Staining of Complement and Modifications of Fluorescent Antibody Procedures

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Abstract
Summary: A modification has been developed of the procedure for conjugating antisera with fluorescein. This involves the use of fluorescein isocyanate impregnated filter discs and labeling is carried out without the use of organic solvents. The quality of the conjugate is determined by spectrophotometric determination of the fluorescein and protein contents. A fluorescein:protein ratio of 3.8–5.5 × 10-3 indicates satisfactory conjugation, whereas lower ratios give less satisfactory staining. It has been shown in a variation of the indirect staining procedure that guinea pig complement can be used in the staining of several antigens in combination with antisera from various animal species. Only one conjugate is, therefore, needed for the visualization of antigens which, in combination with antibody, will fix complement. The role of the various components of complement in the staining reaction was investigated and no evidence was found for the fixation of C′3. C′1, C′2 and C′4, all had to be present simultaneously for full staining to occur and no evidence was found for attachment of midpiece in the absence of endpiece nor of endpiece in the absence of midpiece.
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