Establishment Of Quantitative Detection Method For Magnetic Microparticles Chemiluminescence Of Anti-SSA-60 Antibody

Objectives:The expression vector of SSA-60 protein was constructed by gene recombination technique.The recombinant SSA-60 protein with biological activity was gained by induced expresstion in Escherichia coli Rosetta(DE3)and purified by affinity chromatography.Based on the SSA-60 recombinant protein,a quantitative detection method for the magnetic microparticles chemiluminescence of anti SSA-60 antibody was established,and evaluated its performance.Methods:1.Collect hela to extract RNA,then reverse transcription into cDNA(as a template),SSA protein gene sequence with restriction enzyme cutting site(BamH I and Xho I)was amplified by PCR and subcloned into expression vector pET41 a to construct GST-SSA-6*His-PET41 a recombinant plasmid;The recombinant plasmid was transformed into E.coli OverExpress Rosetta(DE3)and induced with IPTG.Recombinant protein was purified by affinity chromatography.The performance of the recombinant protein was identified by SDS-PAGE,WB and ELISA assay.2.Using the SSA-60 recombinant protein as raw material,the method for quantitative detection of anti-SSA-60 by chemiluminescence method of magnetic particle was established.After optimizing the conditions,the performance of the method was evaluated whether the method met the clinical requirements.Results:1.The target gene was got by PCR amplification.The result of SDS-PAGE shown that the recombinant plasmid was successfully constructed.The recombinant plasmid was transformed into Rosetta(DE3)strain and induced.The recombinant SSA-60 protein was expressed as inclusion body.The purified recombinant SSA-60 protein had enough immunogenicity and good immune reactivity.2.After optimized the conditions and evaluated the performance,the methods detection limit(MDL)was 1.36 RU/mL and the reliable quantitation limit(RQL)was 4.48 RU/mL;The coefficient of variation(CV)was less than 10%;There were no significant cross reactions with other autoantibodies.The linear range was 2-400 RU/mL,which was wider and obviously superior to the traditional ELISA method.For comparison with ELISA,it represented a good correlation(y=1.04x-7.86,R2 =0.9785,P<0.05,)and a high agreement(K=0.949)between two methods.And the standard error(SE)of the differences was 31.3 between two methods.Conclusion:The recombinant SSA-60 antigen with high purity,good specificity and enough protein activity was successfully prepared,which provided a favorable condition for the magnetoparticle chemiluminescence method.Magnetic particle chemiluminescence assay for quantitative detection of anti SSA-60 antibodies had high sensitivity,strong specificity and a wider linear range.Compared with ELISA method,the two methods had good agrenment and correlation.This method had good potential in rapid,high-throughput,quantitative and automatic detection of autoimmune antibodies.What's more,it also laid a foundation for detecting other autoantibodies by this method.