Research On Laboratory Diagnosis And Clinical Application Of Cytomegalovirus And Rubella Virus Infection

Objective: To establish a rapid, useful and convenient laboratory diagnostic method which could be popular applied to detect the HCMV/RV specific immunoglobulin G(IgG) antibody in the scop of primary prevention of birth defects by used two new recombinant antigens rubella virus(RV) and human cytomegalovirus (HCMV). The diagnostic efficiency, safety and practicality in the clinical assay of the two immunoglobulin G antibodies were evaluated.Method: The antigen activity of human cytomegalovirus pp150/p52 protein and rubella virus E1 protein was identified by ELISA. Polystyrene microtiter plates were coated with recombinant HCMV antigen and recombinant RV antigen preparation. The measurement of IgG was conducted by indirect enzyme linked immunosorbent assay (ELISA), which all of the steps and conditions had been optimized. Standard serum and quality controls were calibrated with international standard materials anti RV immunoglobulin provided by World Health Organization and anti HCMV immunoglobulin provided by DiaSorin kit, INC. seperately. In the part of clinical assay, serum of 1000 women of childbearing age were detected by the HCMV and RV specific IgG antibodies in double-blind method. And the efficiency of our methods in clinic were evaluated according to the consequence.Results: The recombinant protein of HCMV pp150/p52 and RV-E1 protein showed a well antigen activity and were significantly better than the most common of antigens. A technical research on the method for detecting IgG was performed. By comparison, the optimal technological flow was defined. The assay used optimal antigen, carbonate buffer (HCMV-Ag), Tris buffer (RV-Ag), 3% defatted milk powder, sample dilution buffer with variable color and special serum preservation used exclusively for our study. In the study, the measurement of anti HCMV-IgG used 0.7 IU/ml of anti HCMV-IgG as Cut-off value. The measurement of anti RV-IgG used 15 IU/ml of anti-RV-IgG as the Cut-off value and defined as minimum immunity criteria of preventable infection. Although WHO rubella standard serum with a Cut-off level at 10 IU/ml, we used 15 IU/ml as the Cut-off of RV IgG, in view of the fact that recurrent infection may also occur due to a lower level of antibody. The methods of establishment were compared with the FDA-approved method of DiaSorin kit(standard methoIVby measuring special IgG in 1000 clinical samples. And then used statistic method to analyse the date of detection, they were showed good correlation. The Kappa value was 0.699 and 0.848 respectively. The p-value for the chi-quare test and McNemar test were all less than 0.05. The positive predicted value, nagetive predicted value and efficiency of diagnosis of our anti HCMV-IgG test was 97.2% (923/950), 86.0%(43/50)and 96.6%(966/1000)respectively. Our anti RV-IgG test was 99.6 %(775/778), 82.4%(183/222) and 95.8%(958/1000) respectively.Conclusion: The prime subjects in our established methods are the women of preconception who are the target of the primary prevention of birth defects. Therefore, we established anti-HCMV IgG and anti-RV IgG ELISA assay, selecting a higher level of Cut-off value, 0.7 IU/ml as the Cut-off and 15 IU/ml as the minimum immunity level. Special IgG assay is applied to distinguish subjects having acquired the disease from those who have not and in detecting the dynamic level of immunity antibodies. Special RV-IgG assay is used for the guidance and monitor of RV vaccine.