Complement fixation assay

Complement fixation assays can be used to look for the presence of specific antibody or antigen in a patient s serum. The test uses sheep red blood cells (SRBC), anti-SRBC antibody and complement, along with specific antigen (if looking for antibody in serum) or specific antibody (if looking for antigen in serum). If antibody... 

FIGURE 6.10 Complement fixation assay. Left Antigen is present in the patient s serum, then the complement is completely utilized and SRBC lysis is minimal. Right Antigen is not present in the patient s serum, then the complement binds anti-SRBC antibody and lysis of the SRBCs ensues. SRBC, sheep red blood cells. 

Antinucleotide sera have been tested also for binding of radiolabeled hapten-protein conjugates or labeled DNA and with quantitative complement fixation assays. Once characterized, specific serum or purified antibodies may be used also in immunohistochemical tests at the level of light or electron microscopy. 

The complement-fixation assay depends on the fact that during the reaction of an antibody with its antigen, added complement is also fixed. The amount of complement fixed is directly proportional to the quantity of antigen which has reacted with the antibody, and therefore, in the presence of an excess of the latter, it is directly proportional to the quantity of antigen added. Another immunological system is... 

The specificity of the complement-fixation system depends directly on the specificity of the antiserum, which should react exclusively with the gonadotropin being estimated. Butt and Lynch (B12) performed experiments designed to compare the specificity of a micro complement-fixation test with that of a radioimmunoassay for FSH. Their results demonstrated that their FSH antibody preparation cross-reacted with LH and HCG in the radioimmunoassay system, and could not be used because, when absorbed with these substances, it no longer reacted with FSH. However, with the same antiserum in the complement-fixation system, neither HCG nor LH fixed complement when present in concentrations ranging from 1 to 6000 ImU/ml, the maximum complement fixation occurring with 30 ImU FSH. It was concluded that their complement-fixation assay was specific for FSH. There are other examples in which complement fixation tests appear to be more specific than the radioimmunoassay systems (see Section 6.1.4.). This may arise from the fact that a system must be multivalent if complement is to be fixed, i.e., the antibody and antigen must have several complementary points... 

Fig. 27. Correlation of results of tests for parietal cell and IF antibodies in pernicious anemia patients, showing their independence. The parietal cell antibodies were tested by complement fixation test (CF) while the IF antibodies were assayed by electrophoretic retention test, i.e., immobilization of the intrinsic factor related Bj2 binder on electrophoresis. From Taylor et al. (T13).

Unlike the other immunological assays which have been used for the estimation of the gonadotropic hormones, complement fixation is not an inhibition reaction. Rather does it rely on the direct reaction between the hormone and its specific antibody. 

Antisera specific for FSH and LH are rare and have invariably been produced by chance. The specificity of such antisera varies depending on such factors as the purified material used as the antigen for testing and the types of assay method employed for the final determination. In general, it may be said that complement-fixation tests are more specific... 

New techniques have also been introduced to increase the sensitivity of the standard cross-match and allow the identification of antibodies that are poor activators of complement. These techniques include extending the incubation times of the standard cross-match (T7) and the addition of an anti-human globulin step (F4, J3). The most sensitive assay, however, is a cross-match using flow cytometry, which does not involve complement fixation (G3). 

Figure 2. Complement fixation immunoassay. Red color released due to the lysis of red blood cells indicates a negative assay, i.e., the absence of the target antigen.

Comparative serology a relative standard of comparison. It may be impractical, technically difficult, or impossible to obtain definitive proof of infection via culture or isolation techniques. In the absence of such a gold standard, less exacting methods must serve as the standard of comparison with the new assay. If the other tests have already established assay performance characteristics (e.g., the Rose Bengal screening test followed by the complement fixation confirmatory test for detection of antibody to Brucella bovis), their results taken together provide a useful composite-based standard by which the new assay may be compared. 

The assay for complement fixation was carried out with a mixture of IgGa and IgGb. 

Techniques for the detection or assay of various substances based on the reaction of those substances with specific antibodies (or vice versa, i.e. the detection and assay of antibodies using antigens). Such techniques include agglutination reactions, automated immune precipitation, complement fixation tests, crossed electrophoresis, counter electrophoresis, double diffusion, enzyme immunoassay, fluoroimmunoassay, haem-agglutination, immunoelectrophoresis, immunofluoresence, radial immunodiffusion, spin immunoassay, immunofixation, immunoradiometric assay and radioimmunoassay. See separate entries for these subjects. 

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