Purification of Anisakis simplex antigen by affinity chromatography

In order to improve the specificity and sensitivity of the techniques for t he 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 wi th A. simplex or Ascaris suum antigens or inoculated with Toxocara canis em bryonated eggs. The IgG-specific antibodies were isolated by means of prote in A-Sepharose CL-4B bead columns. IgG anti-Anisakis simplex, anti-Ascaris sinan and anti-T. canis were coupled to CNBr-activated Sepharose 4B. For th e purification of the larval Anisakis simplex antigens, it was loaded into the anti-A. simplex column and bound antigens were eluted. For the eliminat ion of the epitopes responsible for the cross-reactions, the A. simplex-spe cific proteins were loaded into the anti-Ascaris suum and anti-T. canis col umns. To prove the specificity of the isolated proteins, immunochemical ana lyses by polyacrylamide gel electrophoresis and immunoblotting were carried out. Likewise, immunoaffinity columns were prepared using specific IgG fro m patients with Anisakis simplex sensitization, previously diagnosed by flu oro-enzymo-immunoassay. The protein patterns of antigen after purification by the human columns were similar to those obtained using the rabbit column s.