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 specifi...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 8.2108L/mol. The linear range for free E1 determined by CI-ELISA was 10pg/mL^10ng/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.展开更多
A method was developed for the simultaneous determination of four kinds of estrogens （hexoestrol, diethylstilbestrol, estrone, and 17-beta-estradiol） in feed by gas chromatography-mass spectrometry （GC-MS）. After ...A method was developed for the simultaneous determination of four kinds of estrogens （hexoestrol, diethylstilbestrol, estrone, and 17-beta-estradiol） in feed by gas chromatography-mass spectrometry （GC-MS）. After the sample was extracted by ethyl ether and cleaned-up on HLB phase extraction column, four kinds of estrogens were derived and quantified in gas chromatographymass spectrometry. The results showed that the linear detectable ranged from 2.5 ng· mL-1 to 250 ng· mL-1for hexoestrol and from 5 ng· mL-1 to 500 ng· mL-1 for three other estrogens with the correlation coefficients （R2） were no less than 0.990. The recoveries were in the range of 76.34%-96.33% and the relative standard deviation was no more than 22.7%. The limits of quantitation （LOQ） for all analytics were between 10 ug· kg^-1 and 20 ug· kg^-1. The method was accurate and sensitive and could meet the actual requirements for the analyses of feed samples.展开更多
Hormone Receptor positive (HR+) breast cancer is the most common malignancy in women. New strategies in the treatments have targeted the estrogen biosynthesis pathways including the inhibition of the aromatase and 17...Hormone Receptor positive (HR+) breast cancer is the most common malignancy in women. New strategies in the treatments have targeted the estrogen biosynthesis pathways including the inhibition of the aromatase and 17β-HSD1 enzymes. The present work, describes the study of a new family of 9 hybrid compounds derived from estrone attached to a coumarin fragment, linked through different lengths of hydrocarbon chains. The activity of these compounds was evaluated by molecular docking with two relevant enzymes in breast cancer (HR+). It has been proposed nine compounds as 17β-HSD1 inhibitors and six as aromatase inhibitors. We found important interactions with key amino acids at the orthosteric site of each enzyme and their score values compared to the crystallographic ligand. The in silico analysis showed good score values in the proposed compounds, where the steroidal portion presented important interactions with Met374 and Tyr155 in aromatase and in 17β-HSD1 respectively. Highlighting Compounds 2, 5 and 8 with an aromatic ring at the C4 position of the coumarin moiety, which favored arene-H type interactions essential for protein-ligand recognition. In addition, the results related to the 17β-HSD1 enzyme demonstrated how the length of the linker influences the interaction;the best score was found for derivative 8 with a chain of 8 methylenes.展开更多
基金This work was supported by National Science Foundation of China (Grant no.20075001).
文摘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 8.2108L/mol. The linear range for free E1 determined by CI-ELISA was 10pg/mL^10ng/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.
基金Supported by Fund of Harbin Provincial Education Department(2014AB3BN041)
文摘A method was developed for the simultaneous determination of four kinds of estrogens （hexoestrol, diethylstilbestrol, estrone, and 17-beta-estradiol） in feed by gas chromatography-mass spectrometry （GC-MS）. After the sample was extracted by ethyl ether and cleaned-up on HLB phase extraction column, four kinds of estrogens were derived and quantified in gas chromatographymass spectrometry. The results showed that the linear detectable ranged from 2.5 ng· mL-1 to 250 ng· mL-1for hexoestrol and from 5 ng· mL-1 to 500 ng· mL-1 for three other estrogens with the correlation coefficients （R2） were no less than 0.990. The recoveries were in the range of 76.34%-96.33% and the relative standard deviation was no more than 22.7%. The limits of quantitation （LOQ） for all analytics were between 10 ug· kg^-1 and 20 ug· kg^-1. The method was accurate and sensitive and could meet the actual requirements for the analyses of feed samples.
文摘Hormone Receptor positive (HR+) breast cancer is the most common malignancy in women. New strategies in the treatments have targeted the estrogen biosynthesis pathways including the inhibition of the aromatase and 17β-HSD1 enzymes. The present work, describes the study of a new family of 9 hybrid compounds derived from estrone attached to a coumarin fragment, linked through different lengths of hydrocarbon chains. The activity of these compounds was evaluated by molecular docking with two relevant enzymes in breast cancer (HR+). It has been proposed nine compounds as 17β-HSD1 inhibitors and six as aromatase inhibitors. We found important interactions with key amino acids at the orthosteric site of each enzyme and their score values compared to the crystallographic ligand. The in silico analysis showed good score values in the proposed compounds, where the steroidal portion presented important interactions with Met374 and Tyr155 in aromatase and in 17β-HSD1 respectively. Highlighting Compounds 2, 5 and 8 with an aromatic ring at the C4 position of the coumarin moiety, which favored arene-H type interactions essential for protein-ligand recognition. In addition, the results related to the 17β-HSD1 enzyme demonstrated how the length of the linker influences the interaction;the best score was found for derivative 8 with a chain of 8 methylenes.
