Monoclonal antibody recognizing rabbit IgG (Fab). A specific reagent for second antibody applications

A monoclonal mouse antibody, which recognizes a determinant on the Fab portion of rabbit IgG, has been obtained following fusion of hyperimmune mouse spleen cells with NS-1 plasmacytoma cells. The monoclonal antibody, RIg9C, has a gamma 1 heavy chain and a kappa light chain. RIg9C binds rabbit IgG but not human or bovine IgG. It has been conjugated with horseradish peroxidase for application in an enzyme-linked immunosorbent assay as well as for detection of antigens transferred from polyacrylamide gels to nitrocellulose. In enzyme-linked immunosorbent assays, the RIg9C-peroxidase conjugate was useful for measuring rabbit immunoglobulin either free in solution or in immune complexes. Using the enzyme-linked immunosorbent assay to quantitate antibody-antigen binding, the apparent dissociation constant was found to be 4 X 10(-8) M. Because of its low dissociation constant and specificity, RIg9C will be preferred over other reagents (e.g. second antibody and protein A) where sensitive recognition of only the Fab segment is critical.