Production and characterization of monoclonal antibodies to Fc gamma 2a-binding protein isolated from the detergent lysate of a murine macrophagelike cell line, P388D1

Hybridoma cell lines were produced by fusion of SP2/0 murine myeloma cell line with the spleen cells of Wister rats which were immunized with IgG2a-binding protein isolated from the detergent lysate of a murine macrophagelike cell line, P388D1, by affinity chromatography on IgG-Sepharose 4B. A monoclonal clone (designated as 3A2) out of a total of 13 different antibody-secreting cell lines was found to secrete IgG1 class antibodies, which inhibited more than 70% of the binding of radio-iodinated myeloma IgG2a protein to P388D1 cells. The 3A2 Fab fragments bound specifically to P388D1 cells at 4 degrees C with a KD of 1.9 x 10(-8) M and Bmax of 2.9 x 10(5) per cell. This Fab fragment also specifically bound to Fc gamma 2a receptor (R)-positive T cell line (S49) with a KD of 4.4 x 10(-9) M and a Bmax of 1.0 x 10(4) but did not bind to Fc gamma 2a-negative S49 variant cell line, cyc-. The flow cytometric analysis with the use of fluorescein-isothiocyanate-tagged 3A2 F(ab')2 also showed that this antibody binds to Fc gamma 2aR-positive cells, P388D1 and S49, but not to Fc gamma 2aR-negative cells, cyc-. Monomeric and heat-aggregated IgG2a (13-fold molar excess) inhibited the binding of the radioiodinated 3A2 F(ab')2 to P388D1 cells by 70 and 49%, respectively, whereas the inhibition by monomeric and heat-aggregated IgG2b was 17 and 39%, respectively; 3A2 F(ab')2 (100-fold molar excess) inhibited the binding of IgG2a and IgG2b to P388D1 cells by 90 and 24%, respectively, whereas the inhibition of binding of these IgG to S49 cells was 79 and 49%, respectively. Western blotting analysis showed that 3A2 antibody recognizes a major protein (Mr = 100,000) and a minor component (Mr = 80,000) separated by SDS-PAGE of P388D1 or S49 cell lysates under nonreducing condition, whereas under reducing condition, this antibody recognized a major protein (Mr = 50,000) and two additional minor components (Mr = 40,000 and 35,000). Fc gamma 2aR may thus exist at the cell surface as a disulfide linked dimer of a subunit of Mr of 50,000, which could be partially degraded during the isolation to smaller fragments of 40,000 and 35,000 Mr peptides which are still held together by interchain disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)