The preparation of mouse VH fragments and the characterization of heterologous anti-mouse VH antibodies.

VH fragments were prepared from several mouse IgM molecules by cyanylation. In all cases VH fragments were purified to homogeneity by using Ig light chain affinity columns. Several different anti-VH antisera were prepared in rabbits and the specificities of these antibodies were studied. Two patterns of cross-reactivities were observed: (a) some anti-VH antibodies reacted only with closely related VH molecules, e.g., anti-VH HPC52 anti bodies reacted only with VH of phosphorylcholine-binding myeloma or hybridoma proteins, and concordantly, stained about 4% of mouse spleen B cells; (b) on the other hand, antisera-like anti-VH 104E and 8916 antibodies were very cross-reactive. Binding assays showed that both of these anti-VH antibodies reacted with 50-60% of mouse immunoglobulins. However, they recognized mainly nonoverlapping populations of mouse immunoglobulins, and thus the pool of these antibodies reacted with about 95% of mouse VH regions. Concordantly, anti-VH 104E antibodies stained in the fluorescence-activated cell sorter (FACS) analysis more than 50% of mouse spleen B cells. Cross-reactive anti-VH antibodies ("anti-framework") did not stain T cells nor did they immunoprecipitate VH-like molecules which were synthesized by T cells.