Muller H P, van Tilburg N H, Derks J, Klein-Breteler E, Bertina R M
Blood. 1981 Nov;58(5):1000-6.
Spleen cells of a BALB/c mouse immunized with factor VIII procoagulant activity (VIII:C) (isolated by affinity chromatography) were fused with mouse myeloma cells (P3 x 63 Ag8). After the fusion 12/32 wells produced an inhibitor to VIII:C. Cells from one well (1B3) were subcloned four times in order to isolate the hybridoma that produces the anti-VIII:C antibody. Injection of hybridoma cells in pristane pretreated BALB/c mice results in anti-VIII:C titers of 5000-10,000 Bethesda U/ml. Analysis of the produced immunoglobulin demonstrated heavy chains of IgG1 (produced by the myeloma cell line) and IgG2b subclass. The 1B3 antibody neutralizes VIII:C in LMW FVIII, crysosupernatant, cryoprecipitate, and normal plasma. It was found that binding of the IgG to FVIII results in a delay in its activation and not in an inhibition of its cofactor activity. The antibody removes VIII:C from pooled normal plasma when coupled to Sepharose; when coupled to plastic tubes, it binds VIIICAG from isolated VIII:C, purified FVIII, and pooled normal plasma; it does not bind VIIIR:AG, fibrogen, or serum VIIICAG. The 1B3 antibody can be used successfully in an IRMA for VIIICAG.
用通过亲和层析分离的凝血因子VIII促凝活性（VIII:C）免疫的BALB/c小鼠的脾细胞，与小鼠骨髓瘤细胞（P3×63 Ag8）进行融合。融合后，32个孔中有12个产生了VIII:C抑制剂。来自一个孔（1B3）的细胞进行了4次亚克隆，以分离产生抗VIII:C抗体的杂交瘤。将杂交瘤细胞注射到经 pristane预处理的BALB/c小鼠中，产生的抗VIII:C效价为5000 - 10,000贝塞斯达单位/毫升。对产生的免疫球蛋白的分析表明，其重链为IgG1（由骨髓瘤细胞系产生）和IgG2b亚类。1B3抗体可中和低分子量FVIII、冷上清液、冷沉淀物和正常血浆中的VIII:C。研究发现，IgG与FVIII的结合导致其激活延迟，而非抑制其辅因子活性。当与琼脂糖偶联时，该抗体可从混合正常血浆中去除VIII:C；当与塑料管偶联时，它可结合分离的VIII:C、纯化的FVIII和混合正常血浆中的VIIICAG；它不结合VIIIR:AG、纤维蛋白原或血清VIIICAG。1B3抗体可成功用于VIIICAG的免疫放射分析。
