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目的制备磺胺二甲嘧(Sulfadimidine,SM2)啶单克隆抗体,并对其进行鉴定。方法经小鼠腹腔免疫SM2-BSA抗原,采用常规技术进行细胞融合,间接竞争ELISA法筛选阳性杂交瘤细胞株,有限稀释法进行亚克隆,硫酸铵-辛酸法纯化抗体,并鉴定其抗体效价、杂交瘤细胞染色体数量、相对分子质量、抗体亚型、亲和力和特异性。结果筛选到1株稳定分泌抗体的杂交瘤细胞株3D7,细胞培养液上清和抗体效价分别为1∶6.4×102和1∶1.28×105;纯化后3D7抗体效价>1∶128 000,杂交瘤细胞染色体平均数为50±2对,分泌的抗体属于IgG1亚类,相对分子质量为164 280,亲和常数为6.09×108 M-1,与6种磺胺类药物及青霉素、庆大霉素和泰乐霉素交叉反应IC50大于1 mg/ml,交叉反应率小于0.01%。结论已成功制备了SM2单克隆抗体,该抗体能满足建立免疫学检测方法的需要。
Objective To prepare monoclonal antibody against sulfadimidine (SM2) and to identify it. Methods SM2-BSA antigens were immunized by intraperitoneal injection in mice. Cell fusion was performed by conventional techniques. Positive hybridoma cells were screened by indirect competitive ELISA. Subclones were obtained by limiting dilution method. Antibodies were purified by ammonium sulfate-octanoic acid method. , Hybridoma cell chromosome number, relative molecular mass, antibody subtype, affinity and specificity. Results One stable hybridoma cell line secreting antibody 3D7 was screened. The cell culture supernatant and antibody titers were 1: 6.4 × 102 and 1: 1.28 × 105, respectively. The titer of purified 3D7 antibody was 1:128 000, The average number of chromosomes in tumor cells was 50 ± 2 pairs. The secreted antibodies belonged to IgG1 subclass, with a relative molecular mass of 164 280 and an affinity constant of 6.09 × 108 M-1. The antibodies against six sulfonamides and penicillins, gentamicin Cross-reactivity with tylosin IC50> 1 mg / ml, cross-reactivity rate <0.01%. Conclusion The SM2 monoclonal antibody has been successfully prepared, which can meet the needs of establishing immunological detection methods.