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The following points highlight the top five techniques used for detecting antigens in the serum. The techniques are: 1. Complement Fixation Test 2. Fluorescent Antibody Technique 3. Enzyme-Linked Immunosorbent Assay 4. Radioimmunoassay 5. Radioallergosorbent Test.
Technique # 1. Complement Fixation Test:
In a reaction between an antigen with its cognate antibody present in serum, the complement disappears from the serum because of binding to the antigen-antibody complex. This is known as complement-fixation. This phenomenon has been utilized for developing tests for detecting either the antigen or an antibody in a given sample of serum.
The complement fixation test consists of two parts. In the first part, the test serum is mixed with a known antigen preparation. On incubation, the complement disappears from the mixture only then when the test serum contains antibody specific for the known antigen. Otherwise, the complement remains unaltered in the mixture, because no antigen-antibody reaction has taken place.
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The second part of the complement fixation test consists of detection of complement (or its absence) in the reaction mixture. As an indicator for complement, a mixture of sheep red blood cells and an antibody against sheep RBC is used. A property of this antibody is that it can haemolyse sheep RBC in presence of complement. The antibody is, therefore, called haemolysin.
As a result of haemolysis, haemoglobin is released to turn the mixture red. Now, if haemolysis occurs, it indicates that the original mixture of part one has free complement which, in turn, indicates that the test serum does not have the cognate antibody to form a complex with the known antigen.
If there is no haemolysis of sheep RBC, it means that complement has been fixed because antigen has combined with antibody of the test serum. Thus, absence of haemolysis represents a positive complement fixation test.
The two parts of the test are shown in Fig. 10.50:
A practical application complement fixation had been the Wasserman Reaction (W.R.) for diagnosis of the sexually transmitted disease, syphilis, caused by Treponema pallida. A modified version was based on the observation that serum of a syphilitic patient contained an antibody which immobilized T. pallida spirochaetes in presence of complement. However, these tests are now completely replaced by more modern and sophisticated techniques.
Technique # 2. Fluorescent Antibody Technique:
It is an elegant technique for detecting specific antigens on particulate bodies such as bacterial cells or tissue sections. When such bodies are treated with cognate antibodies tagged to a fluorescent dye, the antibodies bind to the antigens and make them fluorescent under ultraviolet light.
The preparation made on a slide is observed in a fluorescence microscope where the objects stand out in a brilliant contrast. The fluorescent dyes used for tagging antibodies are generally fluorescein which emits a yellow-green fluorescence and rhodamine which emits a reddish-orange fluorescence under UV-light.
The structures of these dyes arc shown in Fig. 10.51:
There are two variations of the fluorescent antibody technique. One is the direct method and the other is an indirect method. In the direct method, the fluorescent antibody treated cells or tissues are examined under a fluorescence microscope. By this method, it is possible to identify a specific microorganism in a mixed population with the help of a known antibody. A specific antigen can also be identified in a tissue section by this procedure.
In the indirect method, the antigen-containing particulate bodies are first treated with the cognate non-fluorescent antibody and then after removing the unbound antibodies by washing, the preparation is treated with an anti-globulin antibody tagged to a fluorescent dye.
The latter binds to the antibody which has combined with the antigen. The indirect method can also be used for detection of a specific antibody in a person’s serum using a known antigen. In this case, the antibody — if present — binds to the antigen and treatment with a tagged anti-globulin antibody binds to the bound antibody making the preparation fluorescent. This technique is now used for diagnosing syphilis in suspected patients by testing the presence of treponemal antibody in the person’s serum.
For diagnosing syphilis, a standard culture of T. pallida is smeared and fixed on a slide and it is flooded with the patient’s serum. The antibody, if present in the serum, binds to the spirochaetes. The unbound serum is then washed away and then fluorescent anti-human globulin antibody is added.
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In connection with Coombs test, these anti-human antibodies are prepared by injecting human immunoglobulin in an experimental animal, generally a rabbit. They can bind to human antibodies. If the patient’s serum contains treponemal antibodies, they bind to fluorescent anti-human globulin antibodies making the preparation fluorescent.
