Screening and Purification of Anti-IpaJ Monoclonal Antibodies

The positive hybridoma clones expressing antibodies against His-IpaJ were screened using the previously established indirect ELISA method, with MBP-IpaJ recombinant protein as the coating antigen (9 (link)). Positive clones were sub-cloned three times by the limiting dilution method. The Ig sub-class of MAbs were identified by using a mouse mAb isotyping kit (Sigma, USA) according to the manufacturer's instruction. The positive hybridoma cell lines secreting anti-IpaJ MAbs were injected intraperitoneally into BALB/c mice to grow and proliferate. Ascites fluids containing abundant anti-IpaJ MAbs were collected from the immunized mice and purified by protein A chromatography (GE Healthcare). The purified MAbs were send to GenScript Biotechnique Company (Nanjing, China) for biotinylation with HRP.