Introduction
Serology is the scientific study of serum and other body fluids. In practice, the term usually refers to the diagnostic identification of antibodies in the serum typically formed in response to an infection, against other foreign proteins, or to one’s own proteins. Serology has been used historically to confirm infections with bacteria, fungi, and viruses that are difficult to detect by other methods. The difficulty with serology is that some immunocompromised patients will not mount an adequate antibody response to infection,
Antigen-Antibody reactions are the methods by which antigens and antibodies are measured. The observable reactions between the two may take various forms (precipitation, agglutination, etc).
An antibody combines with antigen which is
identical to the inducing antigen and not with unrelated antigens. There is formation of a link between the antigen-binding site of the antibody and the chemical groupings which make up the antigenic determinant of the antigen molecule
METHODS OF MEASUREMENT
Serological tests can be used in two ways: a known antibody can be used to detect and measure an unknown antigen, or a known antigen can be used to detect and measure an unknown antibody.
Quantitative results are normally expressed in terms of the titre of the serum, the highest dilution of the serum at which a particular effect can be demonstrated. For a example, a titre of 160 means that in the reaction being studied the serum shows the effect when it is diluted 1 in 160. The titre is thus a measure of the amount of antibody in a unit volume of the original serum.
COMBINATION OF ANTIGEN AND ANTIBODY
The relationship between antigen and antibody has been likened to that between lock and key. Their combining sites are complementary in shape and reactivity. The better the fit, the closer the contact and the stronger the antigen-antibody bond.
The combining forces between the two molecules depend on hydrogen bonding, attraction between oppositely charged groups, hydrophobic bonding and van der Waals forces. The number of combining sites, or valency, of antibodies is two in IgG, two or more in IgA and then ten in IgM. Incomplete antibodies behave as though they were univalent.
In practical serology it is useful to distinguish two types of antibody:
- Classical antibodies. They are detectable by tests such as precipitation and agglutination.
- Incomplete antibodies. Some antibodies
combine with the antigen but cannot be
detected by ordinary tests. These non
precipitating and non-agglutinating incomplete antibodies can be detected only in special ways, e.g. the antiglobulin test.
Antigens are multivalent: the larger the
antigen the higher the valency. Antigen and
antibody combine in varying proportions
depending on their relative concentrations.
The molecules combine to form a network or lattice. As the complex increases in size the hydrophilic groups of the antigen become masked and the complex as a whole becomes susceptible to precipitation and agglutination.
Antigen and antibody combine in varying
proportions. If one reagent in a precipitation
reaction is present in excess the expected
reaction may not take place, i.e. the
combining sites on the reagent present in
smaller amounts are used up before the
complexes are large enough to be precipitated.
If a certain dose of toxin is added to a given
quantity of antitoxin it is exactly neutralized;
but if the same dose is divided into two parts and these are added separately with a short interval in between, the mixture remains toxic (Danysz phenomenon), i.e. when the toxin is added in two fractions the first forms complexes containing a relatively high proportion of antitoxin and insufficient
antitoxin is left over to neutralize the second.
MECHANISMS OF SEROLOGICAL REACTIONS
Antibodies may react with antigens in either
of three ways namely:
- By direct reaction with the antigen
- By activation of the complement system (complement fixation)
- By activation of the anaphylactic system.
1. Direct reaction of antigens and
antibodies
Following the direct binding of antigens and
antibodies, one of several reactions can occur. These include agglutination, precipitation, neutralization, lysis, opsonization and antitoxin reaction.
2. Activation of the complement system
Complement is a system consisting of nine enzymes which are normally present, in a dormant state, in the serum. The enzymes are designated as C’1, C’2, C’3a, C’3b, C’3c, C’3d, C’3e, C’3f and C’4. Following an antigen-antibody reaction, the resulting complex binds with the complement. The complement is then said to be ‘fixed’. Complement fixation is a way of activating the complement system, and the activated
enzymes are capable of digesting or destroying the invading agents (antigens). A single activated enzyme can start a chain of reactions that activate several other enzymes and so enough of active enzymes are produced to attack the invading agents.
The invading agents can be destroyed by enzymes in either of the following ways:
- Lysis: the proteolytic enzymes of the complement system rupture invading bacterial or other cells
- Opsonization and phagocytosis: the complement enzymes make the surfaces of bacterial or other cells more vulnerable to phagocytosis by macrophages and the reticuloendothelial cells. This is called opsonization. The opsonized cells are then quickly taken up by the macrophages and neutrophils.