The direct and indirect fluorescent antibody techniques are shown diagrammatically in Figs. 10.52 A and 52B:
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Besides its use in detection of antigens and antibodies, the fluorescence antibody technique finds application in a highly sophisticated device known as fluorescence-activated cell sorter. It is a modification of a flow cytometer which in its simplest form is an instrument used for rapid counting of cells in a suspension.
A fluorescence-activated cell sorter cannot only count cells, but can also separate different types of cells from a mixture, so that they may be collected separately. It is a highly efficient computerized device, capable of analyzing as many as 1,000 cells/second. Differentiation between cell types is made by tagging with specific antibodies linked to fluorescent dyes.
As different cells have characteristic antigenic determinants, tagging with specific fluorescent antibodies can be used for their separation and quantitation. For example, T-helper and cytotoxic T- cells have on their surface CD4 and CD8 proteins, respectively.
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By fluorescence-activated cell sorter, it is possible to separate and count their relative number in the blood. This becomes necessary to determine the progression of AIDS, because HIV selectively attacks the CD4 cells. The functional principle of the fluorescence-activated cell sorter is briefly described below and illustrated in Fig. 10.53.
A mixture of cells is treated with a fluorescent antibody which binds to only one cell type having a specific antigenic determinant. The mixture is then allowed to pass through a very narrow nozzle to produce micro-droplets, each droplet contains not more than one cell.
A laser beam strikes each droplet and if it contains a fluorescent cell, the fluorescence emitted is measured in a detector. An electrode then makes the fluorescent cell charged, giving it either a positive or a negative charge.
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The droplets fall one after the other between two charged metal plates and the charged droplets move closer towards the charged metal plates depending on the nature of the charge of the droplet i.e. a positively charged droplet is attracted towards a negatively charged metal plate and vice versa. The charged droplets containing the fluorescent-antibody tagged cells and the uncharged droplets containing unstained cells are collected in separate containers.
Technique # 3. Enzyme-Linked Immunosorbent Assay (ELISA):
ELISA is a simple and sensitive technique for detection of either an antigen or an antibody in the serum. There are two major variations of the technique depending on the purpose of the assay. One is a direct method which is employed for detecting a specific antigen in the serum of a person. The other is an indirect method used for detecting a specific antibody in the serum of a patient.
Both the methods require an enzyme-linked antibody. This antibody is chemically attached to an enzyme which is generally horse-raddish peroxidase or alkaline phosphatase. The enzymes react with their respective substrates to yield a coloured product. The antigen or the antibody is adsorbed on an inert surface like that of the wells of a microtitre plate made of plastic material or on to minute polystyrene (latex) particles.
(a) Direct ELISA:
This method is used for detection of a known antigen, e.g. HIV, in the serum with the help of two sets of antibodies, both of which can bind to the antigen. One of these two antibodies is linked to an enzyme having a known catalytic activity. The first antibody (without enzyme) is taken in a buffer solution and allowed to immobilize on the surface of the wells of a microtitre plastic plate or on fine polystyrene beads for several hours. The fluid is then drained off and the wells or beads are thoroughly washed with buffer to remove the un-adsorbed antibody.
Next, the test serum of the patient is added to the wells or beads and incubated. If the serum contains the antigen, it binds to the immobilized antibodies. After thorough washing of the wells or beads to remove completely the unbound antigen, the enzyme-linked antibody is added.
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This antibody binds to the antigen molecules that have already combined with the first immobilized antibody, so that a ‘sandwich’ of antibody-antigen-antibody is formed. If the test serum does not contain any antigen, the second antibody (enzyme-linked) does not bind. The wells or beads are washed and dried.
At the final step, the substrate of the enzyme, linked to the second antibody, is added and if the enzyme is present (i.e. if the enzyme-linked antibody has bound to antigen), a coloured product is formed which indicates that the test serum is ELISA positive.
