| Ovarian cancer is one of the three most common female genital cancers, because of no typical clinical symptoms of the early stages, most patients are already in an advanced stage of illness when treatment. Therefore, how accurate diagnosed in early incidence of ovarian cancer that is particularly important. The study found that as the most common serum tumor markers in gynecological clinical carbohydrate antigen 125(Carbohydrate antigen 125, CA125) has a certain sensitivity and specificity of epithelial ovarian cancer, and has a very positive meaning in the prevention, diagnosis, condition assessment and postoperative effects of ovarian cancer, etc., can be used as the main indicators for the prevention of monitoring ovarian cancer. At present, CA125 detection technology are mainly chemiluminescence and enzyme-linked immunosorbent assay, etc., test kit used is mostly for expensive imported or domestic packaging reagents, and the detection is required to rely on the relevant testing equipment in the laboratory, the testing costs is high, which is not suitable for large-scale screening and bedside quickly seized in primary hospitals. Hence, finding a simple and rapid detection technology provide the basis for clinical diagnosis is urgent need.In this study, octanoic acid- ammonium sulfate precipitation was used to purify CA125 package and label monoclonal antibody, measured at 280 nm UV absorbance with a spectrophotometer after dialysis, calculated the protein concentration of the two antibodies according to the empirical formula, then identified the purity of the two antibodies by SDS-PAGE. Sodium citrate reduction was used to prepare different size of colloidal gold by controling the different amount of trisodium citrate, and selected the optimal of the optimum particle size, preparation of colloidal gold, marked p H, the optimal amount of Labeled protein, the amount of gold standard dilution, nitrocellulose membrane, treatment liquid of sample pad, zoned film solution and optimum coating amount of antibody and so on, and the stability, specificity, sensitivity of test strip were conducted performance studies and laboratory evaluation of clinical specimens, thus prepared colloidal gold test strip to quickly detect CA125 in human serum.Experimental results showed that the purity of the purified antibody obtained was higher, wherein 1mL CA125 coating antibody concentration of 14.93 mg / mL and 1.15 mL labeled antibody concentration of 15.08 mg / mL were obtained from each 3mL antibody ascites purified. 24.5nm gold particles was relatively uniform, and stable performance. The optimal processes prepared colloidal gold were as follows: heat to boiling time was 8 min, the maximum speed was 200 rpm, continued heating time was 5 min after color stability. Colloidal gold test strip technology was established by optimizing reaction pattern: colloidal gold and labeled protein pH was 7.5, the concentration of labeled protein wad 1.4mg / mL, gold dilutions added was 300μL / mL, the selection of NC membrane was TEST NC film, the best formula combination of sample pad treatment solution were 0.03 M p H8.8 Tris, 0.3% Tween-20, 0.5% pvp, 0.3% citric acid, the coat protein concentration of control line and testing line was 1.5mg / mL, the sensitivity of test strip established by this methods was 35 U / mL, and didn’t cross-react with CEA, AFP, CA50, CA15-3, CA19-9, the stability of the test strip showed good after 37 ℃ accelerated destruction experiment for 21 d. Clinical evaluation results showed: the testing positive coincidence rate and the negative coincidence rate were 100/100 and 98/100 of 100 clinical specimens, respectively.This study established CA125 GICA double antibody sandwich detection method successfully, the sensitivity, stability and specificity of the detection method was high. |
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