THE PREPARATION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES TO HUMAN-COMPLEMENT COMPONENT C8 AND THEIR USE IN PURIFICATION OF C8 AND C8 SUBUNITS
THE PREPARATION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES TO HUMAN-COMPLEMENT COMPONENT C8 AND THEIR USE IN PURIFICATION OF C8 AND C8 SUBUNITS
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DOI：
10.1042/bj2510285
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发表时间：
1988-04-01
影响因子：
4.1
通讯作者：
LUZIO, JP
中科院分区：
文献类型：
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作者：
ABRAHA, A;MORGAN, BP;LUZIO, JP
1. 制备了10种抗人补体成分C8的小鼠单克隆抗体。结果发现其中六种抗体与。α反应。-亚基，两个带。的。-亚单位和两个。伽玛。-亚基，在SDS/聚丙烯酰胺凝胶电泳分离C8亚基后进行免疫印迹评估。2. 对10种单克隆抗体进行竞争性结合分析，结果表明6种抗体对。-亚基可分为四个重叠的表位群。抗体。-和。gamma。-亚基与每一个抗原位点结合，但也与-亚基上的抗原位点交叉反应。3. 采用单克隆抗C8免疫亲和柱从新鲜人血浆中纯化C8，制备C8贫血清。免疫亲和纯化的C8在羊和兔红细胞的溶血实验中具有生物活性。4. 用盐洗脱法提纯α和γ。-或。beta。-亚基，当C8分别结合抗-。或反-.alpha。immunoaffinity列。当在溶血试验中加入纯化的亚单位时，重组了c8缺失的血清。
1. Ten mouse monoclonal antibodies to human complement component C8 were prepared. It was found that six of these antibodies reacted with the .alpha.-subunit, two with the .beta.-subunit and two with the .gamma.-subunit, when assessed by immunoblotting after separation of C8 subunits by SDS/polyacrylamide-gel electrophoresis. 2. Epitope analysis of the ten monoclonal antibodies in a competitive binding asssay showed that the six antibodies to the .alpha.-subunit could be classified in four overlapping epitope groups. The antibodies to the .beta.- and .gamma.-subunits bound to a single antigenic site on each, but also cross-reacted with the antigenic sites on the .alpha.-subunit. 3. Monoclonal anti-C8 immunoaffinity columns were used to purify C8 from fresh human plasma and to prepare C8-depleted serum. Immunoaffiity purified C8 was biologically active when assessed by using haemolysis assays of sheep and rabbit erythrocyes. 4. Salt elution was used to purify either .alpha..gamma.- or .beta.-subunits when C8 was respectively bound to an anti-.beta. or anti-.alpha. immunoaffinity column. The purified subunits reconstituted C8-depleted serum when dded together in a haemolysis assay.