The Preparation Of Antibody Against Human3,5,3’-triiodothyronine And Establishment Of Chemiluminescence Immunoassay And Evaluation Methodology

Free3,5,3’-triiodothyronine is a number of thyroid hormone,whichconcentration is not influenced by binding protein and direct reflect thefunction of thyroid function,and is a direct index in diagnosis ofhyperthyroidism.However,the up-to-date determination of serum FT3inclinical is based on import equipments and chemiluminescenceimmunoassay kit,which is restricting the rang of application.Objective:To prepare complete antigen of T3and to prepare rabbit anti-Humanpolyclonal antibodies(mAb) against human triiodothyronine and toestablish a new method of non-equilibrium competitive chemiluminescenceimmunoassay(CLIA) with FITC for serum FT3determination.And toprepare mice anti-human monoclonal antibody against humantriiodothyronine,which could be used to establish other immunoassaymethods and to explore the biological role.Methods: 1. Complete antigen of T3was prepared by the method of EDC，couplingT3with BSAor BGG,respectively.2. Rabbit anti-Human polyclonal antibodies(mAb) against humantriiodothyronine was prepared by immuning T3complete antigen.3. T3polyclonal antibody was labeled with horse radish peroxidase(HRP),and FITC was conjugated to T3analogue-BGG.Microwell was coatedby anti-FITC antibody. In the assay, the calibrators (or samples), FITC--BGG-T3analogue and T3antibody-HRP were added.T3analogue wouldcombine to anti-FITC antibody to form solid phase T3-analogue onmicrowell. T3, if exist in sample, would combine to T3antibody-HRP andcompete with solid phase T3-analogue. The amount of the complex wouldbe measured through enzymatic chemiluminescence immunoassayreaction.Compared with the method of the coating by T3-BGG（non-FITCsystem）,sensitivity、precision、stability and clinic correlation for thismethod were evaluated.This method as well as Roche Elecsys2010Systemwere used in120clinical serum samples for FT3determination.4. Mice spleen cells and myeloma cells were fused after Balb/c micewere immunized by T3complete antigen using50%PEG.By the way ofcloning and dilution method screening,the hybridoma cell lines wereestablished,with which lage-scale monoclonal antibodies in mice asciteswere prepared.Purity、Sensitivity、subset and so on for this method wereevaluated. Result：1. Through ELISAshowed high specificity of the prepared antibody.2. The detection of human serum free triiodothyronine bychemiluminescence immunoassay with FITC system is that linear rangeswere0.25~50pg/mL,and the sensitivity of this method was0.25pg/mL,meanwell,the coefficients of variability among batch were all under5％,which is better than the result of non-FITC system. Compared with theElecsys2010system, the correlation coefficient was0.9622.3. Two hybridoma cell lines were achieved successfully with hybridomatechnique,which could stably secrete specific McAb against T3.After one ofthe hybridoma cell line was injected into mice abdominal cavity,the ascitesabundant in McAb was obtained.After purification the titer of antibody was1:32000,belonging to IgG1type.Conclusions:1. The antibody of high purity and specificity against human T3havebeen successfully prepared.2. The quantitative diagnostic kit for FT3by non-equilibriumcompetitive chemiluminescence immunoassay using FITC system providesa rapid、sensitivity and accurate determination of FT3and it is suitable forapplication to clinical diagnosis.