- Chemotaxis: the complement enzymes cause a massive migration of macrophages, basophils and neutrophils to the region of the antigenic reaction. These powerful
phagocytes will then engulf the invading agents. - Agglutination: the complement enzymes modify the surface of the antigenic agents such that they adhere
to each other to form clumps - Neutralization of viruses: the complement enzymes attack the molecular structure of viruses and so destroy their virulence.
- Inflammatory effect: the complement products cause a local inflammatory reaction leading to hyperaemia,
swelling and coagulation of proteins around the affected tissues, thus preventing movement of the invading agents to other tissues.
3. Activation of the anaphylactic system
Some of the antibodies, particularly, IgE, normally attach to the membrane of certain cells, e.g. mast cells, present in the blood vessels. When these antibodies react with antigens, the reactions cause a rupture of the cells, with the release of a number of
biological chemicals. The chemicals include histamine, slow-releasing substance of anaphylaxis (SRS-A) and chemotaxis factor. Histamine causes a local vasodilatation and increased permeability of blood vessels; SRS-A causes the acute constriction of
certain smooth muscles, particularly the bronchi, and chemotaxis factor causes a massive migration of neutrophils, basophils and macrophages to the vicinity of the antigen-antibody reaction.
These reticuloendothelial cells play a
powerful phagocytic role in protecting the
body against the effects of the antigens.
Although, the primary actions of the various
chemicals are for protection, on occasions,
their effects may manifest as allergies.
Some allergies are very severe and some times fatal. They are described as hypersensitivity reactions or anaphylactic reactions.
Types of serological reaction
- Antitoxin reaction: in this type of reaction, toxins or toxoids are the antigens, and their reactions with antibodies produce a neutralization of the toxins, i.e it is rendered harmless. Since the toxin is an antigen in solution it is also precipitated. Example of the toxin neutralization is the Schick’s test, Nagler reaction. Immunity to diphtheria and tetanus depends on the possession of antitoxins. Toxins themselves are too dangerous to use as immunizing agents, but they can be converted into harmless toxoids by treatment with formalin.
- Nagler reaction: The a-toxin (lethal toxin) of Clostridium perfringens is a lecithinase and is normally identified by an in vitro test. When the organism is grown on plates containing human serum or egg yolk (Nagler medium),
dense opalescent zones of lipid material are deposited around the colonies. C. perfringens ferments lactose. Lactose and an indicator can
be incorporated in the medium to facilitate identification of C. perfringens. The reaction is inhibited by C. perfringens antitoxin. - Agglutination. Antibodies first immobilize motile organisms (antigens) and then aggregate the cells, forming clumps. This type of reaction can only be demonstrated if the antigen is particulate or if the antigen is adsorbed on to the surface of visible particles of uniform size, such as red blood cells or bentonite or polystyrene latex.
- Schick’s test. A standard skin test dose of diphtheria toxin (not toxoid) is injected intradermally into the left
forearm (‘test’) and heat-inactivated toxin into the right (‘control’). The results are read at 48 h and again at 1
week.
-
- Positive: the test arm shows red area (1-5 cm diameter) at 48 h which persists for 1-2 weeks. The control shows no reaction. the patient has insufficient antitoxin to neutralize the toxin and considered ‘susceptible’
- Negative: there is no reaction on either arm. The patient has sufficient antitoxin to neutralize the toxin and is considered ‘immune’.
- Pseudo-reactions: reactions occur on both arms. This indicates a non-specific reaction to some component
other than the toxin.
APPLICATIONS
- One of the classical applications of the agglutination test in diagnostic bacteriology is the Widal test. The widal test is a serological reaction used routinely in the diagnosis of typhoid fever. The aim of the test is to determine the highest dilution of serum which produces agglutination when mixed with the antigenic suspension ‘H’ or ‘O’ of Salmonella typhi, Salmonella paratyphi A or B. Positive ‘H’ agglutination consists of fluffy cotton wool-like clumps that settle at the bottom of the tube. Positive ‘O’ agglutination appears as a fine granular precipitate.
