Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen

Abstract

Objective To prepare monoclonal antibodies (mAbs) against human cytomegalovirus (HCMV) PP65 antigen and characterize their reactivity to HCMV PP65 antigen by immunohistochemistry. Methods The recombinant plasmid PQE30-PP65 was transformed into competent E.coli M15 cells to obtain the recombinant PP65 antigen, which was purified with Ni column affinity chromatography. BALB/c mice were immunized with the purified antigen, and the spleen cells of the mice were isolated and fused with myeloma cells (SP2/0). The mAbs were obtained by HTS screening and purified by protein A affinity chromatography, and their specificity, titer and subtype were identified by indirect ELISA. The performance of the mAbs in immunohistochemical detection of HCMV PP65 antigen was tested on slides of clinical samples. Results Eight highly specific mAbs against HCMV PP65 antigen were obtained. All these mAbs were identified to be IgG1 subtype containing κ light chains, with titers all above 1 :2 048 000. Six of the mAbs were shown to accurately identify an active infection of HCMV by slide immunohistochemistry. Conclusion We successfully prepared HCMV PP65 mAbs, and 6 of these mAbs have good reactivity with HCMV PP65 antigen in leukocytes

Keywords

• human cytomegalovirus
• pp65 antigen
• monoclonal antibodies
• immunohistochemistry