| Alpha-fetoprotein (AFP) found in primary liver cancer is by far the most sensitive and most specific tumor marker,70-95% of primary liver cancer patients showed elevated levels of AFP, AFP levels in the blood for early detection of cancer, the disease progresses, treatment evaluation, monitoring of recurrence and metastasis, etc. have a certain value. In this thesis, the magnetic particles alpha-fetoprotein (AFP) chemiluminescence immunoassay system research and clinical studies, the magnetic particles coated chemiluminescent technology platform and technology platform, combined with double-antibody sandwich immunological response to technology, development a diagnostic kit for the detection of human serum alpha-fetoprotein.The specific studies and conclusions are as follows:1. Kit biological activity of the research process including selection of raw materials, coated magnetic particles, enzyme conjugates, and production technology of calibrators, kit reaction system, the determination of normal reference values and stability. Magnetic particles coated process:The selection of solid phase carrier kit carboxyl magnetic particles, coated two-step method to determine the carboxyl;Reaction kit finally determined to be:The two-step reaction model, the first step: 25μlsample+20μl magnetic particles+50μl diluent solution, and incubated for 15min at 37℃, washed 6 times; Step Two:Add 100μl enzyme conjugate, incubated for 15min at 37℃, washed 6 times; Add the substrate A, B each 50μl,after mixing, measured within 5-10min away from light.Determination of reference values:testing 615 normal samples, determined by percentile the reference value 10.0ng/ml.Study of stability: the study include the accelerated stability and real stability on the product. Ones were placed at 37℃for 10 days and the others were stored at 2-8℃for 14 months. Then testing the kit, the performance indicators are able to meet the requirements of product standards. It is safe and reasonable that the product is determined to be valid for 12 months at 2-8℃.2. For the quantitative detection kit features, including the minimum detection limit, specificity, accuracy, dose-response curve linearity, precision and other aspects of performance evaluation.Accuracy:With the reference material samples for testing, test kit accuracy of the average range in the 0.900～1.100.The limit of detection:The detection limit is not greater than 1.8ng/ml.Dose - response curve was linear:the linear range of 1.8～1000ng/ml, the correlation coefficient r kit should be≥0.9900.Precision:the samples with different concentrations of the repeat test 10 times, and its coefficient of variation (CV) should not exceed 10%.Difference between batches:batch coefficient of variation (CV) should not exceed 15.0%Specificity: human serum albumin (500ng/ml), HCG (1000IU/ml), PRL (500ng/ml) had no cross-reactivity.Interfering substances:50mg/dl bilirubin,81mg/dl hemoglobin,3000mg/dl lipids on the role of non-interference of the kit. 3. On the development and production of AFP quantitative detection kit (magnetic particles chemiluminescence method) to conduct clinical research, also choose the same species that have been listed as control kit, detection of primary liver cancer by preoperative and postoperative. Liver disease and other benign tumors total 1173 cases of patients with clinical laboratory test samples were compared, the results showed that clinical research compared with the control experiment,the negative coincidence rate was 97.85%, the positive coincidence rate was 98.32%, the total compliance rate of 98.04%, correlation coefficient 0.9971. The AFP kits can be used for clinical testing.This research and development of detection system uses magnetic particles chemiluminescence, select the high specificity and sensitivity of the monoclonal antibody, The sensitivity, specificity, stability and other key indicators can be reached domestic leading level, among the ranks of the international advanced level. And the operation is simple, rapid, and easy to automate and better to meet the domestic demand for clinical use. |
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