Maloy W L, Hämmerling G, Nathenson S G, Coligan J E
J Immunol Methods. 1980;37(3-4):287-99. doi: 10.1016/0022-1759(80)90314-2.
Previous studies on the determination of the complete amino acid sequence of H-2 molecules have relied on alloantisera to purify the H-2 molecules by immunoprecipitation. In this study, directed toward the determination of the complete amino acid sequence of the H-2Db molecule, it was found that all H-2.2 alloantisera examined had very low antibody titers when analyzed by immunoprecipitation and that most of these antisera reacted with not only the H-2Db heavy chain but also with a 75,000 dalton virus-associated glycoprotein (gp 75). The problems encountered with conventional alloantisera were surmounted by utilizing a monoclonal antibody from hybridoma B22-249R1 obtained as described by Hämmerling et al. (1979) and maintained in ascites form in (CBA x Balb/c) F1 mice. This antibody reacted with only 45,000 molecular weight material and had the added advantage of requiring only 0.1 ml of hybridoma ascites fluid to bind all of the H-2Db antigen from 3 x 10(8) EK-4 tumor cells. Biochemical examination of the purified H-2Db molecule showed that it possessed overall structural properties similar to other previously characterized H-2 molecules. An NH2-terminal amino acid sequence was determined for the H-2Db molecule for 28 residues and this sequence was compared to the complete NH2-terminal sequences of the H-2Kb and H-2Dd molecules, and the partial NH2-terminal sequence for the H-2Ld molecule; these comparisons indicated 82%, 75% and 100% homology respectively. In addition, these preliminary studies indicated that there was little or no beta-2-microglobulin (beta 2 mu) in the immunoprecipitates with the H2-2Db heavy chain. This lack of beta 2 mu in the immunoprecipitates was found to be true for alloantisera directed against private or public specificities, a xenoantiserum (sheep anti-H-2b), or hybridoma antibody used in the immunoprecipitation.
以往关于确定H-2分子完整氨基酸序列的研究依赖于同种异型抗血清，通过免疫沉淀来纯化H-2分子。在本研究中，为了确定H-2Db分子的完整氨基酸序列，发现通过免疫沉淀分析时，所有检测的H-2.2同种异型抗血清的抗体效价都非常低，并且这些抗血清中的大多数不仅与H-2Db重链反应，还与一种75,000道尔顿的病毒相关糖蛋白（gp 75）反应。利用如Hämmerling等人（1979年）所述获得并以腹水形式保存在（CBA×Balb/c）F1小鼠体内的杂交瘤B22-249R1产生的单克隆抗体，克服了传统同种异型抗血清遇到的问题。这种抗体仅与分子量为45,000的物质反应，并且具有仅需0.1 ml杂交瘤腹水就能结合来自3×10(8)个EK-4肿瘤细胞的所有H-2Db抗原的额外优势。对纯化的H-2Db分子进行生化检测表明，它具有与其他先前表征的H-2分子相似的整体结构特性。确定了H-2Db分子28个残基的氨基末端氨基酸序列，并将该序列与H-2Kb和H-2Dd分子的完整氨基末端序列以及H-2Ld分子的部分氨基末端序列进行了比较；这些比较分别显示出82%、75%和100%的同源性。此外，这些初步研究表明，与H2-2Db重链的免疫沉淀物中几乎没有β2微球蛋白（β2μ）。发现在针对私有或公有特异性的同种异型抗血清、异种抗血清（羊抗H-2b）或用于免疫沉淀的杂交瘤抗体的免疫沉淀物中都不存在β2μ。
