抗雌酮单克隆抗体的制备与鉴定

Production and characterization of anti-estrone monoclonal antibody.

作者信息

Wang Yong-Cheng, Guo Zhen-Quan, Li Yuan-Zong, Chang Wen-Bao

机构信息

College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

出版信息

Biomed Environ Sci. 2002 Jun;15(2):103-12.

PMID:12244751

Abstract

OBJECTIVE

Determination of estrone (E1) levels has a significant meaning in evaluating physiological effect and diagnosing some diseases. In order to detect free E1 in biological fluids, a monoclonal antibody specific for E1 was prepared after the complete antigen of E1 was synthesized. The purified monoclonal antibody was fully characterized for later immunoassay.

METHODS

3-O-carboxymethyl ether derivative of E1 was synthesized and in turn coupled to bovine serum albumin (BSA) to form complete antigen E1-BSA. A monoclonal antibody (McAb) specific for E1 was produced both in vitro and in vivo by a hybridoma anti-E1. Anti-E1 was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse. The McAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western-blotting. The specificity of the immunoassay was investigated by determining the cross-reactions of E1 analogs when free E1 was detected by competitive indirect enzyme-linked immunosorbent assay (CI-ELISA).

RESULTS

Analysis revealed that anti-E1 McAb (E1-McAb) was of the IgG1 type, the molecular weight of E1-McAb was 164,000 daltons. The affinity constant of E1-McAb with coated complete antigen was 8.2 x 10(8) L/mol. The linear range for free E1 determined by CI-ELISA was 10 pg/mL-10 ng/mL. The detection limit was 21.4 pg/mL (defined as twice the standard deviation of the blank).

CONCLUSION

The CI-ELISA developed with E1-McAb was both sensitive and specific. The prepared E1-McAb can be used in some immunoassays.

摘要

目的

测定雌酮（E1）水平在评估生理效应和诊断某些疾病方面具有重要意义。为了检测生物体液中的游离E1，在合成E1的完全抗原后制备了针对E1的单克隆抗体。对纯化的单克隆抗体进行了全面表征，以便后续进行免疫测定。

方法

合成E1的3 - O - 羧甲基醚衍生物，然后与牛血清白蛋白（BSA）偶联形成完全抗原E1 - BSA。通过抗E1杂交瘤在体外和体内产生针对E1的单克隆抗体（McAb）。抗E1是通过将SP2/0鼠骨髓瘤细胞与从免疫的BALB/c小鼠分离的脾细胞融合制备的。通过酶联免疫吸附测定（ELISA）、十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和蛋白质印迹法（Western - blotting）对McAb进行表征。在通过竞争性间接酶联免疫吸附测定（CI - ELISA）检测游离E1时，通过测定E1类似物的交叉反应来研究免疫测定的特异性。

结果

分析表明，抗E1 McAb（E1 - McAb）为IgG1型，E1 - McAb的分子量为164,000道尔顿。E1 - McAb与包被的完全抗原的亲和常数为8.2×10⁸ L/mol。CI - ELISA测定游离E1的线性范围为10 pg/mL - 10 ng/mL。检测限为21.4 pg/mL（定义为空白标准差的两倍）。