Rotblat F, Goodall A H, O'Brien D P, Rawlings E, Middleton S, Tuddenham E G
J Lab Clin Med. 1983 May;101(5):736-46.
C was purified from intermediate-purity concentrate by adsorption on polyelectrolyte E5 and affinity chromatography on Sepharose/anti-VIIIR:Ag. The highly purified VIII:C preparation (sp. act. 1598 U/mg) was used to immunize Balb-C mice. Spleen cells from a mouse with a serum antibody titer of 963 U/ml were fused with P3 NSI mouse myeloma cells. Hybrid clones were screened by a coagulation inhibition assay and by a four-layer antibody adsorption procedure. Nine monoclonal antibodies specific to VIII:C were produced. Five of these antibodies have been cloned and grown in mouse ascitic fluid. Antibody titers from ascitic fluid ranged from 35 to 82,000 BU/ml. The antibodies, when radiolabeled, form a high-molecular-weight complex with antigens present in normal plasma and factor VIII concentrate, but not when incubated with CRM-negative hemophilic plasma. A two-site assay using a combination of monoclonal antibodies is able to detect VIII:CAg in normal plasma and in factor VIII concentrate. Sensitive two-site immunoradiometric assays using monoclonal antibodies as the solid phase have been set up.
通过在聚电解质E5上吸附以及在琼脂糖/抗VIII R:Ag上进行亲和层析，从中间纯度浓缩物中纯化出VIII:C。使用高度纯化的VIII:C制剂（比活性为1598 U/mg）免疫Balb-C小鼠。将血清抗体效价为963 U/ml的小鼠的脾细胞与P3 NSI小鼠骨髓瘤细胞融合。通过凝血抑制试验和四层抗体吸附程序筛选杂交克隆。产生了9种对VIII:C特异的单克隆抗体。其中5种抗体已被克隆并在小鼠腹水中培养。腹水中的抗体效价范围为35至82,000 BU/ml。这些抗体经放射性标记后，可与正常血浆和因子VIII浓缩物中的抗原形成高分子量复合物，但与CRM阴性的血友病血浆孵育时则不会。使用单克隆抗体组合的双位点测定法能够检测正常血浆和因子VIII浓缩物中的VIII:CAg。已建立了以单克隆抗体为固相的灵敏双位点免疫放射测定法。
