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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

REFERENCES

ARAIZA-OROZCO, L. M., AVILA, E., MUNOZ, M. L. & ARIAS-NEGRETE, S. ( 1999). Entamoeba histolytica: surface proteolytic activity and its relationship with in vitro virulence. Folia Parasitologica 46, 161–167.Google Scholar
AVILA, E., SÁNCHEZ-GARZA, M. & CALDERÓN, J. ( 1985). Entamoeba histolytica and E. invadens: Sulfhydril-dependent proteolytic activity. Journal of Protozoology 32, 163–166.Google Scholar
CANTERA, C. S., GOYA, L., GARCIA, R. & SOFIA, A. ( 1999). Proteolytic activity assessment: use of the chromogenic substrate hide powder azure. Journal of the Society of Leather Technologists and Chemists 83, 101–106.Google Scholar
CLARK, C. G., ESPINOSA-CANTELLANO, M. & BHATTACHARYA, A. ( 2000). Entamoeba histolytica: an overview of the biology of the organism. In Amebiasis (ed. Ravdin, J. I.), pp. 1–45. Imperial College Press, London.CrossRef
FLORES DE CASTAÑEDA, M. S. ( 2002). Procedimiento para la preservación de moléculas antigénicas, sin el uso de inhibidores enzimáticos. Mexican Patent 209646.
FLORES DE CASTAÑEDA, M. S. ( 2002). Procedimiento para la preservación de moléculas antigénicas, sin el uso de inhibidores enzimáticos y su aplicación en métodos inmunológicos. Mexican Patent 209648.
FLORES-CASTAÑEDA, M. S. ( 1999). Procedure to preserve antigens of Entamoeba histolytica without enzymatic inhibitors and their use in immunological methods. United States Patent 5 861 263.
FLORES DE CASTAÑEDA, M. S. ( 1995). Procedure to preserve antigens of E. histolytica without enzymatic inhibitors. United States Patent 5459042.
FREITAS, M. A., VIANNA, E. N., MARTINS, A. S., SILVA, E. F., PESQUERO, J. L. & GOMES, M. A. ( 2004). A single step duplex PCR to distinguish Entamoeba histolytica from Entamoeba dispar. Parasitology 128, 625–628.CrossRefGoogle Scholar
HARRIS, E. L. V. & ANGAL, S. ( 1995). Protein Purification Methods: A Practical Approach. IRL Press, Oxford.
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. ( 1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–275.Google Scholar
McLAUGHLIN, J. & FAUBERT, G. ( 1977). Partial purification and some properties of a neutral sulphydril and an acid proteinase from Entamoeba histolytica. Canadian Journal of Microbiology 23, 420–425.CrossRefGoogle Scholar
MERRIL, C. R., GOLDMAN, D., SEDMAN, S. A. & EBERB, M. ( 1981). Ultrasensitive stain for proteins in polyacrylamide gels, regional variations in cerebrospinal fluid proteins. Science 211, 1434–1438.CrossRefGoogle Scholar
PÉREZ-MONFORT, R., OSTOA-SALOMA, P., VELAZQUEZ-MEDINA, L., MONTFORT, I. & BECKER, I. ( 1987). Catalytic classes of proteinases of Entamoeba histolytica. Molecular and Biochemical Parasitology 26, 87–97.CrossRefGoogle Scholar
QUE, X. & REED, S. L. ( 2000). Cysteine proteinases and the pathogenesis of amebiasis. Clinical Microbiology Reviews 13, 196–206.CrossRefGoogle Scholar
RAWAL, S., MAJUMDAR, S., DHAWAN, V. & VOHRA, H. ( 2004). Entamoeba histolytica Gal/GalNAc lectin depletes antioxidant defences of target epithelial cells. Parasitology 128, 617–624.CrossRefGoogle Scholar
RINDERKNECHT, H., GEOKAS, M. C., SILVERMAN, P. & HAVERBACK, B. J. ( 1968). A new ultrasensitivity method for the determination of proteolytic activity. Clinica Chimica Acta 21, 197–203.CrossRefGoogle Scholar
SAÍD-FERNÁNDEZ, S. & FLORES-CASTAÑEDA, M. S. ( 1996). Diagnóstico molecular de amibiasis. Gaceta Médica de México 132, 314–318.Google Scholar
SERRANO-LUNA, J. J., NEGRETE, E., REYES, M. & DE LA GARZA, M. ( 1998). Entamoeba histolytica HM1:IMSS: hemoglobin-degrading neutral cysteine proteases. Experimental Parasitology 89, 71–77.CrossRefGoogle Scholar
TAMEZ-TREVIÑO, E., SEPÚLVEDA-SAAVEDRA, J., TIJERINA-MENCHACA, R. & FLORES-CASTAÑEDA, M. S. ( 2000). Systemic anti-amoeba antibodies induced orally in mice: detection by an ELISA test using preserved Entamoeba histolytica antigens without enzymatic inhibitors. Archives of Medical Research 31 (Suppl.), S28–S29.Google Scholar