In vivo propagation of monoclonal antibodies in ascites fluid produced in BALB/c mice: characterization and diagnostic application for the identification of Vibrio cholerae O1

Abstract

Monoclonal antibodies are powerful tools in biomedical sciences. High affinity of binding domains of monoclonal antibodies towards a specific antigen enables their use in diagnostic assays and kits to identify particular infectious agents. In vitro propagation of monoclonal antibodies is considered expensive, tedious, low yielding and difficult to purify. In vivo ascites methods, however, is more economic for large scale generation of monoclonal antibodies needed to meet the growing demand of diagnostic assays. In this study, in vivo method had been adopted to propagate monoclonal antibody (mAb) against Vibrio cholerae O1 responsible for majority of cholera outbreaks. Hybridoma cells were intraperitoneally injected into pristane primed BALB/c mice and ascites fluid was harvested within 10-14 days. Ascites of 2.05 ± 0.23 ml was obtained from each mouse with IP injection of 1.5-2X106 hybridoma cells. Monoclonal antibody from ascites was purified by affinity chromatography and a concentration of 1.89 ± 0.62 mg/ml of purified antibody was obtained. The purified antibody was investigated for their ability to detect V. cholerae O1 Ogawa and Inaba serotypes by agglutination test. High specificity of the purified antibody against both V. cholerae Ogawa and Inaba serotypes was observed, with a titer upto 1:40-1:80 dilutions for agglutination, while no cross reactivity was found against V. cholerae O139 and other enteropathogenic bacteria. Dot blot immunoassay showed that the purified antibody had binding affinity against lipopolysaccharide antigen from the cell wall of V. cholerae O1 serotypes without possessing any cross-reactivity against lipopolysaccharide of V. cholerae O139. Isotype of the mAb was determined to be IgG3 by ELISA method. This mAb was then conjugated with colloidal gold nanoparticles to develop a simple lateral flow immunoassay for rapid diagnosis of cholera. For this purpose the pH and minimum concentration of mAb for conjugation were optimized as 8.5 and 24 μg/ml, respectively. This rapid diagnostic test warrants further development and evaluation.