Establishment of ELISA method of detecting human cytomegalovirus by use of recombinant antigen pp65 and the comparative analysis with two other methods / 中华检验医学杂志

ABSTRACT

Objective To establish a new ELISA method by use of shorted recombinant antigen pp65 for detection of IgM antibodies to human cytomegalovirus (HCMV).Method According to HCMV pp65 sequence of nucleotide and amino acid, the shorted pp65 was decided by computer soft ware of protein and attained through gene engineering technique. The by computer soft ware of protein and attained through gene engineering technique. The recombinant enzyme- linked immunosorbent assay (REC-ELISA ) method was developed using pp65 recombinant protein as antigen and was applied to detecting anti-HCMV-IgM in sera. It was compared to ELISA by use of whole virus as antigen and Biocheck CMV IgM enzyme immunoassay test kit.Results To detect HCMV IgM by REC-ELISA, the best quantity of pp65 was 3.5?g percent cavity, the best dilution of HRP~*anti-HCMV IgM was 1∶[KG-*2]800 and that of serum was 1∶[KG-*2]100. The positive coefficient of variation (CV) was 9.5% in stability assay. The average CV of Inter- assay and Intra-assay was 4.5% and 9.6% respectively. The positive value of REC- ELISA was 44%, that of ELISA using whole virus was 50% and BioCheck was 45%. REC-ELISA using most suitable condition was concordant with BioCheck, which was 97.0%. Its youden′s index was 92.8% and its specificity (98.2%) was higher than that of ELISA using whole virus as antigen (90.9%), too.Conclusion REC-RLISA had good sensitivity, specificity and reproducibility. The method was easy and quick. Recombinant protein was harmless and easy gained compared with the whole virus. It can be large-scale production and standardization. Therefore, the application value and potential of REC-ELISA was very large.