Qualitative Detecting CMV-IgM In Human Serum Based On A Light-initiated Chemiluminescent Assay

Objective:Cytomegalovirus(CMV)is one of the important pathogenic microorganisms causing congenital and perinatal infection wordwide.And early diagnosis of CMV infection is helpful to eugenics,risk assessment and late-stage antiviral therapy.CMV-Ig M antibody is a common indicator to prevent perinatal CMV infection in pregnant women and congenital CMV infection in newborns.In order to improve the disadvantages of traditional method,such as highly interference,long time,cumbersome process and other shortcomings,this study aimed to establish a complete homogeneous chemiluminescent immunoassay system of CMV Ig M antibody in human serum based on the technology of light-initiated chemiluminescent assay(Li CA).The assay used mouse anti-human Ig M monoclonal antibody,CMV recombinant antigen,Chemibeads and Sensibeads as raw materials,so as to provide a micro,convenient and efficient research platform for clinical detection of CMV Ig M antibody in human serum.Methods:1.The establishment of a light-initiated chemiluminescent assay(Li CA)for determining CMV-Ig M antibody in human serumBased on Li CA,two analysis formats were established to confirm a optimal format for detecting CMV-Ig M antibody in human serum.And then we optimized the reaction conditions,including the assay buffer,the working concentration of antigen,the dilution of antibody and serum,the volume of Sensibeads and reagents input order.(1)Indirect format of CMV Ig M antibody in human serum on Li CACMV antigen-coated with Chemibeads,biotinylated mouse anti-human Ig M monoclonal antibody and diluted serum were added to a micropore and incubated at 37 ℃,forming a compound of Chemibeads-Ag-test serum-anti-Ig M antibody-biotin.And then Sensibeads coated with streptavidin was added to and incubated again.The signals were finally measured on the Li CA analyzer and the results were judged.(2)Capture format of CMV Ig M antibody in human serum on Li CAMouse anti-human Ig M monoclonal antibody with Chemibeads,biotinylated CMV antigen and diluted serum were added to a micropore and incubated at 37 ℃,forming a compound of Chemibeads-anti-Ig M antibody-test serum-Ag-biotin.And then Sensibeads coated with streptavidin was added to and incubated again.The signals were measured and the results were judged.2.Performance characteristics of Li CA for the detection of CMV-Ig M antibody in human serumIn this study,methodological evaluation was carrid out,such as cutoff value,precision,specificity,interference test,rheumatoid factor,the consistency of plasma and serum.Moreover,the clinical application value of the assay was evaluated by the comparison and consistency analysis of 100 clinical serum samples on Li CA and LIAISON.Results:1.The establishment of a light-initiated chemiluminescent assay(Li CA)for determining CMV-Ig M antibody in human serumIndirect format was selected as the final format.The assay was performed by incubating CMV antigen-coated with Chemibeads、biotinylated mouse anti-human Ig M antibody and Sensibeads coated with streptavidin.After optimization,the assy buffer with 0.1 M Tris-HCl(p H 8.0),0.3 M Na Cl,0.01% Tween 20 and 10 mg/ml BSA,2.50 μg/ml of CMV antigen-coated with Chemibeads,a dilution ratio of 1:500 for biotinylated anti-Ig M antibody,a dilution ratio of 1:400 for serum,a volume of 175 μl for Sensibeads coated with streptavidin and one step process of Li CA were the final assay working conditions.2.Performance characteristics of Li CA for the detection of CMV-Ig M antibody in human serumThe primary assay had good analytical performance characteristics.The intraassay and interassay coefficient of variation were both less than 10%,ranging from 5.3% to 6.1% and 6.9% to 9.8%.There was no cross reaction with toxoplasmosis,rubella virus and so on,which proved that the assay had high specificity.Hemoglobin,bilirubin and triglyceride had little effect on the assay results.ROC curve analysis showed that the area under curve,sensitivity and specificity were 0.997,100%,and 98.9%,respectively.Compared with LIAISON,the overall,positive,negative accordance rate were 95.0%,92.5%,96.7%.And the relative sensitivity and specificity were 94.9% and 95.1%,respectively.Moreover,kappa was 0.895 and P was less than 0.01 at consistency test.There was no statistical difference between each other.Conclusion:Indirect Li CA for CMV-Ig M antibody has been established.The assay is simple,rapid,highly sensitive and showed good performance characteristics.This study maybe provide a new,convenient and efficient method for the detection of CMV-Ig M antibody in clinic.And further study and development are expected to promote the clinical application.