suggest two approaches, other than harvesting more bark, by which the supply problem could be addressed.