Studies on the affinity chromatography purification of anti-patulin polyclonal antibodies by enzyme linked immunosorbent assay and electrophoresis

Abstract

Patulin is a mycotoxin produced by fungal species that frequently grow on fruit and vegetables. It presents risks, particularly for children consuming compotes and fruit juices. Thus, it is important to have methods such as immunoassays to screen a large number of samples. In the relevant literature, previous studies on the production of antibodies against patulin derivatives described qualitative tests for a patulin derivative or showed slight responses. The present study reinvestigated the production of polyclonal antibodies against patulin and their purification since crude antiserum could react non-specifically in immunoassays. Patulin-hemiglutarate was synthesized and conjugated to bovine serum albumin as the immunogen for the immunization of five New Zealand white rabbits. The immunoglobulin G (IgG) fraction was isolated twice by affinity chromatography using Sepharose-LS® gel and recombinant G-protein. Classic affinity chromatography using Sepharose-LS gel was unable to eliminate serum albumin from the IgG fraction and the use of recombinant G-protein was efficient to isolate the purified IgG. Titres and specificity were determined by indirect competitive enzyme-linked immunosorbent assay. Patulin-hemiglutarate-ovalbumin gave complete displacement, while patulin displaced 30% of bound antibodies. Thus, a fraction of the antibodies are specific for free patulin. The non-specific binding increased with patulin concentrations. The electrophilic properties of patulin might also induce intermolecular cross-links in vitro that hinder the possibility of responses displacement when free patulin is used.

Keywords:

• Patulin
• polyclonal antibody
• enzyme immunoassay
• affinity chromatography
• recombinant G-protein
• SDS electrophoresis

Acknowledgements

The authors give sincere thanks to Dr Philippe Delahaut and his team at the Laboratoire d’Hormonologie de Marloie, Belgium, for their contribution to the production of the antibodies.