Establishment A New ELISA Method For Serum T-PSA And Development A New Intergration Detection Method For F-PSA/t-PSA

Objective1.To establish a detection method for total prostate specific antigen in serum with indirect package ELISA.2.To establish a new intergration detection method for f-PSA/t-PSA and discuss the feasibility of replacing the concentration ratio(f-PSA/t-PSA)by electrochemical with the absorbance ratio(ODf-PSA/ODt-PSA)by indirect package ELISA.Method1.The establishment of immunological detection method for serum t-PSA with indirect package ELISA:(1)To prepare BBA reacting plates coated with Biotin-BSA-Avidin;(2)To determine the concentration of the biotin-labeled t-PSA antibody and the horseradish over peroxidase-labeled t-PSA antibody in test reacion;(3)To optimize the response patterns of the new immunological detection method;(4)Evaluation of the indirect package ELISA to detect serum t-PSA.2.To Explore a new intergration detection method for f-PSA/t-PSA:(1)The determination of the specific antibodies for the new intergration detection method;(2)To determine the concentration of the biotin-labeled f-PSA antibody;(3)The establishment and precision evalution of the intergration detection method;(4)The correlation between absorbance ratio(ODf-PSA/ODt-PSA)by the intergration detection method and concentration ratio(f-PSA/t-PSA)by chemoluminescence method.Result1.The establishment of immunological detection method for serum t-PSA with indirect package ELISA:we determined the optimum concentration of antibodies by board titration.The optimum concentration of the biotin-labeled t-PSA antibody and the horseradish over peroxidase-labeled t-PSA antibody were 1:8000 and 1:4000 respectively.Also,by the comparison of different reaction conditions,we determined the optimum reaction mode was one-step method.The method had better constancy;When the range of the testing was 0-100 ng/ml,the sensitivity for t-PSA was 0.17 ng/ml,the recovery rate was 95-105%,the intraassay coefficient of variation was below 10%.the interassay coefficient of variation was below 11%.and there was no cross-reaction with AFP,CEA,CA125 and Fer..The correlation coefficient was r =0.869 between the indirect package ELISA and the chemoluminescence method.2.To Explore a new intergration detection method for f-PSA/t-PSA:the cross-reaction was detected between the selected biotin-labeled f-PSA antibody and t-PSA.and the crossactivity was 2%,however,the crossactivity would decrese to 0.5%between the purified f-PSA antibody and the t-PSA;the optimum concentration of the biotin-labeled f-PSA antibody was 1:1500;both of the intraassay and the interassary coefficient of variations were below 15%;the correlation coefficient between absorbance ratio(ODf-PSA/ODt-PSA)by the intergration detection method and concentration ratio(f-PSA/t-PSA)by chemoluminescence method was 0.8542,they were highly correlated.Conclusion1.Here,we established a new ELISA method for serum t-PSA.We introduced biotin-avidin system to improve the utilization of antibodies and reduce the '"hook effect".Moreover,reacting plates not directly coated with antibodies can be used to detect other target.Besides,it is sensitivity and specificity,and the correlation with chemiluminescence is r2 = 0.869,which can be applied not only in clinical but more to lot examination testing.2.We developed a new intergration detection method for f-PSA/t-PSA and discussed the feasibility of replacing the concentration ratio(f-PSA/t-PSA)by electrochemical with the absorbance ratio(ODf-PSA/ODt-PSA)by the new intergration method.The presion of the new method is good,and it was highly correlated with chemoluminescence method.However,because of the insufficient in specimens,we still have to do lots of work to prove its clinical value.