Production and characterization of anti-estrone monoclonal antibody.
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).


CONCLUSIONS
The CI-ELISA developed with E1-McAb was both sensitive and specific. The prepared E1-McAb can be used in some immunoassays.
Wang
Yong-Cheng
YC
College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China. ychwang@chemms.chem.pku.edu.cn
Guo
Zhen-Quan
ZQ
Li
Yuan-Zong
YZ
Chang
Wen-Bao
WB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D015153:Blotting, Western; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004970:Estrone / Q000276:immunology*; D005260:Female; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D004970:Estrone