Preparation and identification of methylene blue polyclonal antibody

Abstract

Methylene blue and its metabolites (azure A, azure B and azure C) residues in fishery products were dangerous for human health and it still remains a big challenge in China. However, there are few available assays to detect specifically methylene blue and its metabolites residues in fishery products. In order to establish an enzyme-linked immunosorbent assay (ELISA) method for the detection of methylene blue and its metabolites residues in fishery products, methylene blue metabolite azure C with similar structure as methylene blue was selected as the hapten of methylene blue. The amino group on azure C was coupled with the carboxyl group on protein by glutaraldehyde method to prepare the immunogen azure C-BSA and coating antigen azure C-OVA. Immunogen and coating antigen were identified by ultraviolet scanning (UV) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). BALB/c mice were immunized with azure C-BSA as immunogen at a dose of 0.2 mg for one mouse, and then the titer, sensitivity and specificity of the obtained polyclonal antibody were detected. Results showed that the artificial antigen of azure C-BSA and azure C-OVA was synthesized successfully, with the coupling ratio of the prepared artificial antigen azure C-BSA and azure C-OVA was about 31∶1 and 52∶1, respectively. The titer of polyclonal antibody obtained from mouse was above 16 000, and the NO.2 mice had the highest antibody titer with the half inhibitory concentration (IC₅₀) of 43.9 ng·mL^-1. The antibody cross reacted with the methylene blue metabolites azure A, azure B and azure C, and the cross reaction rates were 127.6%, 84.7% and 138.1%, respectively. There was no cross reaction with other common dyes, indicating that the antibody was of good specificity. This experiment successfully synthesized methylene blue artificial antigen and obtained mouse-derived polyantiserum with good immunological properties, which laid the foundation for the preparation of monoclonal antibody and the development of rapid immunological detection method.