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Preparation of Entamoeba histolytica antigens without enzymatic inhibitors

Published online by Cambridge University Press:  19 May 2005

M. S. FLORES ,
E. TAMEZ-TREVIÑO ,
F. CASTAÑEDA ,
R. TIJERINA-MENCHACA ,
L. GALAN-WONG  and
R. RANGEL
M. S. FLORES
Affiliation:
Departamento de Microbiología, Facultad de Medicina, UANL Apdo. Postal 4355-H, Monterrey N.L. C.P. 64000, México Facultad de Ciencias Biológicas, UANL Instituto de Biotecnología, Av. Pedro de Alba y Manuel L Barragán, Ciudad Universitaria c.p. 66450 San Nicolás de los Garza, N.L. México
E. TAMEZ-TREVIÑO
Affiliation:
Departamento de Microbiología, Facultad de Medicina, UANL Apdo. Postal 4355-H, Monterrey N.L. C.P. 64000, México
F. CASTAÑEDA
Affiliation:
CIDETEQ, San Fandila, Querétaro Qro. México
R. TIJERINA-MENCHACA
Affiliation:
Departamento de Microbiología, Facultad de Medicina, UANL Apdo. Postal 4355-H, Monterrey N.L. C.P. 64000, México
L. GALAN-WONG
Affiliation:
Departamento de Microbiología, Facultad de Medicina, UANL Apdo. Postal 4355-H, Monterrey N.L. C.P. 64000, México
R. RANGEL
Affiliation:
Departamento de Microbiología, Facultad de Medicina, UANL Apdo. Postal 4355-H, Monterrey N.L. C.P. 64000, México Department of Immunology, Unit 902, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA
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Abstract

The goal of this work is to report a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors. A great reduction of proteolytic activity in the insoluble chloroform/methanol and heated amoebic fraction (IC[ratio ]MC) was obtained by this method, even in the presence of sodium dodecyl sulphate and 2-mercaptoethanol. The substrates azo-casein and the hide powder azure were used to determine the reduction of proteolytic activity of IC[ratio ]MC compared with the activity of crude extract and crude extract with iodoacetamide. The IC[ratio ]MC SDS-PAGE pattern shows a higher quantity of bands than extract with the inhibitor iodoacetamide or than crude extract. In addition, anti-Entamoeba histolytica antibodies from amoebic liver abscess patients recognized a richer antigenic Western blot pattern in the IC[ratio ]MC fraction than in crude extract alone or with inhibitor. The described method has proved to be suitable to preserve amoebic antigens for its use in diagnostic tests and it can be used for immunological response studies against E. histolytica antigens. Furthermore we propose that this method to obtain the IC[ratio ]MC fraction can be applied for the study of other microorganisms or cells with high enzymatic content.

Keywords

Entamoeba histolyticaantigen preservationproteolytic activityenzymatic inhibitorscellular enzymes
Type
Research Article
Information
Parasitology , Volume 131 , Issue 2 , August 2005 , pp. 231 - 236
Copyright
2005 Cambridge University Press

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Footnotes

U.S.A. Patents 5,459,042 and 5,861,263, Mexican Patents 209646 and 209648.

References

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