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目的制备抗DcR3单克隆抗体(mAb),鉴定其生物学特性,并应用于ELISA、Western blot、Flowcytometry(FCM)检测。方法以纯化的可溶性DcR3(sDcR3)免疫Balb/c小鼠,采用杂交瘤技术制备抗DcR3 mAb。用Ig亚类ELISA试剂盒鉴定抗DcR3mAb的亚类。用ELISA方法测定抗DcR3 mAb与sDcR3结合的特性,SDS-PAGE鉴定抗DcR3 mAb与SW480细胞上清中DcR3结合的特性,以鉴定mAb的特性。用间接ELISA法检测腹水mAb的效价、亲和常数并进行表位分析。Western blot检测mAb的特异性及应用,并用所获抗DcR3单克隆抗体(mAb)通过流式细胞仪检测肿瘤细胞表面DcR3的表达水平。结果获得4株可分泌DcR3 mAb的杂交瘤细胞系ZZ-393、ZZ-394、ZZ-151和ZZ-268。其中DcR3 mAb ZZ-268(下文简称为ZZ-268)的Ig亚类为IgG1(κ型);腹水效价为1×10-5;亲和常数为1.28×109水平;ZZ-268和ZZ-151可识别与其他2种抗体不同的抗原表位;Western blot证实,获得的ZZ-268可特异性地识别DcR3;通过流式细胞术可敏感地检测到不同肿瘤细胞表面DcR3的表达水平。结论获得4株抗DcR3的mAb,其中ZZ-268效价高、特异性强,将此抗体用于膜DcR3与sDcR3的检测分析。
Objective To prepare anti-DcR3 monoclonal antibody (mAb) and identify its biological characteristics, and apply it to ELISA, Western blot and Flowcytometry (FCM). Methods Balb / c mice were immunized with purified soluble DcR3 (sDcR3) and anti-DcR3 mAb was prepared by hybridoma technique. A subclass of anti-DcR3 mAb was identified using an Ig subclass ELISA kit. The binding properties of anti-DcR3 mAb to sDcR3 were determined by ELISA and the binding of anti-DcR3 mAb to DcR3 in SW480 cell supernatant by SDS-PAGE to identify the characteristics of mAb. The titer, affinity constant and epitope analysis of ascites mAb were detected by indirect ELISA. The specificity and application of mAb was detected by Western blot. The expression level of DcR3 on the surface of tumor cells was detected by flow cytometry using anti-DcR3 monoclonal antibody (mAb). As a result, four hybridoma cell lines ZZ-393, ZZ-394, ZZ-151 and ZZ-268 secreting DcR3 mAb were obtained. The Ig subclass of DcR3 mAb ZZ-268 (hereinafter abbreviated as ZZ-268) was IgG1 (κ type); the ascites titer was 1 × 10-5; the affinity constant was 1.28 × 109; the level of ZZ-268 and ZZ- 151 could recognize different antigenic epitopes from the other two kinds of antibodies. Western blot confirmed that ZZ-268 could specifically recognize DcR3. The expression level of DcR3 on different tumor cells could be detected by flow cytometry. CONCLUSIONS Four mAbs against DcR3 were obtained. ZZ-268 was highly potent and specific, and was used for the detection of DcR3 and sDcR3.