Lengweiler S, Smyth S S, Jirouskova M, Scudder L E, Park H, Moran T, Coller B S
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA.
Biochem Biophys Res Commun. 1999 Aug 19;262(1):167-73. doi: 10.1006/bbrc.1999.1172.
To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from beta3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.
为了获得针对血小板蛋白的小鼠特异性单克隆抗体（mAb），用洗涤过的小鼠血小板免疫一只亚美尼亚仓鼠。然后将免疫脾细胞与一种不分泌的小鼠骨髓瘤细胞系融合，并利用酶联免疫吸附测定（ELISA）筛选产生的异源杂交瘤，该ELISA中靶抗原是在凝血酶存在下吸附于微量滴定板上的小鼠血小板。二次筛选试验包括使用小鼠纤维蛋白原或来自β3整合素基因敲除小鼠的血小板进行ELISA检测、流式细胞术、免疫印迹、免疫沉淀以及一项功能测定，以鉴定抑制血小板 - 纤维蛋白原相互作用的抗体。鉴定出了产生针对小鼠糖蛋白（GP）IIb/IIIa、纤维蛋白原、CD9和其他血小板整合素的仓鼠mAb的杂交瘤细胞。对在免疫沉淀分析中与GPIIb/IIIa复合物反应的两种杂交瘤（1B5和9C2）进行了两次亚克隆。通过聚集和黏附试验进行的功能分析表明，1B5完全抑制血小板 - 纤维蛋白原相互作用，而9C2不影响血小板聚集或血小板黏附。
