Establishment of an indirect ELISA method for detection of human cytomegalovirus

Abstract

Objective To establish an indirect ELISA method for the detection of human cytomegalovirus (HCMV) based on HCMV IE1/pp65/pp150 recombinant protein. Methods From June to December 2024, 5 serum samples from HCMV IgG-positive patients, 50 serum samples from HCMV IgG-negative patients, as well as 8 serum samples each from the patients with positive herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), and Epstein-Barr virus (EBV), and 349 serum samples from volunteers undergoing physical examination were collected from Qingdao Municipal Hospital. An indirect ELISA method for HCMV was established by optimizing the detection conditions such as serum dilution ratio and the type of blocking solution, and the cut-off value of this method was determined; coefficient of variation (CV) was calculated to verify the repeatability of the method; the sensitivity and specificity of the method were assessed by detecting the serum samples from the patients with positive HCMV IgG, HSV-1, HSV-2, and EBV at various dilution ratios; the consistency rate between HCMV indirect ELISA and cytomegalovirus IgG ELISA kit was assessed by analyzing the serum samples of 349 healthy volunteers undergoing physical examination at the outpatient service. Results The optimal antigen-coating concentration of HCMV indirect ELISA assay was 3.0 mg/L, with an optimal serum dilution ratio of 1∶200, an optimal blocking solution of 5% bovine serum albumin, an optimal blocking time of 2 h, an optimal serum incubation time of 90 min, an optimal dilution ratio of the second antibody of 1∶8 000, an optimal incubation time of the second antibody of 30 min, and an optimal color development time of 15 min. The cut-off value of HCMV indirect ELISA assay was 0.360, with an intra- or inter-batch CV of <10% and a sensitivity of 1∶3 200, and the absorbance values of serum samples from HSV-1, HSV-2, and EBV-positive patients were all below the cut-off value. The overall consistency rate between HCMV indirect ELISA and cytomegalovirus IgG ELISA was 94.0%. Conclusion An indirect ELISA method is successfully established for detecting HCMV with HCMV IE1-pp65-pp150 recombinant protein as the coating antigen, and this method has good repeatability, specificity, sensitivity, and consistency rate.

Keywords

• cytomegalovirus|immediate-early proteins|phosphoproteins|recombinant proteins|serologic tests|enzyme-linked immunosorbent assay