The essential features of this procedure are shown in Fig. 10.54:
(b) Indirect ELISA:
The indirect method is employed to detect a specific antibody in the serum of a suspected patient e.g. for detection of HIV-specific antibody in a person suspected to be suffering from AIDS.
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The first step in this procedure is to immobilize the known antigen, e.g. HIV, on the wells of a microtitre plate or onto polystyrene beads. After incubation, the excess is removed and the wells or beads are washed and dried.
The next step is to add the test sample of serum to the wells or beads. If the complementary antibody is present, it binds to the immobilized antigen in the wells or beads. After incubation, the unbound antibody is removed and the wells and beads are thoroughly washed and dried.
In the next step, an enzyme-linked anti-human globulin antibody is added to the wells or beads. This antibody can bind to the first immobilized antibody. After incubation, the wells or beads are thoroughly washed and dried.
In the final step, the substrate of the enzyme is added, to produce a coloured product indicating a positive ELISA.
The essential features of the indirect ELISA are shown in Fig. 10.55:
Technique # 4. Radioimmunoassay (RIA):
RIA is a highly sensitive method of estimation of antigens or haptens. The method uses known antigens or haptens labelled with a radioactive element and complementary antibodies. Hence the name radioimmunoassay. It was first developed by Yalow and coworkers in the 1960s for estimation of insulin in serum.
The technique has since been developed to estimate a variety of substances including antigens like hepatitis viruses, hormones, like glucagon, testosterone, estradiol, prostaglandin etc. as well as several drugs like morphine, digitalin etc. Its main usefulness lies in its high sensitivity. It is possible to estimate quantities in the order of 10-9 g (nanogram).
The underlying principle of RIA is different from other methods. The basis of RIA is a competition between a radioactive antigen or a hapten (a ligand) and a non-radioactive counterpart of the same antigen or hapten for binding to the sites of the cognate antibody molecules. As the antibodies bind either the labelled or unlabeled ligands without bias, the probability of binding of a labelled or unlabeled ligand depends on their relative concentration in the mixture of the two components.
In an assay mixture of RIA, a known quantity of the radioactive ligand having a known concentration is mixed with a known amount of the cognate antibody and an unknown concentration of the non-radioactive ligand. Both types of ligands compete with each other for the available binding sites of the antibody molecules and form antigen-antibody complexes.
After incubation, the antigen-antibody complexes are separated and radioactivity determined. Also, the radioactivity left in the mixture (after removal of antigen-antibody complex) is measured. From these two measurements, the ratio of bound radioactive ligand to the free radioactive ligand left over in the reaction mixture is calculated.
This ratio is inversely proportional to the concentration of non-radioactive ligand in the reaction mixture at the beginning. The primary objective of RIA is to measure the quantity of the non-radioactive ligand in a test sample. The higher the concentration of the non-radioactive ligand in the initial test mixture, lower is the ratio of bound to free radioactive ligand.
The concentration of the non-radioactive ligand can be found out from a standard curve as shown in Fig. 10.56:
Technique # 5. Radioallergosorbent Test (RAST):
This test is employed for detection of immunoglobulin E (IgE) specific for a particular allergenic antigen. IgE is a class of heat-labile antibody which is involved in manifestation of allergic reactions. RAST is performed by adsorbing a specific antigen prepared from the allergenic substance to be tested on to a suitable carrier, like sephadex particles.
The adsorbed antigen is then mixed with the serum which may contain the IgE antibody complementary to the antigen. After incubation, the unbound antibodies are removed by washing of the carrier particles. Next, a radioactive anti-IgE antibody is added to the carrier particles. This antibody can bind to IgE antibodies. In case the serum contains IgE specific for the known antigen, it binds to the antigen and becomes immobilized. The anti- IgE antibody then bind to these immobilized IgE molecules.
After incubation of the mixture, the sephadex particles are washed to remove unbound radioactive anti-IgE antibody and the radioactivity retained by the sephadex particles is measured. If radioactivity is found, it indicates a positive RAST which means that the serum tested contains IgE specific for the antigen used.
The principle of RAST is schematically shown in Fig. 10.57:
