IMMUNOLOGY - Roitt A. - Mir 2000

Chapter 29. Immunological Methods

COMPLEMENT ASSAY

The simplest way to measure Complement activity is to determine the serum concentration that causes 50% lysis of Cells in a standard preparation of antibody-sensitized erythrocytes (EA). The assay is performed in test tubes or microplates. For an approximate assessment of complement activity, single radial hemolysis is more convenient. This method is similar to single radial immunodiffusion, except that the test serum is placed into the wells, and the gel contains EA. A zone of hemolysis forms around the well containing active complement, the size of which is proportional to The amount of complement in the test serum. This method allows the total activity of the classical and lytic pathway components (C1–C9) to be determined; however, if complement deficiency is detected in the serum, this method cannot determine which specific component is missing.

Methods also exist for assaying various individual complement components based on their concentration or functional activity. This is an important distinction, since a component may be present in normal amounts yet be functionally inactive. The concentration (level) of individual complement Proteins is usually determined by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), using Antibodies specific for the protein in question. To measure functional activity, all complement components necessary for lysis, except for the protein being tested, are added to a suspension of sensitized erythrocytes (Fig. 29.18).

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Fig. 29.18. These tests are used to measure individual complement components in the test serum. The assay involves adding 1) a specific complement reagent containing all components necessary for lysis except one, and 2) the test serum to a suspension of sensitized erythrocytes. For example, to assay for component C4 in the test serum, guinea pig serum deficient in C4 (C-reagent) is added to antibody-sensitized erythrocytes. If the test serum contains C4, hemolysis will occur; conversely, in the absence of C4, there will be no hemolysis. The table shows the combinations of Reagents used to assay each complement component. Erythrocytes are sensitized with antibodies so as to achieve a specific degree of hemolysis upon interaction of EA with complement. Reagents may be sera that naturally lack one of the complement components or those subjected to Physical and Chemical Treatment to remove or inactivate a specific component. Quantitative determination of components can be performed using, for example, single radial hemolysis or the 50% hemolysis tube assay. (EA — antibody-sensitized erythrocytes; EAC — antibody- and complement-sensitized erythrocytes; EGTA — Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid.)