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Western blot antibody determination in sera from patients diagnosed with Anisakis sensitization with different antigenic fractions of Anisakis simplex purified by affinity chromatography

Published online by Cambridge University Press:  01 September 2007

M. Rodero ,
C. Cuéllar ,
T. Chivato ,
J.M. Mateos  and
R. Laguna
M. Rodero
Affiliation:
Departamento de Parasitologia, Facultad de Farmacia, Universidad Complutense, 28040 MadridSpain
C. Cuéllar*
Affiliation:
Departamento de Parasitologia, Facultad de Farmacia, Universidad Complutense, 28040 MadridSpain
T. Chivato
Affiliation:
Servicio de Alergia e Inmunología, Hospital del Aire, 28007 Madrid, Spain
J.M. Mateos
Affiliation:
Servicio de Alergia e Inmunología, Hospital del Aire, 28007 Madrid, Spain
R. Laguna
Affiliation:
Servicio de Alergia e Inmunología, Hospital del Aire, 28007 Madrid, Spain
*
*Fax: +34 91 3941815, E-mail: [email protected]
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Abstract

Using Western blot techniques, the specificities of crude and purified (PAK and PAS) Anisakis simplex antigens were compared against 24 sera from patients diagnosed with Anisakis sensitization. All patients recognized a 60 kDa protein against the A. simplex crude extract, while 37.5% and 12.5% reacted with proteins of 40 and 25 kDa, respectively, when IgG was tested. In the case of IgE determination, 41.6% of sera were negative, while 12.5% and 20.8% appeared to cross-react against Toxocara canis and Ascaris suum, respectively. When the PAK antigen (A. simplex antigen purified by means of a column of IgG anti-A. simplex) was tested, immune recognition towards the 60, 40 and 25 kDa proteins increased in 83.3%, 16.7% and 4.2%, respectively, when the Ig antibodies were tested. In the case of the PAS antigen (PAK antigen purified by means of a column of IgG anti-A. suum), the reaction against the 40 and 25 kDa proteins increased to 45.8% and 25%, respectively, when Ig antibodies were used. Finally, when the EAS antigen (eluted from the anti-A. suum column after PAK purification) was tested, 83.3% of the assayed sera reacted against the 14 kDa protein, when the Ig antibodies, IgG and IgM immunoglobulins were measured. With the IgE determination, the reactions were observed in 41.7% of patients with proteins between 60 and 35 kDa against the PAS antigen. With the EAS antigen, reactive bands of 184, 84 and 14 kDa appeared. In conclusion, in the purification process of the A. simplex larval crude extract, the proteins implicated in cross-reactions with Ascaris and Toxocara were eliminated, with an important concentration of proteins responsible for the induction of specific responses.

Type
Short Communication
Information
Journal of Helminthology , Volume 81 , Issue 3 , September 2007 , pp. 307 - 310
Copyright
Copyright © Cambridge University Press 2007

