Anisakis simplex check for this species in other resources - antigen purification - affinity chromatography - ELISA - rabbit antisera"/>
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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060
EISSN: 1678-8060
Vol. 97, No. 2, 2002, pp. 247-252
Bioline Code: oc02045
Full paper language: English
Document type: Research Article
Document available free of charge

Memórias do Instituto Oswaldo Cruz, Vol. 97, No. 2, 2002, pp. 247-252

 en Evaluation by ELISA of Anisakis simplex check for this species in other resources Larval Antigen Purified by Affinity Chromatography
M Rodero; A Jiménez & C Cuéllar

Abstract

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex check for this species in other resources third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum check for this species in other resources antigens or inoculated with Toxocara canis check for this species in other resources embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of 1/400 and 1 μg/ml of antigenic concentration.

Keywords
Anisakis simplex check for this species in other resources - antigen purification - affinity chromatography - ELISA - rabbit antisera

 
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