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The following points highlight the six important serological reactions used in laboratories. The serological reactions are: 1. Precipitation Test 2. Agglutination Test 3. Fluorescent-Antibody Technique 4. Radioimmunoassay 5. Enzyme-Linked Immunosorbant Assay 6. Complement Fixation Test.
Serological Reaction # 1. Precipitation Test:
When soluble antigens and its homologous antibody molecules react, they sometimes form large polymeric macromolecules terminating into visible precipitate (Fig. 41.19).
The precipitation occurs in two stages: first, the antibodies bind to antigens forming antibody-antigen-complex within a few seconds or minutes, then the “constant regions” of antibodies of the complexes bind to each other within some hours resulting in the formation of visible precipitate.
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The formation of precipitate is dependent on the proper relative concentrations of the antigen and antibody molecules in a specific region called the zone of equivalence or zone of precipitation, i.e., the zone of equivalence (or precipitation) defines the region wherein the concentrations of antigen and antibody molecules reach almost equivalence.
Precipitation-tests are performed either in fluid-fluid precipitation, or gel-gel precipitation. In the former, the solutions of antigen and antibody are layered over each other in a thin tube, whereas the diffusion of antigen and antibody takes place through a semisolid gel (such as agarose).
In the latter, mainly applied in the laboratory diagnosis of bacterial infections of humans and important animals, the precipitation tests are also used for many other purposes such as:
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(i) Identification of blood or seminal fluid in stains on clothing, weapons, etc.;
(ii) Post-mortem diagnosis of anthrax from a dead or decomposed animal;
(iii) Detection of food adulteration and
(iv) Determination of the kind of an animal a mosquito as recently fed on, to help preventing spread of arthropod-borne diseases.
Serological Reaction # 2. Agglutination Test:
Agglutination-test is that in which visible clumping or aggregation of cells or particles takes place due to the reaction of surface-bound antigens of such cells or particles with homologous antibodies (Fig. 41.20).
Pathogens causing many diseases like typhoid fever (Salmonella typhi), gonorrhoea (Neisseria gonorrhoeae), rickettsial diseases are detected by agglutination-tests; it is probably best known for its use in human blood typing (haemagglutination).
Four types of human blood (A, B, AB, O) are recognized on the basis that the human red blood cells (RBCs) possess either type A or type B polysaccharide antigens, or both type A and type B polysaccharide antigens, or neither of these two antigens on their surface, respectively.
Blood types are determined by mixing known antisera (anti-A and anti-B antibodies) with a blood sample. An agglutination reaction indicates the presence of the corresponding antigen.
Type A blood possesses type A but not type B antigens on the surface of RBCs; type B blood has the type B but not type A antigens on the surface of RBCs; type AB blood possesses both type A and type B antigens on the surface of RBCs; and, type O blood has neither type A nor type B antigens on the surface of RBCs.
In those cases where the antigens are not present or cell on particle surfaces and remain free in soluble- state, the direct agglutination-tests normally fail. For detection of such antigens, passive agglutination-test is employed.
In passive agglutination-test (Fig. 41.21), the soluble antigens are taken out, are first attached to the surface of one of the carriers like latex beads, polystyrene, particle, red blood cells, and then mixed with patient’s serum. Homologous antibodies present in serum attach with antigens present on the surface of the carrier forming antigen-antibody-complexes that agglutinate.
An excellent example of passive agglutination-test using latex beads as carrier is one of the modern pregnancy test; other examples are the detection of pathogens like Haemophilus influenzae (meningitis), Streptococcus pneumoniae (pneumonia), Neisseria meningitis (meningococcal meningitis), Treponema pallidum (Syphilis), Rubella virus (German measles), etc.
Serological Reaction # 3. Fluorescent-Antibody Technique (Immunofluorescence):
The fluorescent-antibody technique (immunofluorescence) is often used to identify unknown antigen. The technique is based on the behaviour of certain dyes which fluoresce (glow) when exposed to certain wavelengths of light. Such dyes are: fluorescenin isothiocyanate which emits an apple-green glow, and rhodamine isothiocyanate which emits orange-red light.
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Fluorescent-antibody technique may be direct or indirect. In direct method, known antibody molecules are conjugated (labelled) with a fluorescent dye; the dye-labelled antibodies combine with those microbes that possess complementary antigens on their surface.
The mixed microbial population is viewed under fluorescence microscope with an excitation wavelength appropriate for the dye; only dye-labelled antibody attached microbial cells fluoresce and become visible (Fig. 41.22A). In indirect method, the initially applied antibody is not labelled (conjugated) with the dye.
Instead, a second labelled antibody is applied which binds the fluorescent label to the specific antibody that has already reacted with its complementary antigen present on the surface of the microbial cells in the mixed population (Fig. 41.22B).
Fluorescent-antibody technique particularly helps identifying specific strains of microorganisms within a mixed microbial population; it is also useful in identifying those pathogenic microbes that are difficult or impossible to culture in vitro.
Serological Reaction # 4. Radioimmunoassay (RIA):
Radioimmunoassay (RIA) is a widely accepted and highly sensitive serological test in which one of the reactants—antibody, antigen or hapten—is radiolabeled with radioactive isotopes of elements like iodine (125I) or hydrogen (3H) are detected in situ by radioautography.
This technique was developed in 1960s to detect hormones such as insulin, and is now used to quantify very low levels of polypeptides, steroids, thyroid hormones, vitamin B12, and viruses.
The steps involved in radioimmunoassay are:
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(i) A sample containing an unknown quantity of antigenic substance and its specific antibodies that react to form antigen-antibody-complex, is taken.
(ii) A known amount of radiolabeled antigenic substance is added which combines with unreacted antibody molecules in the sample forming radiolabeled antigen-antibody complex,
(iii) The radiolabeled antigen-antibody-complex is separated from the sample and its amount is determined,
(iv) The concentration of the unknown antigenic substance in the sample is calculated (Fig. 41.23).
The basis of radioimmunoassay is the competition between a known amount of an antigenic substance that is radiolabelled and an unknown amount of the same antigenic substance that is non-radiolabelled for the same antibody.
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The relative amounts of radiolabeled and non-radiolabelled antigenic substance that bind with the antibody molecules indicates the levels of the antigenic substance in the sample. High levels of antigen- antibody-complex (radiolabelled) indicates a low level of unknown antigenic substance whereas low level of radiolabelled antigen-antibody-complex indicates a high level of unknown antigenic substance in the sample.
Serological Reaction # 5. Enzyme-Linked Immunosorbant Assay (ELISA):
ELISA has been pioneered by two groups of scientists, one in Sweden by Engvall and Perlmann, and the other in Holland by Van Weeman and Schurs in 1972, and developed by Clark and Adams in 1977. ELISA is based on, as the name suggests, enzyme-linked antibodies adsorbed on some solid surface.
The most commonly employed enzyme is alkaline phosphatase (other enzymes used are peroxidase, glucose oxidase, p-galactosidase, malate dehydrogenase, etc.) and the solid surface is that of micro-ELISA plates (Fig. 41.24) having shallow walls (depressions, capacity 0.4 ml) and made up of polystyrene which has the property to bind with antigen or antibody covalently.
There are two method of ELISA: indirect- ELISA used for the detection and measurement of antibody and direct-ELISA used for the detection of antigen.
1. Indirect-ELISA:
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Indirect-ELISA is performed involving the following steps (Fig. 41.25):
(i) The wells (depressions) of the micro-ELISA plates are first filled with antigen which adsorb to the well surface, the wells are then emptied and washed so that free antigens are removed.
(ii) Test-antiserum is added to these walls and allowed to incubate; if the antibodies in the antiserum are homologous, they bind with adsorbed antigens forming antigen- antibody complex. The wells are again washed to remove free antibodies, if any.
