Cresswell P, Ayres J L
Eur J Immunol. 1976 Feb;6(2):82-8. doi: 10.1002/eji.1830060203.
Papain-solubilized HLA antigens giving only two bands of 34 000 mol.wt. and 11 000 mol.wt. by sodium dodecyl sulfate (SDS) gel electrophoresis and isolated from the cultured human lymphoblastoid cell line RPMI 4265, have been used to prepare antisera in rabbits. Antisera were raised against soluble products of the A (or 1 st) series of HLA, bearing the determinant HLA-A2 (A2 substance), or against a mixture of products of the B (or 2nd) series of HLA, bearing the determinants HLA-B7 and HLA-B12 (B7-12 substances). Rabbit antisera to A2 substance reacted primarily with A2 substance on Ouchterlony analysis, showing an apparent spur of cross-reactivity with B7-12 substances. Rabbit antisera to B7-12 substances reacted primarily with B7-12 substances, giving a spur of cross-reactivity with the A2 material. Neither antiserum precipitated beta-2microglobulin. Both types of sera reacted with membrane molecules of 43 000 mol.wt. and 11 000 mol.wt. by immune precipitation and gel electrophoresis in SDS of detergent-solubilized radiolabeled membranes from cultured cell lines, as predicted for sera directed towards HLA antigens. F(ab')2 fragments of the antibodies blocked the complement-mediated cytotoxicity of all HLA alloantisera tested for human peripheral blood lymphocytes. After absorption with B7-12 substances, F(ab')2 fragments of antisera to A2 substance only blocked the cytotoxicity of HLA alloantisera to A series specificities. After absorption with A2 substance, F(ab')2 fragments of rabbit antisera to B7-12 substances only blocked the cytotoxicity of HLA alloantisera to B series specificities. The results prove the existence of shared antigenic determinants between all members of the same series. These findings support the genetic evidence that A series HLA antigens are allelic products of a single locus, while B series HLA antigens are allelic products of a separate locus, by establishing some invariance of structure, presumably amino acid sequence, between members of the same series. The apparent cross-reactivity between A2 substance and B7-12 substances, and the ability of the unabsorbed F(ab')2 preparations to block the cytotoxicity of all HLA alloantisera, suggests that some determinants are common to both HLA loci. This may be considered to support the hypothesis that the two loci arose by gene duplication.
木瓜蛋白酶可溶解的HLA抗原，通过十二烷基硫酸钠（SDS）凝胶电泳仅呈现出两条分子量分别为34000和11000的条带，这些抗原从培养的人淋巴母细胞系RPMI 4265中分离得到，并用于在兔体内制备抗血清。抗血清是针对带有决定簇HLA - A2的HLA A（或第1）系列的可溶性产物（A2物质），或针对带有决定簇HLA - B7和HLA - B12的HLA B（或第2）系列的产物混合物（B7 - 12物质）产生的。在双向免疫扩散分析中，兔抗A2物质的抗血清主要与A2物质反应，与B7 - 12物质呈现出明显的交叉反应刺。兔抗B7 - 12物质的抗血清主要与B7 - 12物质反应，与A2物质产生交叉反应刺。两种抗血清均未沉淀β2微球蛋白。通过对培养细胞系中去污剂溶解的放射性标记膜进行免疫沉淀和SDS凝胶电泳，这两种血清均与分子量为43000和11000的膜分子发生反应，正如针对HLA抗原的血清所预期的那样。抗体的F(ab')2片段阻断了所有检测的针对人外周血淋巴细胞的HLA同种异体抗血清的补体介导的细胞毒性。用B7 - 12物质吸收后，抗A2物质抗血清的F(ab')2片段仅阻断了HLA同种异体抗血清对A系列特异性的细胞毒性。用A2物质吸收后，兔抗B7 - 12物质抗血清的F(ab')2片段仅阻断了HLA同种异体抗血清对B系列特异性的细胞毒性。结果证明了同一系列所有成员之间存在共同的抗原决定簇。这些发现通过确立同一系列成员之间结构（推测为氨基酸序列）的某种不变性，支持了遗传证据，即A系列HLA抗原是单个基因座的等位基因产物，而B系列HLA抗原是另一个独立基因座的等位基因产物。A2物质与B7 - 12物质之间明显的交叉反应，以及未吸收的F(ab')2制剂阻断所有HLA同种异体抗血清细胞毒性的能力，表明某些决定簇在两个HLA基因座中是共有的。这可以被认为支持了两个基因座由基因复制产生的假说。
