A solid-phase assay for antiactin antibody and actin using protein-A

A method has been developed for the detection of an immunocomplex between an agaroselinked antigen and its antibody using ¹²⁵I-labeled protein-A. This procedure has been used to follow the appearance of antiactin antibody in rabbits injected with 200 μg of sodium dodecyl sulfate (SDS)-denatured and electrophoretically purified murine skeletal muscle actin on Days 0 and 20. Significant immunoglobulin G (IgG) binding to agarose-actin was detected at about Day 50 and the titer was maximum at about Day 100. The antibody binding reaction is not affected by bovine serum albumin, ovalbumin, or a bacterial extract, but can be blocked by soluble actin. Murine skeletal muscle actin (0.1 mg/ml) prevents about 80-90% of the binding, with 50% inhibition observed at about 10 μg/ml. Using an SDS extract of neuroblastoma cells, only about 60-70% inhibition was obtained, suggesting that the nonmuscle actin in this preparation does not react with all the antibodies which bind to muscle actin. The specificity and general usefulness of this assay are also suggested by the fact that antiactin antibodies do not bind to agarose-BSA, agarose-ovalbumin, or agarose-uterine myosin, while antibodies to the two latter proteins bind preferentially to their respective antigens. Thus, this method can potentially be used to assay any antigen immunologically active in the solid phase, the antibody to that antigen, and also crossreacting antigens.