Development Of A HCMV Pp65 Specific Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay For Diagnosis Of Active HCMV Infection

Objective: To develope an IgM capture enzyme-linked immunosorbent assay(ELISA) for detection HCMV pp65 specific IgM and assess active HCMV infection, which provides a sound basis for development commercial kit. Methods: HF cells were infected with HCMV ADI69 Strain. HCMV antigen was extracted. HCMVpp65 antigen was screened by SDS-PAGE and western-blotting. Its avidity with HCMV specific IgM was evaluation by indirect ELISA. HCMVpp65 McAb was labeled with HRP by NalCu method. IgM capture ELISA was developed through coating mircotiter with anti-human u chain, then adding serum and compound of HCMV pp65 antigen and HRP- McAb. 88 serum samples (30 from babies, 30 from pregnant and 28 from immunodeficiency patients) were detected by this method. Same serum samples were detected by the DIESSE enzywell CMV IgM kit. Results: HCMV pp65 antigen is a single strap that localized to 65KD marker on PAGE. Double immunodiffusion titer of HRP-pp65 McAb is 1:32. The appropriate dilution of anti-human u chain is 0.75 u g/well, serum sample is 1:100, pp65 antigen is 1:100, HRP-pp65 McAb is 1:20. Positive rate of three groups serum samples'is 6.7%, 13.3% and 10.7% with HCMV-pp65 specific IgM capture ELISA, respectively, which is 10%, 13.3% and 14.3% with the DIESSE enzywell CMV IgM kit. The total coincidence rate is 91%. Conclusions: It was first time to develope an IgM capture ELISA for detection HCMV pp65 specific IgM. It is convenient, fast and high specificity and sensitivity. The total coincidence rate between this method and the DIESSE enzywell CMV IgM kit is 91%. It can be used to diagnosis active HCMV infection of children, pregnant and immunodeficiency patients. It is suitable to widely use in foot layer hospital. Keyword:human cytomegalovirs;pp65;ELISA;Immunoglobulin M...