Study of heart-reactive antibody in antisera and hybridoma culture fluids against group A streptococci

Abstract

Heart-reactive antibody is known to be produced in animals immunized with group A streptococci. However, the detection and quantitation of these antibodies and their respective antigens have been limited to immunofluorescence or immunoprecipitation tests. In this study a more sensitive technique was used to detect heart-reactive antibody, i.e., the enzyme-linked immunosorbent assay (ELISA). Countercurrent immunoelectrophoresis and agar gel immunodiffusion were also used to confirm the reactivity of rabbit antisera to group A streptococci and human heart tissue. Human heart tissue antigen was prepared by Triton X-100 extraction, and checkerboard titrations of heart tissue antigen versus streptococcal antisera revealed optimal concentrations of each for the ELISA. When immune rabbit serum was reacted with heart tissue antigen, a 5- to 10-fold increase was observed over the reactions of preimmune sera controls. Streptococcal antiserum was found to have a six- to eightfold greater reactivity with heart tissue antigen than with similar concentrations of kidney and skeletal muscle antigens. Heart tissue antigen was partially purified by DEAE-cellulose chromatography, and quantities of greater than 10 to 100 ng (dry weight) were shown to react with streptococcal antisera in the ELISA. Heart, kidney, and skeletal muscle antigens were subjected to electrophoresis in polyacrylamide gel slabs and blotted onto a nitrocellulose filter. These filter blots reacted with streptococcal antisera and confirmed the tissue antigen specificity observed with the ELISA. In addition, the ELISA was found to be an effective method for the detection of heart-reactive antibodies produced by murine hybridomas that were producing antibody to group A streptococci.