| Objective:To establish simple, fast and sensitive immunoassay analytical methods for detecting sodium estrone sulfate (ESS) with preparation of polyclonal antibodies (PcAb) for sodium estrone sulfate. Key techniques such as colloidal gold immunochromato graphic test and ELISA methods would help to develop new tools for detection on great quantity of ESS from pregnant mare urine (PMU) more quickly in fields. Methods:1) ESS and bovine serum albumin (BSA) as key raw materials,. The conjugations of sodium estrone sulfate-17-(O-carboxymethyl)oxime with proteins as a complete antigen were prepared by the mixed acid anhydring method. And the conjugations were analyzed by ultraviolet scanning and SDS-PAGE New Zealand white rabbits were immunized by above immunogen of hapten-BSA according to the immune protocol. Then antiserum was collected in order to prepare polyclonal antibodies of sodium estrone sulfate.2) Tri-sodium citrate with aqueous gold chloride were prepared to make gold sol. Colloidal gold were labeled with ESS-PcAb and then were dropped onto conjugate pad after the conjugate were purified. At the same time ESS-PcAb and sheep anti-rabbit IgG were coated ESS-PcAb and sheep anti-rabbit IgG on Nitrocellulose membrane with different dilutions. Nitrocellulose membrane, gold conjugate pad, sample application pad and absorbent pad were assembled onto a plastic back and cut into individual strips (3m m×5.5mm).3) A sandwich competition ELISA was developed for detection ESS from PMU. 40ug/ml ESS-PcAb was coated on Use microplate as a capture and HRP conjugate ESS-PcAb as a conjugate, In order preparation sandwich competition ELISA method. And the tested samle was PMU. Results:1) sodium Sodium estrone sulfate-17-(O-carboxymethyl) oxime was identified by the infrared spectrometry(IR), nuclear magnetic resonance(NMR) and ESI-mass spectroscopy. The optimal conjugation ratio of im-munogen was 25.74, and dilution rate of antiserum could up to 1.28×105 through an indirect enzyme-linked immunosorbent assay.2) The type HF13504 of NC membrane made by Millipore Corporation were used to prepare the strip, which with the 2mg/ml dilution of sheep anti-rabbit IgG as a control line, and the 1.6mg/mL of ESS-PcAb as a test line. The materials of Millipore were used as the sample pad and conjugation pad. The sensitivity of strips for ESS standards was 30 ng/mL.3) We observed that the optimum coating condition for sandwich ELISA was hatching overnight at 4℃with CB as capture buffer, the optimum block condition was hatching 2 hours at room temperature with 1% BSA, and the optimum conjugated antibody reactive condition was cultured 1 hours at room temperature; the substrate of a solution of fresh prepared 3,3',5,5'-tetramethyl-benzidine was added and at last stopped by 2 mol/L sulfuric acid. Conclusion:The preparation methods of sodium estrone sulfate-17-(O-carboxymethyl) oxime and poly-clonal antibodies of sodium estrone sulfate was established. And initial easy to use and accurate colloidal gold immunochromatographic test and ELISA method for detecting conjugated estrongen were developed and basically useful in the laboratory. |
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