The Development Of Quantitative Detection Kit Of Human Thyroid Peroxidase Antibody

Objective:TPO antibody is an important diagnostic indexe of thyroid autoimmune diseases.This study to establish the natural TPO antigen and TPO antibody preparation process,and use them as raw materials to establish a new enzyme-linked immunosorbent assay(ELISA)kit and homogeneous chemiluminescent immunoassay(LICA)kits for the quantitative detection of TPO antibodies.We investigate the kits clinical application performance which provide detection reagents for the diagnosis of antoimmune thyroid disease.Methods:1.TPO antigen preparation:Collect thyroid tissue of Graves' patients which were surgically removed.After tissue homogenates,sonication,and repeated centrifuge,trypsin hydrolysis,dialysis and other methods of preparation of the TPO antigen.The crude extracts go through affinity chromatographycolum and then get the purified TPO antigen.The protein purity and immunological activity were identified by SDS-PAGE and Western Blot.2.TPO antibody preparation:Collect clinical Specimens:TPO antibody>1000 IU/mL,mix them.Get gamma globulin by 50%ammonium sulfate saturation solution precipitation.Get IgG through the Sephadex 4B-Potein A affinity chromatography.The activity of TPO antibody was identified by Abbott company AXSYM microparticle enzyme immunoassay.Determine the concentration and prepare the standare stock solution.3.New enzyme-linked immunosorbent assay(ELISA)kit:The microtiter plate was coated with biotinylated bovine serum albumin,then add streptavidin,get the BBA microtiter plate;Get the biotinylated TPO antigen by labeled TPO antigen with biotin;Determined the working concentration of biotinylated TPO antigen and enzyme labeled anti-human-IgG antibody by checkerboard titration Optimize the reaction conditions and identify the main methodological parameters.4.TPO antibody homogeneous luminescence immunoassay(competition method)Kit:.The TPO antibody was coated on receptor particles;Prepare the biotin TPO antigen;Optimize the reaction conditions such as reaction order,the dilution of the receptor particles labeled with TPO antibody,the addition of donor particles,the volume ratio of the measured antibody and the receptor particles labeled TPO antibody,as well as the reaction time of the first step and finally did method evaluation.Results:1.TPO antigen preparation:We obtained the purified TPO antigen from thyroid tissue.A simple protein band whose molecular mass is about 55KD is visible on SDS-PAGE,Western Blot shows that the protein band can react with TPO antigen.2.TPO antibody preparation:The purified IgG composition of TPO antibody was extracted from the positive clinical specimens.The total protein is 512 mg;The activity of protein is 4408 IU/ml.3.New enzyme-linked immunosorbent assay(ELISA)kit:According to the results of chessboard titration,the optimal dilution of biotinylated TPO antigen is 1:6000,the optimal dilution of IgG-HRP is 1:8000.The optimal temperature of the method is 37?;the optimal reaction time is 30 min.The result of method evaluation shows that the detection sensitivity of the assay was 0.165 IU/ml,the coefficient of variations(CV)of inter-assay between high and low concentration of quality control serum were 9.2%?9.0%,CV of intra-assay were 4.6%?5.6%,the recovery rate was between 96%?104%,the kit remained stable for 5 days in 37?.4.TPO antibody homogeneous luminescence immunoassay(competition method)Kit:This study uses the balance method.The optimize dilution ration of the microspheres coated with TPO antibody is 1:100;The sample amount of donor microspheres coated with avidin is 150 ul;The volume ratio of test antibody and receptor microspheres coated with TPO antibody is 2:1;The optimal reaction time is 30min.The sensitivity of this method is 1.35IU/ml,intra-assay CV is 6.9%?8.0%,inter-assay CV is 3.4%-6.2%,the recovery rate is 5.6%?103.3%,the kit remained stable for 5 days in 37?.Conclusion:1.In this study,We extracted TPO antigen from the thyroid tissue successfully and established a set of relatively perfect extraction and purification process,;obtained higher purity TPO antigen.Purity,specificity and immunological activity of TPO antigen were identified by SDS-PAGE,Western blot and Abbott microparticle enzyme immunoassay.which lay the foundation for the development of new enzyme-linked immunosorbent assay(ELISA)kit kits for quantitative detection TPO antibody and light induced chemiluminescent immunoassay(LICA)kits for quantitative detection(competition method)of TPO antibody.2.In this study,We purified TPO antibody from the patients' serum and extracted the IgG component,exclude the interference from other components to this experiment.The immunological activity identification and quantitative of TPO antibodies were done by the Abbott ASCYM microparticle.On the one hand,we prepared the TPO antibody standard preparation,on the other hand,we used purified TPO antibody from natural human serum to coat the receptor particles in TPO antibody homogeneous luminescence immunoassay kit(competition method).3.The new enzyme-linked immunosorbent assay(ELISA)kit is suit to detect TPO antibodies quantitatively,which can reduce dosage of TPO antigen,and cut down the cost.The new ELISA kit is sensitive and fit for basic-level hospital.4.The light induced chemiluminescent immunoassay(LICA)kits for quantitative detection(competition method)of TPO antibody don't need separation and washing steps,high precision,and save testing time.Because of the method of chemical luminescence,it has a very strong ability of anti-interference,lower background signal,and a high degree of automation,and fit to large hospital.