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References

Águila, C., Cuéllar, C., Fenoy, S. & Guillén, J.L. (1987) Comparative study of assay detecting circulating immuno-complexes and specific antibodies in patients infected with Toxocara canis. Journal of Helminthology 61, 196–202.CrossRefGoogle Scholar
Arrieta, I., del Barrio, M., Vidarte, L., del Pozo, V., Pastor, C., González, J., Cardaba, B., Rojo, M., Minguez, A., Cortesano, I., Gallardo, S., Aceituno, E., Palomino, P., Vivanco, F. & Lahoz, C. (2000) Molecular cloning and characterization of an IgE-reactive protein from Anisakis simplex: ani s1. Molecular and Biochemical Parasitology 15, 263–268.CrossRefGoogle Scholar
Cuéllar, C., Fenoy, S., del Águila, C. & Guillén, J.L. (1990) Evaluation of chemotherapy in experimental toxocarosis by determination of specific immune complexes. Journal of Helminthology 64, 279–289.CrossRefGoogle ScholarPubMed
del Rey, A., Valero, A., Mayorga, C., Gómez, B., Torres, M.J., Hernández, J., Ortiz, M. & Lozano, J. (2006) Sensitization to Anisakis simplex s.l. in a healthy population. Acta Tropica 97, 265–269.CrossRefGoogle Scholar
Hames, B.D. (1986) An introduction to polyacrylamide gel electrophoresis. pp. 21–57in Hames, B.D. & Rickwood, D. (Eds) Gel electrophoresis in proteins. Oxford, IRL Press.Google Scholar
Iglesias, R., Leiro, J., Santamarina, M.T., Sanmartin, M.L. & Ubeira, F.M. (1997) Monoclonal antibodies against diagnostic Anisakis simplex antigens. Parasitology Research 83, 755–761.CrossRefGoogle ScholarPubMed
Ishikura, H., Kikuchi, K., Nagasawa, K., Ooiwa, T., Takamiya, H., Sato, N. & Sugane, K. (1993) Anisakidae and Anisakiosis. pp. 43–102in Sun, T. (Ed.) Progress in clinical parasitology. New York, Springer-Verlag.CrossRefGoogle Scholar
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680.CrossRefGoogle ScholarPubMed
Lorenzo, S., Iglesias, R., Audícana, M.T., García, R., Pardo, F., Sanmartin, M.L. & Ubeira, F.M. (1999) Human immunoglobulin isotype profiles produced in response to antigens recognized by monoclonal antibodies specific to Anisakis simplex. Clinical and Experimental Allergy 29, 1095–1101.CrossRefGoogle ScholarPubMed
Lorenzo, S., Romaris, F., Iglesias, R., Audícana, T., Alonso, J., Leiro, J. & Ubeira, F.M. (2000) O-glycans as a source of cross-reactivity in determinations of human serum antibodies to Anisakis simplex antigens. Clinical and Experimental Allergy 30, 551–559.CrossRefGoogle ScholarPubMed
Moneo, I., Caballero, M.L., Gómez, F., Ortega, E. & Alonso, M.J. (2000) Isolation and characterization of a major allergen from the fish parasite Anisakis simplex. Journal of Allergy and Clinical Immunology 106, 177–182.CrossRefGoogle Scholar
Nakata, H., Yamamoto, Y. & Yamamoto, Y. (1990) Analysis of antigens defined by anti-Anisakis larvae antibodies of IgE and IgG type in the sera of patients with acute gastrointestinal anisakiasis. Nippon Shokakibyo Gakkai Zasshi 87, 762–770.Google ScholarPubMed
Perteguer, M.J. & Cuéllar, C. (1998) Isotype-specific immune-responses in murine experimental anisakiasis. Journal of Veterinary Medicine 45, 603–610.CrossRefGoogle ScholarPubMed
Rodero, M., Jiménez, A., Chivato, T., Laguna, R. & Cuéllar, C. (2001) Purification of Anisakis simplex antigen by affinity chromatography. Parasitology Research 87, 736–740.Google ScholarPubMed
Rodero, M., Jiménez, A. & Cuéllar, C. (2002) Evaluation by ELISA of Anisakis simplex larval antigen purified by affinity chromatography. Memorias do Instituto Oswaldo Cruz 97, 247–252.CrossRefGoogle ScholarPubMed
Rodero, M., Chivato, T., Muro, A. & Cuéllar, C. (2005) Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography. Memorias Instituo Oswaldo Cruz 100, 293–301.CrossRefGoogle ScholarPubMed
Sakanari, J.A. & McKerrow, J.H. (1989) Anisakiasis. Clinical and Microbiological Reviews 2, 278–284.CrossRefGoogle ScholarPubMed
Welch, J.S., Symons, M.H. & Dobson, C. (1983) Immunodiagnosis of parasitic zoonoses: purification of Toxocara canis antigens by affinity chromatography. International Journal for Parasitology 13, 171–178.CrossRefGoogle ScholarPubMed