Monoclonal Antibodies Against Cardiac Myosin Heavy Chain

Abstract

Three mouse IgG1 monoclonal antibodies (MAbs), named FA1, FA2, and FA3, against cardiac myosin heavy chain (MHC) with high specificity have been obtained. The immunogen used to generate these MAbs was the high-salt- and detergent-insoluble fraction of adult rat myocardial tissue. Western blots showed that these MAbs reacted with a 200 kD protein band, which comigrated with the heavy chain of purified rat cardiac myosin in SDS-PAGE. Immunofluorescence microscopy revealed that the antigen recognized by these MAbs was localized at the A-band of isolated myofibrils. The tissue-, species-, and isoform-specificities of these MAbs were examined by Western blots on various muscle samples. FA2 recognized fish, frog, chicken, rabbit, bovine, mouse and rat cardiac MHC, as well as rabbit skeletal and rat aorta smooth muscle MHC. This antibody reacted equally well with both alpha- and beta-isoforms of MHC. FA1 did not crossreact with any MHC tested so far but with rat cardiac MHC. It appeared to react only with alpha-isoform of MHC. FA3 recognized only rat, bovine and rabbit cardiac MHC with the specificity to bovine and rabbit atrial MHC. Elisa competition assay revealed that different epitopes on the antigen molecules were recognized by these three MAbs, although there was a partial overlap between the epitopes for FA1 and FA2. These anti-MHC MAbs will be most useful in investigating the expression of MHC during myocardial development.