Purification of Anisakis simplex antigen by affinity chromatography.

Abstract

In order to improve the specificity and sensitivity of the techniques for the diagnosis of human anisakidosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG-specific antibodies were isolated by means of protein A-Sepharose CL-4B bead columns. IgG anti-Anisakis simplex, anti-Ascaris suum and anti-T. canis were coupled to CNBr-activated Sepharose 4B. For the purification of the larval Anisakis simplex antigens, it was loaded into the anti-A. simplex column and bound antigens were eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex-specific proteins were loaded into the anti-Ascaris suum and anti- T. canis columns. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis and immunoblotting were carried out. Likewise, immunoaffinity columns were prepared using specific IgG from patients with Anisakis simplex sensitization, previously diagnosed by fluoro-enzymo-immunoassay. The protein patterns of antigen after purification by the human columns were similar to those obtained using the rabbit columns.