Characterization of an anti-H-2 monoclonal antibody and its use in large-scale antigen purification

A rat anti-mouse monoclonal antibody (MAb), M1/42, has been found to react with H-2 antigens from cells of the a, b, d, j, k, s, and u haplotypes (all haplotypes tested). This antibody, when bound to cells and reacted with FITC-conjugated anti-rat Ig, could be used to quantitate H-2 expression on several cell types. The antibody was also useful in comparing the H-2 products precipitated from a variety of haplotypes. M1/42-coupled Sepharose-4B beads were used to purify H-2(d) antigens by affinity chromatography. Pure H-2 molecules eluted from the column in 0.5% DOC, 0.65 M NaCl, 20 mM Tris, pH 8.0, yielding 110 to 180 μg H-2(d)/10¹⁰ P815 tumor cells. This antibody, when used in series with H-2K(k)-specific MAb 11-4.1, allowed purification of D(k) and D(d) from RDM-4 and YAC cells, respectively. H-2(d) purified by column chromatography or M1/42 was found to be serologically and biologically active, as determined by MAb rebinding, inhibition of cell lysis by alloantisera plus complement, and ability to stimulate alloreactive CTL. This antibody and the described protocols should be useful in the preparation of relatively large quantities of a number of H-2 antigens.