Petersen J S, Dyrberg T
Hagedorn Research Laboratory, Gentofte, Denmark.
J Immunol Methods. 1992 Jul 6;151(1-2):15-26. doi: 10.1016/0022-1759(92)90102-y.
To study the expression of HLA-DQ beta chain alleles associated with type 1 diabetes, mAbs were generated from mice immunized with synthetic peptides representing allelic HLA-DQw7 and HLA-DQw8 beta chain sequences. The splenocytes from immunized mice were fused with myeloma cells, either immediately after or following additional in vitro boosting with peptide. Peptide-specific mAbs, predominantly of the IgG isotype, were isolated only from in vitro boosted splenocytes. Immunoblot analysis showed that several of the mAbs cross-reacted with DQ beta chain molecules. One mAb to a peptide representing DQw8 beta position [49-60] specifically recognised the DQw8 beta chain. Three mAbs to a peptide representing DQw8 beta position [39-52] specifically recognised an epitope consisting of Gly-Val-Tyr in position 45-47, i.e., all DQ beta alleles except DQw7 beta (position 45-47: Glu-Val-Tyr) and DQw2 beta (position 45-47; Gly-Glu-Phe). In FACS analysis these mAbs bound lymphocytes with the same specificity as found by immunoblotting analysis. Thus, by combining in vivo and in vitro immunization we have generated a number of epitope specific monoclonal IgG antibodies that distinguish closely related HLA-DQ beta chain alleles in predetermined positions.
为研究与1型糖尿病相关的HLA - DQβ链等位基因的表达，用代表等位基因HLA - DQw7和HLA - DQw8β链序列的合成肽免疫小鼠，制备单克隆抗体（mAb）。免疫小鼠的脾细胞与骨髓瘤细胞融合，融合操作可在免疫后立即进行，也可在体外再用肽刺激后进行。仅从体外刺激的脾细胞中分离出主要为IgG同种型的肽特异性mAb。免疫印迹分析表明，几种mAb与DQβ链分子发生交叉反应。一种针对代表DQw8β链[49 - 60]位肽段的mAb特异性识别DQw8β链。三种针对代表DQw8β链[39 - 52]位肽段的mAb特异性识别由45 - 47位的甘氨酸 - 缬氨酸 - 酪氨酸组成的表位，即除DQw7β链（45 - 47位：谷氨酸 - 缬氨酸 - 酪氨酸）和DQw2β链（45 - 47位；甘氨酸 - 谷氨酸 - 苯丙氨酸）外的所有DQβ等位基因。在荧光激活细胞分选（FACS）分析中，这些mAb与淋巴细胞结合的特异性与免疫印迹分析结果相同。因此，通过体内和体外免疫相结合，我们产生了一些表位特异性单克隆IgG抗体，这些抗体能够区分预定位置上密切相关的HLA - DQβ链等位基因。
