Production and characterization of monoclonal antibodies to Fc gamma 2a-binding protein isolated from the detergent lysate of a murine macrophagelike cell line, P388D1.
Production and characterization of monoclonal antibodies to Fc gamma 2a-binding protein isolated from the detergent lysate of a murine macrophagelike cell line, P388D1.
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从鼠类巨噬细胞系 P388D1 的去污剂裂解液中分离出 Fc gamma 2a 结合蛋白的单克隆抗体的制备和表征。
DOI：
10.1002/jlb.45.4.311
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发表时间：
1989
影响因子：
5.5
通讯作者：
Suzuki,T
中科院分区：
文献类型：
--
作者：
Kagami,M;Funatsu,Y;Suzuki,T
Mybridoma 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-rbinding 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 IgG1class antibodies, which inhibited more than 70% of the binding of radio-iodinated myeloma IgG2aprotein to P38SD1cells. The 3A2 Fab fragments bound specifically to P3S8D1cells at 4° with a KDof 1.9 × 10-8M and Bmax of 2.9 × 105per cell. This Fab fragment also specifically bound to Fcγ 2a receptor (R)-positive T cell line (S49) with a KDof 4.4 × 10-9M and a Bmax of 1.0 × 104but did not bind to Fcγ 2a-negative S49 variant cell line, cyc-. The flow cytometric analysis with the use of fluorescein-isothiocyanate-tagged 3A2 F(ab’)2also showed that this antibody binds to Fcγ 2aR-positive cells, P388D1and S49, but not to Fcγ 2aR-negatlve cells, cyc-. Monomeric and heat-aggregated IgG2a(13-fold molar excess) inhibited the binding of the radioiodinated 3A2 F(ab’)2to P388D1cells by 70 and 49%, respectively, whereas the inhibition by monomelic and heat-aggregated IgG2bwas 17 and 39%, respectively; 3A2 F(ab’)2(100-fold molar excess) inhibited the binding of IgG2aand IgG2bto P38SD1cells 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 P388D1or 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). Fcγ 2aR may thus exist at the cell surface as a disulfide linked dimer of a subunit of Mrof 50,000, which could be partially degraded during the isolation to smaller fragments of 40,000 and 35,000 Mrpeptides which are still held together by interchain disulfide bond. Furthermore, 3A2 antibody-binding protein isolated from P388D1cell membrane lysate was found to exhibit casein kinase activity, which was previously found to be associated with IgG2a- but not with lgG2b-binding protein isolated from a similar cell lysate.
