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The Establish and Clinical Application of Osteocalcin Test by Chemiluminescence Immunoassay

Author: ZhangZhongHe
Tutor: LiMu
School: Shandong University
Course: Surgery
Keywords: osteocalcin chemiluminescence immune analysis bone metabolism
CLC: R914
Type: Master's thesis
Year: 2011
Downloads: 50
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Abstract

Objective:The research is aimed to establish a stable, sensitive and specific chemiluminescence immune analysis for detecting osteocalcin, which can be used to provide objective clinical diagnosis evidence for accurate diagnosis, prediction and prognosis of diseases as well as taking corresponding measures to prevent or delay the development process of diseases; make preliminary observations on serum osteocalcin level of several bone metabolism diseases, and investigate the clinical significance of detecting the changes of osteocalcin concentration in bone metabolism through chemiluminescence immune analysis.Approaches:At the stage of establishing methodology, research was done mainly on technical formulation, the optimization and conformation of reaction system, the analysis of methodological performance indexes and observations of its stability; and then in the course of preliminary application to the clinical practice, more attention was paid to the preliminary observations on serum osteocalcin level in several bone metabolism diseases, and investigate the clinical significance of detecting the changes of osteocalcin concentration in bone metabolism through chemiluminescence immune analysis.Result:1. Research on technical formulation the research is based on the principle of using double antibody sandwich ELISA to detect osteocalcin, and making technical formulas and sealing technical formulas for solid phase peridium, select 0.05M of carbonate buffer solution with its PH between 9.5 and 9.7 as solid phase peridium, incubate 20-22 hours at 2-8℃; wash plate once before removing loose and free components; and then add inert protein buffer solution and incubate for 18-20 hours at 2-8℃to seal spare non-specific locus, after that, empty the sealing solution; dry it 3-7 hours under the circumstances of relative humidity at 30%, lastly, seal it in aluminum foil bag and preserve at 2-8℃2. Research on reactive systemBy referring to the reactive mode of similar products, the research is also centered on the reactive system of the newly established chemiluminescence immune analysis of osteocalcin. And Established its reactive mode as:add 50μl of calibrator, quality control and specimen to be tested into the reactive hole, then add 100μl more to test the combination of osteocalcin and enzyme, mix them evenly and react them at room temperature for 60 minutes, following washing the plate four times, wash it once more with distilled water, and then pat it dry; add 100μl into each hole with 50μl of chemiluminescence substrate liquid A and 50μl of liquid B, and then detect the luminescence value(0.1 s/hole) with chemiluminescence immune analyzer, and then read the result.3. Research on analysis performanceAfter finishing the research on technical formulation and reactive system, the research is shifted to the analysis performance of the osteocalcin chemiluminescence immune analysis, which includes the analysis on sensitivity, specificity, the linear of dose-response curves, its accuracy and precision. The standard is set on the basis of the statistics and analysis on the result of the performance evaluation of the finished products made at different time and in various batches, which helps control the continuous stability of the operation. In the end analysis sensitivity or the minimum detection limit is set not higher than 0.20ng/ml; analysis on specificity:the cross response rate with calcitonin, parathyroid hormone and insulin should be below 0.01%; The linear correlation coefficient r of the dose-response curves ought to be not lower than 0.9900; determination of accuracy:the ratio of the measured value and the labeled value of quality control should be between 0.90-1.10; non-precision of the same batch represented with coefficient of variation(CW%) should be not higher than 10.0%, the non-precision from different batches represented with coefficient of variation(CW%) should be not higher than 15.0%.4. Preliminary clinical applicationThe osteocalcin chemiluminescence kit is also put to preliminary clinical use, including observing different kinds of diseases and how serum osteocalcin level differ between age groups.Conclusion:Synthesizing the whole development process and results of the preliminary clinical application, the application of the kit of the osteocalcin chemiluminescence immune analysis developed in the research which is used in detecting osteocalcin showed its sensitivity, accuracy, linearity, precision, specificity, stability, clinical sensitivity and specificity well meet the clinical demands, thus establishing a stable, sensitive and specific chemiluminescence immune analysis, which helps provide objective clinical diagnosis evidence for accurate diagnosis, prediction and prognosis of diseases as well as taking corresponding measures to prevent or delay the development process of diseases; preliminary observations on serum osteocalcin level in several bone metabolism diseases further confirmed the clinical significance of the application of chemiluminescence immune analysis to detecting the concentration changes of osteocalcin in bone metabolism diseases. On the whole, the expected goal of the research has been achieved successfully.

The Establish and Clinical Application of Osteocalcin Test by Chemiluminescence Immunoassay

Abstract

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