Production and Purification of Monoclonal and Polyclonal Antibodies Against Cholera Toxin

Abstract

The aim of this study was to produce monoclonal and polyclonal antibodies against cholera toxin (CT). Hyperimmune ICR mice produced polyclonal antibodies (PAbs) after injection with 0.5 mL of pristane and were injected with NS-1 myeloma cells 2 weeks later. Hyperimmune Balb/c mice were used for the production of monoclonal antibodies (MAbs). After these mice were immunized four times and given a final boost, their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG 1500. The fused cells were then selected in the hypoxanthine, aminopterin, and thymidine (HAT)-RPMIX medium. Anti-CT antibody-secreting hybridoma cell lines with high titer were cloned by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in 15% fetal bovine serum (FBS) HT-RPMIX medium. Eleven murine hybridoma producing anti-CT MAbs were obtained and designated CT-A2, CT-B4, CT-B11, CT-C7, CT-D7, CT-E8, CT-F4, CT-F2, CT-F8, CT-E3, CT-E6. Isotypes of MAbs were identified as IgM heavy chain and all were λ light chain. Hitrap rProtein A and Hitrap IgM purification columns were used for the purification of PAbs and MAbs, respectively.