The plates were read in a Bio-Rad ELx800 microplate reader (USA) at 450?nm. Indirect ELISA was used to identify the purified mAbs. patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were Astragaloside A developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner. Keywords: Human cytomegalovirus, Glycoprotein B, Monoclonal antibody, Antigen detection, Transplantation Introduction Human cytomegalovirus (HCMV), which belongs to the subfamily Betaherpesvirinae, continues to be an important pathogen in immunocompromised individuals (1) despite a decrease in the morbidity and mortality of post-transplant HCMV disease with routine antiretroviral therapy. Glycoprotein B (gB), one of the most important envelope glycoproteins of HCMV, is implicated in virus entry, cell-to-cell spread (2), and the fusion of infected cells (3). Therefore, detecting gB antigen in patients with HCMV infection may facilitate the monitoring of the infection. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients. In long-standing past research, gB has been demonstrated to be a significant target for both humoral and cellular immune responses (4-6). Antibodies against gB have been of interest because of their therapeutic potential for neutralization (7,8). However, limited information is available about the diagnostic potential of gB monoclonal antibodies (mAbs). In this study, two potential diagnostic mAbs to gB were generated. The HCMV strains Towne and AD169 were used to analyze the specificity of the mAbs prepared. Specimens from patients submitted to allogeneic hematopoietic stem cell transplantation (allo-HSCT) were used to demonstrate the diagnostic efficacy of the mAbs. Material and Methods Animals BALB/c mice (6-8 weeks old) were purchased from the Shanghai Laboratory Animal Center of the Astragaloside A Chinese Academy of Sciences (Shanghai, China) and housed under pathogen-free conditions. All experiments were conducted according to guidelines of the Animal Ethics Committee of Zhejiang University. Peptides Hydrophilic sequences of favorable immunogenicity containing 14 amino acids of HCMV gB were screened. Peptides (HuaAn Biotechnology Co., China) ST (amino acids 27-40 of the Towne strain gB: STRGTSATHSHHSS, GenBank accession No. M60929), and SH (amino acids 81-94 of the AD169 strain gB: SHATSSTHNGSHTS, GenBank accession No. X04606) were synthesized and partly cross-linked with keyhole limpet hemocyanin (KLH) to enhance their immunogenicity. The molecular masses of the peptides were 3998.2 and 1513.5?Da, respectively, and their purity was 95.1 and 90.3%, respectively. Cell culture and lysate preparation MRC-5 cells (American Type Culture Collection, USA) were cultured to confluence, and then scraped and lysed at 4C for 40?min with cell lysis buffer (Cell Signaling Technology, Astragaloside A USA) containing 20?mM Tris-HCl, pH 7.5, 150?mM NaCl, 1?mM Na2EDTA, 1?mM EGTA, 1% Triton, 2.5?mM sodium pyrophosphate, 1?mM -glycerophosphate, 1?mM Na3VO4, and 1?g/mL leupeptin. All insoluble material was removed by centrifugation at 8000 for 10?min. Protein concentration was determined using a BCA protein assay kit (Pierce, USA) with bovine serum albumin (BSA) as the standard. To prepare samples of viral protein, MRC-5 cells were infected with HCMV strains Towne and AD169 (American Type Culture Collection) at a multiplicity of infection (MOI) of 1 1.0 for 3 days at 37C (9), after which the proteins were purified as described above. Generation and purification of monoclonal antibodies BALB/c mice were immunized subcutaneously four times (100?g/mouse) at 2-week intervals. The peptides were Rabbit Polyclonal to KR2_VZVD mixed with an equal volume of Freund’s complete adjuvant (Sigma, USA) in the initial immunization, and with Freund’s incomplete adjuvant (Sigma) in the second and third immunizations. The 2 2 mice with the highest antibody titers specific for ST and SH, as determined by indirect enzyme-linked immunosorbent assay (ELISA), were boosted by a tail vein injection 3 days before cell fusion. Feeder layer.