Bonnefoy J Y, Guillot O, Spits H, Blanchard D, Ishizaka K, Banchereau J
UNICET, Laboratory for Immunological Research, Dardilly, France.
J Exp Med. 1988 Jan 1;167(1):57-72. doi: 10.1084/jem.167.1.57.
Two hybridomas that produce the mAbs 135 and 449 B4 were obtained that inhibited the binding of IgE to the Fc epsilon RL/CD23 on the EBV-transformed B cell line RPMI 8866. mAb 135 was obtained from a mouse immunized with RPMI 8866 cells, whereas mAb 449B4 was obtained from a mouse immunized with a partially purified preparation of Fc epsilon RL/CD23 obtained as the eluate of an IgE immunoabsorbent loaded with a soluble extract of RPMI 8866 cells. These two mAbs bound to Fc epsilon RL/CD23- cell lines and precipitated two polypeptides with 36,000 Mr and 28,000 Mr, which were the HLA-DR alpha and beta chains, respectively. Immunoprecipitation with mAb 135 of NP-40 lysates from dithio-bis(succinimidyl propionate) (DSP) crosslinked 125I-labeled RPMI 8866 or normal B cells incubated with rIL-4 showed three polypeptides with 42,000, 36,000, and 28,000 Mr. The 42,000 Mr polypeptide is identical to the Fc epsilon RL/CD23 since it could be precipitated by the anti-Fc epsilon RL/CD23 mAb 25 after resolubilization from the SDS-PAGE gel. Immunoprecipitations of the crosslinked cell extracts carried out with the anti-Fc epsilon RL/CD23 mAb 25 yielded the same three polypeptides. Furthermore, when RPMI 8866 or rIL-4 preincubated normal B cells were solubilized with a digitonin buffer, which prevents the dissociation of noncovalently linked polypeptide complexes, mAb 135 and mAb 25 precipitated complexes composed of three molecules with 42,000, 36,000, and 28,000 Mr. The well-characterized anti-HLA-DR mAb L243 was unable to block the binding of either IgE or mAb 135 to RPMI 8866 cells, although it could immunoprecipitate the complex (HLA-DR-Fc epsilon RL/CD23) from crosslinked cell lysates. Since mAb 135 and L243 were able to both bind the RPMI 8866 cells, it demonstrates that they bind to different epitopes of the HLA-DR complex, the mAb 135 epitope of the HLA-DR molecule being close to the IgE binding site of the Fc epsilon RL/CD23. These data demonstrated that the Fc epsilon RL/CD23 and HLA-DR antigens are spatially associated on the B cell membrane.
获得了两种产生单克隆抗体135和449 B4的杂交瘤，它们可抑制IgE与EB病毒转化的B细胞系RPMI 8866上的FcεRL/CD23的结合。单克隆抗体135是从用RPMI 8866细胞免疫的小鼠中获得的，而单克隆抗体449B4是从用部分纯化的FcεRL/CD23制剂免疫的小鼠中获得的，该制剂是从用RPMI 8866细胞的可溶性提取物加载的IgE免疫吸附剂的洗脱物中获得的。这两种单克隆抗体与FcεRL/CD23阴性细胞系结合，并沉淀出两条多肽，分子量分别为36,000和28,000，分别为HLA-DRα链和β链。用单克隆抗体135对来自二硫代双（琥珀酰亚胺基丙酸酯）（DSP）交联的125I标记的RPMI 8866或与rIL-4孵育的正常B细胞的NP-40裂解物进行免疫沉淀，显示出分子量为42,000、36,000和28,000的三条多肽。42,000分子量的多肽与FcεRL/CD23相同，因为从SDS-PAGE凝胶中重新溶解后，它可以被抗FcεRL/CD23单克隆抗体25沉淀。用抗FcεRL/CD23单克隆抗体25对交联的细胞提取物进行免疫沉淀，得到相同的三条多肽。此外，当用洋地黄皂苷缓冲液溶解RPMI 8866或rIL-4预孵育的正常B细胞时，该缓冲液可防止非共价连接的多肽复合物解离，单克隆抗体135和单克隆抗体25沉淀出由分子量为42,000、36,000和28,000的三个分子组成的复合物。特征明确的抗HLA-DR单克隆抗体L243不能阻断IgE或单克隆抗体135与RPMI 8866细胞的结合，尽管它可以从交联的细胞裂解物中免疫沉淀复合物（HLA-DR-FcεRL/CD23）。由于单克隆抗体135和L243都能够结合RPMI 8866细胞，这表明它们结合到HLA-DR复合物的不同表位上，HLA-DR分子的单克隆抗体135表位靠近FcεRL/CD23的IgE结合位点。这些数据表明FcεRL/CD23和HLA-DR抗原在B细胞膜上在空间上相关联。
