Homogeneous Chemiluminescence Assay Research At The CA125 Detection Kit And The Application Of JAK1 Protein Interaction Analysis

This thesis was aiming to establish the CA125 detection reagent basing Homogeneous chemiluminescence assay(HCLA),the quantitative evaluation of the performance indicators.Constructed recombinant plasmids pENTER-IL-2RB and pENTER-IL-10RA,transfection to 293T cells,using co-immunoprecipitation and luminescence immunoassay technology to detect the interaction between JAK1-IL-2RB,JAK1-IL-10RA.Method:1 In this thesis,HCLA kit was developed for detecting CA125 basing on antibody-sandwich method.1.1 Standards:The series standrads were carried out by diluting antigen CA125 in the buffer.The antigen CA125 was diluted at series concentrations of 1,12,34,118,310 and 670 U/mL.The standards prepared were cryopreserved at-20 ?,1 mL per tube,4 ? after melting preservation.1.2 The coupling of antibodies and acceptor beads:200 ug CA125 labelled antibody was added in the ultrafiltration tube,and centrifuged for 6 min at 9,000 rpm.The antibody was washed 5 times.Then 1 mg acceptor beads were respectively coupled with 30,50,70 and 100 ?g antibody.The solution was added 10 ?L of 25 mg/mL NaBH3CN and 1.25?L of 20%Tween-20,and then incubated at 37 ?for 48 h in the dark,the total volume of the reaction solution was 200 ?L.In order to block the unconjugated sites,10 ?L 65 mg/mL CMO solution was added in the reaction solution and incubated for an hour at 37 ?.Unbound protein was removed by centrifugation(15,000g,15 min,4 ?)and microspheres successfully conjugated to anti-CA125 antibody were stored in the storage buffer at a concentration of 5 mg mL.1.3 Biotin conjugation:CA125 coated antibody 100 ug was added in the ultrafiltration tube,and centrifuged for 6 min at 9,000 rpm.The antibody was washed 5 times.0.5 ?L NHS-D-biotin dissolved in DMSO(22 mg/mL)was added in the antibody solution,the total volume of the reaction solution was 200?L.And incubated for 2 h in the room temperature.The unconjugated biotin was filtered with ultrafiltration tube.The biotinylated antibody was stored at 5 mg/mL.1.4 Evaluation of assay performance:including the calibration curve,sensitivity,specificity,analytical recovery,interference and comparison.2 Expression of eukaryotic vector Human human interleukin 2 receptor ? and interleukin 10 Receptor a.2.1 Human total RNA was extracted from Hela cells,then IL-2RB gene was amplified by RT-PCR and inserted into pENTER-His.2.2 Following series of double digestion,T4 DNA ligase enzyme,randomly selected the positive clones,the pENTER-IL-2RB and pENTER-IL-10RA plasmids were constructed.The plasmids were verified by PCR,double enzyme digestion using AsiSI and MIuI and sequencing.2.3 293T cells were transfected with the recombined plasmid pENTER-IL-2RB and pENTER-IL-10RA.' The express level of IL-2RB and IL-10-RA were determined by indirect immunofluorescence and western blotting.2.4 The determination of interaction proteins between JAK1 and IL-2RB,JAK1 and IL-10RA were verified by Co-immunoprecipitation.2.5 The detection of interaction proteins JAK1 and IL-2RB,JAK1 and IL-10RA by HCLA.Results1 The sensitivity of the HCLA kit for CA125was 0.46 U/mL.Calibration was carried out using log-log regression and the best-fit calibration was determined to be described by the following equation,log(Y)= 1.06 log(X)+ 1.66,with a correlation coefficient of 0.999.The intra-assay coefficients of variation and the inter-assay coefficients of variation were 4.4%-6.8%and 9.9%-12.7%.The HCLA kit and commercial Roche kit had good correlation,the correlation coefficient was 0.991.2 Restriction analysis and sequencing proved the accuracy of the recombinant plasmid,and the target protein band IL-2RB and IL-10RA were identified by Western Blotting.3 Based on CA125 HCLA assay,when the acceptor beads conjugated 30 ug antibodies,the interaction proteins JAK1 and IL-RB,JAK1 and IL-10RA could also be successfully detected.ConclusionsThe results demonstrated that the HCLA reagent for CA125 was developed and could meet the demand for clinical application.Recombined plasmid pENTER-IL-2RB-His and pENTER-IL-lORA-His were successfully constructed and expressed effectively in 293T cells.The interaction between JAK1 and IL-2RB,JAK1 and IL-10RA were validated by co-immunoprecipitation and HCLA,which laid a foundation of mechanism research on protein-protein interaction of JAK1 and IL-2RB,JAK1 and IL-10RA.