The following points highlight the five main types of serological reactions used for detection of antibodies. The reactions are:- 1. Complement Fixation Test 2. Fluorescent Antibody Techniques 3. Radioimmunoassay 4. Radioallergosorbent Test 5. Enzyme Linked Immunosorbent Assay.
Reaction # 1. Complement Fixation Test:
The complement fixation test was devised by Jules Bordet and Octave Gengou in 1901. It was later adapted for syphilis by August von Wassermann in 1906, and for many decades it remained a mainstay for syphilis diagnosis. Modern technologists now use it for detection of antibodies against a variety of viruses, fungi, and bacteria.
The test is performed in two parts. The first part, the test system, utilizes the patient’s serum, a preparation of antigen and complement derived from guinea pigs. The second part, the indicator system, requires sheep red blood cells and hemolysin (antibodies against sheep red cells). Hemolysins cause lysis of red blood cells in the presence of complement.
The first step in the test is to heat the patient’s serum to 56°C for 30 minutes. This destroys any complement present in the serum and allows the laboratory technician to regulate its amount. Next, carefully measured amounts of antigen and guinea pig complement are added to the serum.
This test system is then incubated at 37°C for 90 minutes. During this period, if antibodies specific for the antigen are present in the serum, an antibody-antigen reaction takes place and the complement is used up, or fixed. However, there is no visible sign of whether a reaction has occurred.
After the incubation period, the indicator system (sheep red blood cells and hemolysin) is added to the tube, and the tube is reincubated at 37°C for an additional two hours. If the complement was previously fixed, none would be available to the hemolysin, and lysis of the sheep red blood cells could not take place.
The blood cells would therefore remain intact and, when the tube is centrifuged, the technician observes clear fluid with a “button” of blood cells at the bottom. Conclusion – the serum contained antibodies that reacted with the antigen and fixed the complement.
If the complement was not fixed in the test system, it would still be available to the hemolysin and the hemolysin-complement mixture would lyse the sheep red blood cells. When the tube is centrifuged, the technician sees red fluid, coloured by the hemoglobin of the broken blood cells, and no evidence of blood cells at the bottom of the tube. Conclusion – the serum lacked antibodies for the antigen tested.
The complement fixation procedure is valuable because it may be adapted by varying the antigen. In this way, tests may be conducted for such diverse diseases as encephalitis, Rocky Mountain spotted fever, meningococcal meningitis, and histoplasmosis. The versatility of the test together with its sensitivity and relative accuracy have secured its continuing role in diagnostic medicine.
Reaction # 2. Fluorescent Antibody Techniques:
The fluorescent antibody technique is a slide test performed by combining particles containing antigens with antibodies and a fluorescent dye. When the three components react, the dye causes the complex to glow on illumination with ultraviolet light under a fluorescent microscope. Two co2mmonly used dyes are fluorescein, which emits an apple-green glow, and rhodamine, which gives off orange-red light.
Fluorescent antibody techniques may be direct or indirect.
i. Direct Method:
In the direct method, the fluorescent dye is linked to known antibody molecules. The antibodies are then combined with particles such as bacteria that may contain complementary antigens. If the presumption is correct, the tagged antibodies accumulate on the particle surface and the particle glows under the microscope. In this way, an unknown antigen or unknown organism can be identified.
ii. Indirect Method:
The indirect method is illustrated by the FTA-ABS diagnostic procedure used for detecting syphilis antibodies in the blood of a patient. A sample of commercially available syphilis spirochetes is placed on a slide, and the slide is then flooded with patients serum.
Next, a sample of fluorescein-labeled antiglobulin antibodies is added. Antiglobulin antibodies are antibodies that unite with human antibodies. They are produced by an animal injected with human antibodies. The slide is then observed under the fluorescent microscope.
The test is interpreted as follows. If the patient’s serum contains syphilis antibodies, the antibodies bind to the surfaces of spirochetes and the labeled antiglobulin antibodies are attracted to them. The spirochetes then glow from the dye.
However, if no antibodies are present in the serum, nothing accumulates on the spirochete’s surface, and labeled antiglobulin antibodies also fail to gather on the surface. The labeled antibodies remain in the fluid and the spirochetes do not glow.
Fluorescent antibody techniques are adaptable to a broad variety of antigens and antibodies and are widely used in serology. Antigens may be detected in bacterial smears, cell smears, and viruses fixed to carrier particles. The value of the techniques is enhanced because the materials are sold in kits and are readily available to small laboratories.
