| Objective:In this study, the conditions required to prepare a rapid colloidal gold strip capable of specifically detecting male testosterone were determined.Method:The quality of colloidal gold was checked. The optimal pH and concentration of anti-testosterone monoclonal antibody for saturating 1mL colloidal gold was determined by visual inspection and microplate reader.Diluting the coating antigens with different conjugation molecular ratios, the appropriate one was selected by observing the color of test line. Then dilute the coating antigen by 1:50 and choose the optimal diluent for coating antigen by the color of test line. The optimal gold-labeled antibody diluent and glass fiber treating fluid were screened by the color of test line and control line, releasing rate and residue of gold-labeled antibody. The flow rate of gold-labeled antibody on NC membrane and color of test line determined the optimal NC membrane. Observe the amount of red blood cells filtered and coagulation of blood then could find the best sample pad and plastic packaging shell. According to orthogonal experiment, the concentration of coating antigen and gold-labeled antibody were found. The colorimetric card was made with a series of testosterone standards and professional color software. At last, performance tests about strip were taken.Results:(1)The diameter of colloidal gold was 30nm, which was suitable to the test. (2) The optimal pH was 8.3 and the best condition of saturating 1mL colloidal gold needed the anti-testosterone antibody was 24ug. (3) The conjugation ratio of coating antigen was 25:1. The optimal concentration of coating antigen was 0.465ug/mL. The best dilution of gold-labeled antibody was 4 times. (4) The results of solution screening were as follows:coating antigen:0.01M pH9.6 CBS. Gold-labeled antibody: 0.01M pH7.4 PBS (containing0.1%BSA、5% sucrose、0.05%Tween-20).glass fiber:0.01M pH7.4 PBS (containing5% sucrose、0.5%Tween-20 0.1%NaN3). (5)The results of materials screening:NC membrane:WhatmanAE99, glass fiber:Ahlstrom8964, whole blood separator:GF2, absorbent pad:H5072, whole blood plastic packaging shell: A-12. (6) The range of the strip for testing testosterone standard was 300ng/dl-900ng/dl and the concentration interval was 100ng/dl.7) It had no significant cross reaction with analogues but had good precision. The strip could be preserved for one month at 4℃ with dry condition. Comparing with the results of Elisa, coincidence rate was 98%.Conclusion:A colloidal gold strip of semi-quantitative detection for male testosterone is established and its testing arrange is 300ng/dl-900ng/dl. It will be a convenient and quick detection method with high specificity and precision. |
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