- Blood grouping: clean the finger with 70 % alcohol, dry and stab. Allow 2 drops of blood to fall into 2 ml of 1% sodium citrate in normal saline and mix. Sodium citrate acts to prevent coagulation whereas normal saline preserves the osmotic tension of the
blood cells. On 3 slides, place separately:- a) A drop of anti A serum (containing antibody to substance A)
- b) A drop of anti B serum (containing antibody to substance B)
- c) A drop of normal saline (control) Add to each, a drop of the blood suspension. Mix with separate clean glass rods, and rock the slides gently for about 5 minutes. Watch for agglutination.
- Result: A positive result is indicated by large, brick red clumps; a negative result appears as a smooth suspension of cells without clumps. Positive result (clumps) means the patient’s blood is not compatible with the test serum but negative result implies compatibility. If both test sera give positive results, that means the patient belongs to AB blood group. if both sera give negative results, that means the patient is in the O blood group.
- Coombs’ test: The presence of antibody globulin on a red cell may not result in direct agglutination of the cells, for example in some Rhesus negative mothers with Rh-positive infants. It is possible to show that the red cells are coated with antibody by using an antiglobulin serum (produced in the rabbit by injecting human globulin) which will bring about agglutination of the cells.
- Precipitation: Antibodies react with
antigen molecules in solution forming an insoluble complex, which precipitates out. This type of reaction can only be demonstrated if the antigen is soluble. The reaction is quickest and the precipitate most heavy when antigen and antibody are present in optimal proportions. Excess of antigen and antibody may inhibit the formation of the precipitate. - Ring test: A solution of antigen is layered on the surface of the antibody in a small tube. A narrow ring of precipitate occurs near the
junction of the two fluids. - Gel-diffusion (double diffusion) precipitation: Antigen and antibody are allowed to diffuse towards each other in an agar medium. Where antigen and antibody meet in optimal proportions they produce a thin line of precipitate. Usually, there are several lines because the serum contains several distinct antibodies and the antigen is a mixture of antigens. There are controls.
- Single radial immunodiffusion: The antigen is placed in a well cut in an agar gel containing suitably diluted antibody. A ring of precipitate
forms where the reactants meet in optimal proportions. The higher the concentration of antigen, the greater is the diameter of the ring. - Immunoelectrophoresis: This consists of electrophoresis followed by gel-diffusion precipitation. An electric current is used to separate the components of an antigen mixture on an agar-coated slide. A trough is cut in the agar parallel to the axis of electrophoresis and filled with antibody.
- Countercurrent immunoelectrophoresis (CIE): This is a rapid and more sensitive variant of the double diffusion method in which an electric current is used to drive the antigen towards the antibody.
Lysis and bactericidal action
When an antibody combines with an antigen
which forms part of the surface of certain cells and provided complement is also present the cells are lyzed, i.e. dissolved.
Complement fixation test
The complement fixation test involves two
stages:
- a test system in which antigen and
antibody are allowed to react in the presence of a limited amount of complement and - a haemolytic system in which it is determined whether or not complement has been fixed.
Since human sera contain a variable and
unknown quantity of complement, all sera
used in the test are heated (55°, 30 min) and a known amount of complement is provided in the form of fresh or specially preserve guinea pig serum.
- Test system: Antigen, antibody and
complement are mixed, incubated and
allowed to react. If the antigen encounters its specific antibody they combine and complement is fixed. If the antigen does not encounter its specific antibody no reaction takes place and the complement remains.
When the reaction has had time to go to completion, the presence or absence of complement in the test mixture is determined in a separate experiment by using the haemolytic system. - Haemolytic system: This consists of a
suspension of sheep red cells sensitized with anti-sheep red cell serum. The suspension is mixed with the test mixture and incubated. If antigen has combined with specific antibody, complement is fixed and is not available for haemolysis: the red cells remain intact and the result is positive. If antigen has not combined with specific antibody, complement is still present and haemolysis can take place: the red cell suspension clears with release of haemoglobin into solution and the result is negative.
The classical complement fixation test is the Wassermann’s reaction used in the diagnosis of syphilis. The test system consists of Wassermann antigen mixed with dilutions of the patient’s serum in the presence of guinea-pig complement.
