Antigen is a substance which when introduced into the tissues of an animal by any route provokes an immune response after a latent period which can be demonstrable by, (i) production of antibody in blood and other body fluids which reacts with antigen, (ii) specific cell mediated immunity, (iii) increased reactivity known as hypersensitivity.
Antibody is the substance produced in response to introduction of antigen with which it reacts specifically in some observable way.
Antigen and antibody by definition combine with each other specifically and in an observable manner. These reactions can be used for the detection and quantitation of either antigens or antibodies. Antigen-antibody reactions in vitro are known as serological reactions.
- Precipitation reaction:
- When a soluble antigen combines with its antibody in the presence of electrolytes (NaCl) at a suitable temperature and pH, the antigen-antibody complex forms an insoluble precipitate. When instead of sedimenting the precipitate remains suspended as floccules, the reaction is known as flocculation.
Antigen antibody reaction in agarose gel is commonly performed in the clinical laboratory by a technique called double immunodiffusion. In this technique, cylindrical holes or wells are cut out of an agarose gel in a small petri dish and spaced appropriately. The specimen containing the unknown soluble antigen is placed in one well, and a known antibody-containing solution is placed in an adjacent well. The antigen and antibody molecules in solution diffuse out of the well and through the porous agarose. If antigen specific for the known antibody is present, the two components combine and produce a visible precipitin band, or line of precipitation, at a point of optimal concentration of each component. Diffusion is a slow process and is not generally amenable to rapid diagnosis. It is most commonly used to detect fungal exo-antigens or serum antibodies.
CIE is a modification of the principle of immunodiffusion that dramatically quickens the migration of soluble antigens and antibodies by applying an electric current.
- The antigen formerly used in the VDRL test was a colloidal suspension of tissue cardiolipin, which in the presence of cholesterol and lecithin, reacted with cardiolipin autoantibodies to form floccular type agglutination. Today chemically synthesized cardiolipin is used as the sole antigen.
- The VDRL test uses serum that has been heat treated for 30 minutes at 560 C. The serum must be allowed to cool to room temperature before being tested. The test is usually performed as a slide technique. A measured drop of diluted antigen suspension is added to a measured volume of serum on a slide. After rotating the slide for 4 minutes, the preparation is examined microscopically for clumping of antigen particles.
- Positive sera are reported as “reactive” if the clumps are of medium or large size, or as “weakly reactive” if clumps are small. All reactive or weakly reactive sera require diluting to estimate the antibody titre.
The antigen used in the RPR card test is similar to that used in the VDRL test. The antigen particles, however, are either carbon containing or dyed to enable the reaction to be read macroscopically on a card. No heating of the patient’s sera is required and plasma or serum can be used for the RPR card test. A reactive serum shows definite clumping of the antigen particles or slight roughness. A reactive serum requires serial diluting to estimate the antibody titre.
- Agglutination reaction:
- When a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated
- The Widal test performed reliably and interpreted with care, can be of value in the diagnosis of typhoid fever in endemic areas when facilities for culture are not available.
- The patient’s serum is tested for O and H antibodies against the antigen suspensions of S. typhi O9, 12 & S. typhi Hd for diagnosis of typhoid fever.
- The Widal test is reported by giving the titre for both O and H antibodies. The antibody titre is taken as the highest dilution of serum in which agglutination occurs. The type of agglutination seen with O reaction is granular while that seen with H reaction is more uneven type of clumping usually described as floccular.
- If no agglutination occurs the test should be reported as:
- “S. typhi O titre less than 1 in 20,
- S. typhi H titre less than 1 in 20”.
- Interpretation of the Widal test:
- The agglutination titre will depend on the stage of the disease. Agglutinins usually appear by the end of the first week. The titre increases steadily till the third or the fourth week, after which it declines gradually.
- The result of a single test should be interpreted with caution. It is difficult to lay down levels of significance though it is generally stated that titres of 1/100 or more for O agglutinins and 1/200 or more for H agglutinins are significant.
- Demonstration of a rise in titre of antibodies, by testing two or more serum sample is more meaningful than a single test.
- Complement fixation:
- Complement system, the normal component system of plasma proteins is necessary for complete action of lytic antibodies. Antigen-antibody complex fixes the complement. Complement is absorbed during the combination of antigens with their antibodies. In presence of appropriate antibodies, complement lysis erythrocytes, kills or lyses bacteria, immobilize motile organisms, promotes phagocytosis and immune adherence and contributes to tissue damage in hypersensitivity.
Complement fixation test:
- The CFT is a technique that has been used to detect and quantify antibody that does not agglutinate or precipitate when reacted with its antigen but can be demonstrated by its use, or fixation, of complement.
- When complement takes part in antigen antibody reactions it is bound, or fixed, to the antigen antibody complexes. When these complexes are on bacteria, red cells or other cells the complement brings about the lysis of the cells involved. In the CFT, the patient’s inactivated serum is serially diluted and reacted with known antigen in the presence of complement. If the corresponding antibody is contained in the serum it will combine with the antigen and use up the complement. This will leave no complement to hemolyse the antibody coated red cells that are added. The highest dilution of serum that prevents hemolysis is the antibody titre. If patient’s serum however does not contain the corresponding antibody, the complement will not be used and will be available to fix and hemolyse the antibody coated red cells.
- Most complement fixation tests are specific but not always very sensitive. CFTs are still used in the diagnosis of rickettsial infections and several viral and parasitic infections.
- Neutralization reaction:
- When an antitoxin combines with a toxin, the biological effects of the toxin are neutralized rendering it harmless to animals. Since toxin is an antigen in solution it is also precipitated. Toxin-antitoxin neutralization can be measured in vivo and in vitro.
Elek’s gel precipitation Test:
- Toxigenic strains of C. diphtheriae are demonstrated by the Elek’s gel precipitation technique, an in vitro immunodiffusion test.
- A rectangular strip of filter paper soaked in antitoxin (1000 units/ml) is placed on the surface of a serum agar plate containing 20% horse serum while the medium is still in fluid form. When the medium solidifies, the testing strain is streaked across the plate at right angles to the filter paper strip and then incubated at 370 C for 24 to 48 hours. Toxin produced by bacterial growth diffuses in agar medium and produces line of precipitation where it meets antitoxin molecules in optimum concentration. The presence of such arrowhead lines of precipitates indicates that the strain is toxigenic.
Various immunoassays are now used for diagnosis of infectious diseases.
- Radioimmunoassays are specialist techniques with high specificity and sensitivity and performed in reference laboratories that have equipment to measure radioactivity and facilities for the safe storage and handling of radioactive materials.
- In the radioimmunoassays the radioactivity of a specific isotope labelled antibody or antigen is used to detect and quantify antigen in test specimen.
- Immunofluorescent tests are widely used in the serological diagnosis of bacterial, viral, fungal, and parasitic diseases. They are usually sensitive.
- Fluorescent dyes (fluorochromes) illuminated by ultra-violet (UV) light are used to show the specific combination of an antigen with its antibody. The antigen antibody complexes are seen fluorescing against a dark background.
- Immunofluorescence tests are referred to as fluorescent antibody test (FAT). They are of two types, direct and indirect.
Direct fluorescent antibody test: A direct FAT is used to detect and identify unknown antigen in specimen, for example viral, bacterial, and parasitic antigens. It is called a direct test because the fluorescent dye is attached, or labelled, directly to the antibody. In the direct FAT, the specimen is placed on a microscope slide. Fluorescent-labelled specific antibody is added and time allowed for the antigen and antibody to react. The preparation is then washed leaving on the slide only the labelled antibody that has combine with antigen. When examined with fluorescent microscopy using correct filter the antigen antibody complexes are seen fluorescing due to the fluorochromes attached to the antibody.
Indirect fluorescent antibody test: In the indirect FAT, unlabelled antibody combines with antigen and the antigen antibody complexes are detected by attaching a fluorescent-labelled anti-species globulin to the antibody.
Enzyme linked immunosorbent assays (ELISA):
- The enzyme linked immunosorbent assay uses an enzyme system to show the specific combination of an antigen with its antibody.
- The enzyme system consists of: An enzyme, which is labelled, or linked, to a specific antibody or antigen. A substrate is a substance which is added after the antigen antibody reaction. This substrate is acted on by the enzyme attached to the antigen antibody complexes, to give a colour change. The intensity of the colour gives an indication of the amount of the bound antigen or antibody.
- There are two main ways to perform ELISA:
- Detection of antigen: Specific antibody is coated on the wall of a microtiter plate and the specimen is added. After a period of incubation during which the antibody takes up the antigen form the specimen, the well is washed leaving the antigen attached to the antibody. Enzyme labelled specific antibody is added to detect the presence of the antigen. After a further period of incubation during which the enzyme labelled antibody combines with the antigen, the well is washed and a substrate is added. The enzyme acts on the substrate to give a colour change in the fluid. The enzyme activity is stopped by altering the pH of the reaction or denaturing the enzyme. By measuring the colour produced, the amount of attached antibody and therefore of antigen in the specimen can be estimated.
- Detection of antibody: Known antigen is attached to the inside surface of the well and patient’s serum is added. After incubation and washing, enzyme labelled anti-human globulin is reacted with the antibody that has been attached to the antigen. The uncombined labelled enzyme is washed from the well and a substrate is then added. The presence and concentration of antibody that has reacted with the antigen is shown by a change in colour of the substrate. Indirect ELISA is used in the diagnosis of several parasitic, bacterial, viral and fungal infections.
- Western blotting:
- It is used to identify the bacterial antigen in the patient’s serum. Proteins in the patient’s serum are separated by a process called electrophoresis. The proteins are than transferred to a filter by blotting. Next, antibodies tagged with a dye are washed over the filter. If the specific antigen is present in the serum, the antibodies will combine with it and will be visible as a coloured band on the filter. Western blotting is used to diagnose HIV infection.