| Parathyroid Hormone is an important hormone that regulates calcium and phosphorus metabolism in the body. The quantitative determination of PTH plays important roles in the auxiliary diagnostic and treatment monitoring of hyperparathyroidism, hypoparathyroidism, hypercalcemia, hypocalcemia, chronic renal failure and renal osteopathy. In this thesis, a quantitative determination assay kit for parathyroid hormone is developed and evaluated. With good performance, the newly developed kit is capable of quantitative determination of PTH in human serum or plasma.The studies and conclusions are as follows:1. Study of main raw materials:BALB/c mice are immunized with recombinant full length human PTH,36 monoclonal antibodies are prepared. The binding sites of these antibodies are determined by PTH fragment,4 antibodies against PTH (1-34) and 5 antibodies against PTH (39-84) are selected out of them, one antibody pair is chosen for the kit development, then the quality criterion of raw materials is established.2. Preparation technique of kit components:Magnetic particles are prepared with a double step technique using carboxyl modified particles, coating concentration is 0.3μg/T, activation and coating time are respectively 0.5 hour and 2 hours. The formula of enzyme conjμgate is a pH7.40.05M Tris buffer with 1% BSA in it, with a working concentration of 1/750 to 1/500. The formula of calibrators is a pH7.4 0.05M Tris buffer with 1% BSA and 1% EDTA in it, the calibrators are traceable to international reference material WHO 95/646.3. Study of reaction parameters:Serum and plasma are the acceptable sample types, EDTA plasma is recommended.20mg/dl bilirubin,200mg/dl hemoglobulin, 3000mg/dl triglyceride show no interference to the results under a criterion of ±10%. The sample volume needed for one test is 50μl, incubation time is 30 minutes. Measurement of signals can be carried at anytime between 1 and 13 minutes after adding the substrate.The assay uses a 4 parameter logistic curve fit method to generate calibration curve.4. Study of analytical performance:The within-run CV, between-run CV and between-lot CV is not more than 10%. 1000ng/ml osteocalcin,500ng/ml calcitonin, 500ng/ml β-CTX, 10000pg/ml PTH (1-34) and 10000pg/ml PTH (39-84) is assayed respectively, neither results are higher than 2.0pg/ml. Functional sensitity of the assay is 5.0pg/ml, assaying 500000pg/ml PTH will get a result of not less than 3000pg/ml. Dilution recovery is between 0.90-1.10, Fortified Recovery is between 0.85-1.15.5. Study of stability:Accelerated stability, transport simulation stability, real time storage stability and consumer use stability are tested. The stability of components and whole kit under 37℃ for 10 days meets the defined criterion. After stored at 2-8℃ for 12 months, there is no significant change in the performance of the kits. The kit can be used within 28 days after first use. Serum stored at room temperature after 24 hours shows a loss of 12%, it is recommended that samples should be kept not more than 12 hours at room temperature and not more than 48 hours at 2-8℃.6. Establish of reference interval:Serum of 148 healthy individuals are assayed, central 95% of the reference range is defined as the reference interval, the lower limit and upper limit of the referen interval is calculated to be about 11.0 and 81.0 using SPSS statistical software, the reference interval of the kit is setted to be 11.0-81.0pg/ml.7. Clinical application study:214 cilinical samples are assayed using the evaluated kit and reference kit, it is shown that the evaluated kit has a positive coincidence rate of 97.73%, a negative coincidence rate of 96.34%, a total coincidence rate of 97.20% with reference kit, there is no significant difference between two kits. The test results of samples are highly correlated between two kits with a correlation coefficient of 0.9940, the kit can be used to detect PTH in clinical laboratory.Conclusion:In this thesis, a quantitative determination assay kit for parathyroid hormone (Chemiluminescence magnetic particles immunoassay) is developed, the sensitivity, precision, specificity, accuracy and stability is studied. Reference interval is established and clinical application study is carried out. It is shown that the developed kit has high clinical coincidence rate, good accuracy. Its performance has reached international industry technical level.With high quality and low cost, this kit will be good choice for clinical laboratory. |
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