After the antigen and patient’s serum have had time to react and take up the limited amount of complement available in the system, the indicator system is added to show whether or not there is free complement. Complement fixation tests are used routinely for detecting viruses in tissue cultures which have been inoculated with specimens of blood or tissues fluids from humans with probable virus
Capsule swelling
When a specific antibody combines with an
antigen which forms the capsule of an
organism the capsule appears to swell. The
phenomenon is also referred to as the
Neufeld Quellung (swelling) reaction or the
specific capsular reaction. It can be used for
serological typing of capsulated organisms
such as pneumococci, Klebsiella and H.
influenzae
Virus neutralization
When an antibody combines with an antigen which is part of a virus the virus is neutralized, i.e. rendered non-infective.
Suitable mixtures of virus and antibody are introduced into a living host system which will serve as an indicator of the presence or absence of active virus, e.g. animals (especially the mouse), the embryonated hen’s egg, tissue cultures and, in the case of phage, cultures of bacteria. The neutralization test is widely used in virology to estimate antibodies and to identify viruses. The antibodies it measures are not always the same as the antibodies detected by complement fixation and other methods.
Immune electron microscopy
When viruses in suspension are exposed to specific antibody they form clumps and become much more readily detectable when viewed in the electron microscope. In this way it is possible to demonstrate viruses which are not detectable by ordinary methods, e.g. hepatitis A virus can be identified in faeces by using convalescent serum to aggregate the particles. The technique is of value in identifying new viruses and investigating their role in diseases.
Radioimmunoassay (RIA)
When an antibody reacts with a mixture of an antigen and the same antigen labelled with a radioactive marker, the unlabelled and labelled antigens compete for the binding
groups of the antibody. When the reaction is complete, free antigen and antigen-bound antigen can be separated and the radioactivity of each fraction determined. By comparison with suitable standards, unlabelled antigen can be measured by the extent to which it prevents the binding of labelled antigen. RIA is a highly sensitive technique and can be used to measure protein hormones, enzymes, complement
components, immunoglobulins, viral antigens and, by using antisera produced against haptens, small molecules such as
antibiotics (especially aminoglycosides), steroids, thyroxine, digoxin and morphine.
Other radioactive binding techniques
- Solid-phase immunoradiometric assay:
Unlabelled antibody is first firmly attached to a solid phase such as the surface of a well in a plastic tray. The test sample containing antigen is allowed to react with this antibody-coated surface. The amount of antigen bound to the surface is then measured by means of a labelled antibody. The technique can be used to detect hepatitis A virus and measure it’s antibody. - Radioallergosorbent test (RAST): This
technique is used to measure IgE antibodies to specific antigens (‘allergens’) in patients with atopic hypersensitivity. The allergen obtained from pollens, mites, etc., is bound to
a solid phase support such as a paper disc and is allowed to react with the patient’s serum. IgE binds to the allergen. After washing, the disc is allowed to react with a radiolabelled anti-human IgE antibody which binds to the allergen-IgE complexes. After further washing, the residual radioactivity gives a measure of the amount of IgE in the patient’s serum. - Enzyme-linked immunosorbent assay (ELISA): Antibodies can be labelled by linking them to a readily detectable enzyme. In one commonly used ELISA technique for measuring antibodies the antigen is first firmly attached to the surface of a well in a plastic tray and is then allowed to react with the test serum. Excess serum is removed by washing and an antiglobulin serum conjugated with a suitable enzyme is added. The conjugate becomes attached to the antibody bound to the original antigen. After Ofurther washing the amount of conjugate retained is measured by adding enzyme substrate and determining enzymic activity by means of a colour change.
Diseases Associated with Antigen Antibody Reaction
Humoral and cellular immunological responses as well as hypersensitivity (allergic) reactions are based on the ability of the body mechanisms to distinguish the host’s own tissues (‘self’) from foreign materials (‘non self’). The body does not normally produce antibodies against its own tissue components. This fact is called immunological tolerance. The differentiation of ‘self’ from ‘non self’ is an important homeostatic function of the body. In some rare occasions, however, this specific function fails, and the body rejects self. This results in a number of conditions known as autoimmune diseases. This include:
- Multiple sclerosis
- Chronic thyroiditis
- Systemic lupus erythematosus,
- Chronic active hepatitis
- Allergic encephalitis
- Autoimmune haemolytic anaemia
- Rheumatoid arthritis
- Polyarteritis nodosa
- Scleroderma and
- Dermatomyositis