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Detection of Bound C3 by a New Immunochemical Method

From Biology Branch, Immunochemistry Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014

Abstract

C3 bound to sheep erythrocytes (EC3) reacts with anti-C3 antibody. The C3-anti-C3 complex fixes complement and can be detected quantitatively by either 1) the C1 fixation and transfer test or 2) the amount of lysis which occurs when whole complement is added to the erythrocyte-C3-anti-C3 complex. The hemolytic activity of anti-C3 antibody is inhibited by fluid phase C3 or by C3 bound to immune complexes. We have devised a quantitative test for C3 antigen based on the inhibition of anti-C3 antibody by C3. The C3 to be measured (in either bound or fluid phase form) is added to a standard amount of anti-C3 antibody. Residual antibody activity is then determined by adding the absorbed antiserum to sheep EC3 indicator cells and measuring lysis of the cells which occurs after addition of whole guinea pig complement. By comparing the observed lysis with an inhibition-of-lysis curve obtained with known C3 antigen, the amount of test antigen can be estimated. The method is simple, rapid and reproducible and can detect nanogram amounts of bound C3.