ABSTRACT
Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on 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 developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Antibodies, Viral/immunology , Cytomegalovirus Infections/diagnosis , Cytomegalovirus/immunology , Viral Envelope Proteins/immunology , Antibodies, Monoclonal/immunology , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Hematopoietic Stem Cell Transplantation , Immunohistochemistry , Immunoprecipitation , Mice, Inbred BALB C , Sensitivity and SpecificityABSTRACT
Objective To investigate JC virus(JCV) infection in kidney transplant recipients and its influence on graft function and also initially explore JCV infection factors. Methods A total of 49 kidney transplant recipients and 24 health examination persons were enrolled in our study, JCV DNA was measured using nested qualitative polymerase chain reaction assays of urine, while CMV DNA was measured by common qualitative polymerase chain reaction assays of urine. JCV infection factors, such as age, male, immunosuppressive therapy, cytomegalovirus(CMV) infection were analyzed by Binary Logistic Regression, and glomerular filtration rate(GFR) was selected as a index of kidney function and the difference of GFR between JCV-infected and non-infected patients was compared using t test. Results JCV was detected in 42.9% of kidney transplant patients and 4.2% health examination persons. CMV infection and Pred + MMF + CsA triple immunosuppressive regimen were found to be the risk factors of JCV infection. No difference of GFR was observed between JCV infected and non-infected patients (86.470 ± 29.990 and 84.060 ± 33. 729 for each; t =0. 259, P =0.797). Conclusion JCV is frequently detected in kidney transplant recipients. CMV infection and using of Pred + MMF + CsA triple immunosuppressive regimen can significantly increase the risk of JCV infection. While, graft function was not influenced by JCV infection in kidney transplant patients.