Microcomplement fixation

A number of immunological techniques have been used in comparative studies.8,9 The most important of these is microcomplement fixation (MC F), a quantitative technique that has played a key role in many classic studies of molecular evolution and molecular systematics. By selecting proteins with different rates of evolution, a broad range of divergences can be examined. The cost of the technique is moderate, but biochemical expertise is required and the labor involved is substantial. Protein must be purified from some or all taxa for antibody production, and, for those taxa, a sizable tissue or serum sample is needed. Antibody production itself is usually done in rabbits, so an animal care facility must be available. Like isozyme electrophoresis, the large body of immunological distance data already available ensures the continued value of this technique for certain investigations. 

For over 25 years, the microcomplement fixation method1 has allowed rapid estimation of the approximate degree of sequence difference between monomeric, globular proteins from many different species.2,3 With this immunological method and two other ways of comparing proteins, elec-... 

Fig. 1. Confidence limits and prediction intervals for immunological comparisons of five proteins. For each protein the heavy central line is the regression line through the origin relating immunological distance in the microcomplement fixation test to percent difference in amino acid sequence, and the shaded region portrays the 95% confidence limits for that line. The outer two lines in each graph define the boundaries of the intervals for one prediction made at the 90% level of confidence.

It is clear that the degree of immunological cross-reaction between various antigens varies with the type of assay used. Nakai and Parlow raised an antiserum to highly purified LH and, using a microcomplement fixation test, were able to show that HCG did not cross-react with this antiserum. However, the same antiserum did react in both the hemagglutination-inhibition and radioimmunoassay systems (Nl). Schuurs et al. 

Since serum from unimmumzed rabbits also interfered with the fixation of complement, it was necessary to employ IgG for microcomplement fixation tests. In a typical experiment, 6 xg of anti-GAD IgG and 5-100 ng of GAD was used in each tube Isotonic veronal buffer containing Ca and Mg and 0.1% gelatin was used as a diluent (Mayer, 1961). Sensitization of sheep blood cells with an optimal amount of hemolysin was performed as described (Mayer, 1961). 

Wasserman E, and Levine L (1961) Quantitative microcomplement fixation and its use in the study of antigenic structure by specific antigen-antibody inhibition. /. Immunol. 87, 290-295... 

---