丙氨酸氨基转移酶单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibodies against alanine aminotransferase

目的制备特异性抗人丙氨酸氨基转移酶(ALT)单克隆抗体(McAb)并鉴定其特性。方法用纯化的ALT抗原(20μg/次)免疫BALB/C小鼠5只,取小鼠脾细胞与SP2/0骨髓瘤细胞(按细胞数5∶1比例)融合,采用间接ELISA筛选杂交瘤细胞,阳性孔经3次有限稀释法克隆。用ELISA检测腹水及上清的抗体效价和相对亲和力,用Western blotting检测McAb的特异性,用秋水仙素阻断法进行染色体分析。结果得到1株杂交瘤细胞株(7D1),其染色体数目为95—106,其分泌的抗体为IgG1,轻链为κ型;上清和腹水抗体效价分别为10-4和10-6;SDS-PAGE显示该抗体在55和25 kD附近各有1条带,与预计的分子量大小一致。纯化抗体的亲和常数为2.2×1011。PAGE后Western blot检测显示该McAb可与人血清中的ALT结合。结论所制备的杂交瘤细胞株能分泌抗人ALT的特异性McAb。

Objective To obtain the hybfidoma secreting ALT monoclonal antibodies , and to prepare monoclonal antibodies against ALT and to identify the ALT monoclonal antibodies. Methods Five Balb/c mice were immunized subcutaneously four times with 20μg of ALT in 50% Freund＇s adjuvant at a interval of 2 weeks, and titers in immunized mice sera were determined by indirect ELISA . The splenic lymphocytes from the immunized mouse were fused to myeloma Sp2/0 cells （5：1 ）and cultured selectively with HAT and HT. Each supematant was screened by its reactivity to ALT. One of antibody-producing wells was selected for further use through following successive subelonings by limiting dilution method. The selected hybridoma cells were proliferated and injected into Balb/c mice for generation of ascites. The antibodies in the ascites were purified by the method of acetic acid amino sulphate precipitation. The titers and affinities of ascites and were determined by indirect ELISA. The chromosome of hybridoma cells was analyzed by the method of Colehicine treatment. The subtype of the antibodies was identified by SBA Clontyping System/HRP. The obtained ALT monoclonal antidodies were analyzed by SDS-PAGE. The specificity of the monoclonal antibody was assessed by Westem blotting. The application was studied by a Sandwich ELISA which was established after the ALT monoclonal antibodies were labeled with Horseradish peroxidase. Results One hybridoma cell line（7Dl ） secreting monoclonal antibodies against ALT was obtained. The chromesome number of 7D1 hybfidoma cells varied from 95 to106. The subtype of 7D1 monoclonal antibodies was IgG1. The titer of ascites and supernatant are 1 ： 10^4 and 1 ： 10^6 , respectively. SDS-PAGE of ALT monoclonal antibodies showed that heavy and light chain with a relative molecular weight of 55KDa and 23Kda, respectively. The purified monoclonal antibodies showed good affinity and specificity against ALT in Western blotting. The biochemistry value of ALT was consistent with the result of Sandwich ELISA. Conclusion In present study, one strain hybridoma cell was obtained, which could stably secret ALT monoclonal antibodies. The antibodies that prepared showed good affinity and could recognize ALT specially without cross-reactions with other proteins in human serum, which provide a potential value for establishing the colloidal gold immunochromatography assay.