Preparation Of Monoclonal Antibody Of Sodium Estrone Sulfate And Study Immunoassay Specific For Sodium Estrone Sulfate

Objective: To establish anti-sodium estrone sulfate monoclonal antibody(ESS-Mab), and to establish a quick, simple, sensitive colloidal gold immuno-chromatographic test by using ESS Mab. Develop a tool for detection thequantity of ESS from pregnant mare’s urine (PMU) to simplify the detection work.Methods:1. BALB/c female mice of6-8weeks immunized with the completeESS antigen by routine method.Immunological methods,dose of antigen given,times having effects on immune were considered.2.Preparation immune mice forcell fusion by selecting6BALB/c female mice with the dose of200/100μg/per,immune four times.The blood serum potencies were measured by indirectenzyme-linked immunosorbent assay.3.Cell fusion was carried by using PEGmethod.With the help of PEG1450the concentration of which is50%, the spleencells, which had higher blood serum potencies, were fused with SP2/0myelomacells (the ratio is10:1). The fused cells were cultured in the cell culture plateswhich had owned feeder layer cells for24hours. The selected solution HAT wasused to culture the fusion cells so as to select hybridoma cells.The hybridomacells was selected and identified by ELISA.4.Ascites with monoclonal antibodieswere purified by caprylic acid-saturated and protein A. The protein concentrationof purified ascites were checked.5.Tri-sodium citrate with aqueous gold chloridewere prepared to make the gold sol.Colloidal gold were labeled with ESS-MAband then were dropped onto conjugate pad after the conjugate were purifiedproperly. Results:1.Hybridoma technology was adopted to do the fusion of mousespleen cells and myeloma cells,the fusion rate was90.2%; ELISA screening positivehybridoma rate was4.4%. Finally two cell lines2C8and8A7with good specificity andsensitivity were obtained.The protein concentration of purified ascites monoclonalantibodies2C8and8A7were3.12mg/ml,3.57mg/ml respectively,and ELISA titer were1:1.02×10~5,1:2.05×10~5;IC50values were12.5ng/ml.With other estrogens,8A7hada little degree of cross-reaction with sodium equilin sulfate,and no cross-reaction withother estrogens.2.The colloidal golds of19.76nm prepared by tri-sodium citrate withaqueous.The optimal amount of antibodies was determined as8μg/ml,and the bestpH8.2. With the2mg/ml dilution of sheep anti-rabbit IgG as a control line, and the0.5mg/mL of ESS-MAb as a test line.The detection limit of ESS is50ng/mL.With otherestrogens, it had a a little degree of cross-reaction with sodium equilin sulfate,and nocross-reaction with other estrogens. Conclusions: The hybridoma technique was able toprepare monoclonal antibody of anti-sodium estrone sulfate successfully. A initialaccurate colloidal gold immuno-chromatographic test for detecting conjugated estrogenswere developed and basically useful in laboratory.