Reaction # 3. Radioimmunoassay (RIA):
Radioimmunoassay (RIA) is an extremely sensitive serological procedure used to measure the concentration of low molecular weight antigens such as haptens. Since its development in the 1960s, the process has been adapted for quantitating hepatitis antigens, as well as reproductive hormones, insulin, and certain drugs. One of its major advantages is that it can detect trillionths of a gram of substances.
The radioimmunoassay procedure is based upon the competition between radioactive-labeled antigens and unlabelled antigens for the reactive sites on antibody molecules. A known amount of the radioactive (labeled) antigens, is mixed with a known amount of specific antibodies, and an unknown amount of unlabelled antigens.
The antigen-antibody complexes that form during incubation are then separated out, and their radioactivity is determined. By measuring the radioactivity of free antigens remaining in the leftover fluid (unbound labeled antigen), one can calculate the percentage of labeled antigen bound to the antibody. This percentage is equivalent to the percentage of unlabeled antigen bound to the antibody because the same proportion of both antigens will find spots on antibody molecules.
The concentration of unknown unlabelled antigen can then be determined by reference to a standard curve. The curve is constructed from data obtained by allowing varying amounts of unlabeled known antigen to compete with antibody and determining the percentage of bound to free antigens.
Radioimmunoassay procedures require substantial investment in sophisticated equipment and carry a certain amount of risk because radioactive isotopes are used. For these reasons, the procedure is not widely used in routine serological laboratories. However, immunologists with access to radioimmunoassay have discovered a wealth of information.
Reaction # 4. Radioallergosorbent Test (RAST):
The radioallergosorbent test (RAST) is an extension of the radioimmunoassay. Another sophisticated procedure, it may be used to detect IgE, other antibodies, or a variety of small antigens.
To detect IgE against penicillin, penicillin antigens are attached to a suitable particle. Serum that may contain penicillin IgE is then added. If the antibody is present, it will combine with the penicillin antigens on the surface of the particle. Now another antibody, one that will react with human antibodies, is added. This antiglobulin antibody carries a radioactive label.
The entire complex will therefore become radioactive if the antiglobulin antibody combines with the IgE. By contrast, if no IgE was present in the serum, no reaction with the antigen on the particle surface will take place, and the radioactive antibody will not be attracted to the particle. When tested, the particles will not show radioactivity.
The RAST is commonly known as a “sandwich” technique. There is no competition for an active site as in RIA, and the type of unknown antibody as well as its amount may be learned by determining the amount of radioactivity deposited.
Reaction # 5. Enzyme Linked Immunosorbent Assay (ELISA):
Enzyme linked immunosorbent assay (ELISA) has virtually the same sensitivity as radioimmunoassay and RAST, but does not require expensive equipment or radioactivity. The procedure involves attaching antibodies or antigens to a solid surface and combining (immunosorbing) the coated surfaces with the test material.
An enzyme system is then linked to the complex, the remaining enzyme is washed away, and the extent of enzyme activity is measured. This gives an indication that antigens or antibodies are present in the test material.
An application of ELISA is found in the hour-long laboratory test used to detect antibodies against human immunodeficiency virus (HIV). A serum sample is obtained from the patient and mixed with a solution of plastic or polystyrene beads coated with antigens from HIV. Antibodies present in the serum will adhere to the antigens on the surface of the beads.
The beads are then washed and incubated with antiglobulin antibodies chemically tagged with molecules of the enzyme horseradish peroxidase. The preparation is washed and a solution of substrate molecules for the peroxidase enzyme is added. Initially the solution is clear, but if enzyme molecules react with the substrate, the solution will become yellow-orange in colour.
The enzyme molecules will be present only if HIV antibodies are present in the serum. If no HIV antibodies are in the serum, no enzyme molecules could concentrate on the bead surface, no change in the substrate molecules could occur, and no colour change would be observed.
ELISA procedures may be varied depending on whether one wishes to detect antigens or antibodies. The solid phase may consist of beads, paper disks, or other suitable supporting mechanisms, and alternate enzyme systems such as the alkaline phosphatase system may be used.
In addition, the results of the test may be quantitated by noting the degree of enzyme-substrate reactions as a measure of the amount of antigen or antibody in the test sample. Availability of the ELISA tests as inexpensive kits has brought the procedure into the doctor’s office and routine serological laboratory, and broad applications of the test are expected in the future.