Development Of Monoclonal Antibody Against HCMV Pp65 And Identification Of Neutralizing Activity In Vitro

Objective We produced neutralizing monoclonal antibody against HCMV infection and identified its biological character.We explored McAb effection in clinical diagnoses and therapy. Methods Balb/c mice were immunized by antigen of HCMV for five or six times. HGPRT" SP2/0 was fused with mice spleen B lymphocytes. The hybridoma cell was cultured in the HAT selective medium .The positive clone was screened by indirect ELISA(IELISA). We gathered supernatant of hybridoma and identified cell with IELISA .We finally got the four hybridoma strain which secreted antibody against HCMV. McAb class and subclass were identified by agarose immunity double diffusion technique. We inoculated the hybridoma into Intraperitoneal mice and hydroperitoneum was purified with salt precipitation. The protein concentration was detected by Lowry. We took micronutralization experiment in HE D7 hybridoma strain McAb was labeled by HRP. Results We produced monoclonal antibody against HCMV .The McAbs were demonstrated to combined HCMV antigen by IELISA and agarose double diffusion. Its possess high specificity. The protein concentration was 11.2mg/ml and 13.8mg/ml. The McAb which D7 hybridoma strain secrete was demonstrated specifically to bind HCMV pp65 antigen by IELISA and Western-blotting. Microneutralization also confirm the McAb character which has neutralizative antibody. The titer of McAb that was labeled by HRP was 1:128. Cone I us i ons HCMV pp65 monoclonal antibody which was produced by hybridoma technique can neutralize CPE of HCMV, which extended the way that monoclonal antibody were regard as drug.