(iii) Enzyme- conjugated (labelled) antibodies are now added which link to the antigen-antibody-complex formed in the previous step. Unlinked enzyme-conjugated antibodies are washed away.
(iv) A substrate that reacts with the enzyme is added. The substrate is degraded (hydrolysed) as a result of its reaction with enzyme and a coloured product is resulted in.
(v) The concentration of the antibody present in the test antiserum can be estimated by measuring the intensity of the colour using spectrophotometer. It is because the intensity of the colour is associated with amount of substrate degradation which is directly proportional to the amount of enzyme-linked antibody which, in turn, is proportional to the amount of antibody present in the test-antiserum.
2. Direct-ELISA or Double-Antibody Sandwich ELISA:
Direct-ELISA (or double-antibody sandwich ELISA) is performed involving the following steps (Fig. 41.26):
(i) The wells of the micro-ELISA plate are filled with antiserum, the antibodies present in the antiserum adhere to the surface of each well. The wells are then emptied and washed so that free antibodies are removed.
(ii) The test-antiserum is added and, if the antigens are homologous, they bind with absorbed antibodies forming antigen-antibody-complex. The wells are again washed to remove free antigens, if any.
(iii) Enzyme-conjugated (labelled) antibodies specific to the antigen are then added. These antibodies link to the antigen already fixed by the first antibody; this results in an antibody (with enzyme)-antigen-antibody “sandwich”. Washing helps removing unbind enzyme-conjugated antibodies at this stage.
(iv) Enzyme substrate is added which is degraded (hydrolysed) as a result of its action with the enzyme, and results in colour change that can be visualized or measured using spectrophotometer.
(v) The intensity of colour is proportional to the enzyme action, which is directly proportional to the quantity of enzyme-conjugated antibodies present, and that in turn is proportional to the amount of the test-antigen.
ELISA is advantageous over other methods of serology because of its simplicity, less expensiveness, sensitivity, and accuracy similar to that of radioimmunoassay (antigens and antibodies detectable at levels of about 10-10 g/ml or part in ten billion), stability of reagents (reagents remain fully functional both immunologically and enzymatically throughout the process), and most importantly, the lack of radiation hazards as the radioisotopes are not used. ELISA, however, is a time saving device and can be completed within hours even in laboratories with rudimentary facilities if prepared enzyme-conjugated antibodies are available.
Serological Reaction # 6. Complement Fixation Test:
Complement fixation refers to the ability of antigen-antibody complex to bind complement so that the latter becomes “fixed” and “used up” (Fig. 41.27). The complement fixation is used in complement fixation test (CFT), which is very versatile and sensitive and can be used to detect extremely small ; mount of an antibody (as little as 0.04 μg) for a suspect microorganism in an individual’s serum.
Complement fixation test is performed in two stages. In stage 1, test serum (for the detection of antibody) and the antigen are mixed carefully in a test tube. If the test serum contains antibody then antigen-antibody complexes (immune complexes) are formed. Now a measured amount of complement is added to the mixture and the latter is incubated at 37°C for one hour. The complement is fixed and used by the immune complexes.
In stage 2, sensitized indicator cells, usually sheep red blood cells previously coated with complement-fixing antibodies, are added to the mixture; the indicator cells help determining whether the complement has been fixed and consumed in stage 1 reaction or not.
One of the two, positive and negative, tests are found at this stage. If the complement has been fixed and consumed during stage I reaction, insufficient amount of complement will be available to lyse the indicator cells and; therefore, a positive test is obtained.
On the other hand, if the complement has not been fixed and consumed due to absence of antibodies during stage 1 reaction, the unused complement results in lysis of the indicator cells and, therefore, a negative test is obtained. Absence of lysis of indicator cells (positive test) shows that specific antibodies are present in the test serum, whereas presence of lysis (negative test) shows that specific antibodies are absent in the test serum.
Complement fixation test was once used in the diagnosis of syphilis and is currently employed in the diagnosis of certain viral, rickettsial, chlamydial, protozoan, and fungal diseases.
