Preparation of Entamoeba histolytica antigens without enzymatic inhibitors.
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: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:MC compared with the activity of crude extract and crude extract with iodoacetamide. The IC: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: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:MC fraction can be applied for the study of other microorganisms or cells with high enzymatic content.
Flores
M S
MS
Departamento de Microbiologia, Facultad de Medicina, UANL Apdo. Postal 4355-H, Monterrey N.L. C.P. 64000, Mexico. maflores@fcb.uanl.mx
Tamez-Treviño
E
E
Castañeda
F
F
Tijerina-Menchaca
R
R
Galan-Wong
L
L
Rangel
R
R
D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000302:isolation & purification* / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D008101:Liver Abscess, Amebic / Q000276:immunology; D010447:Peptide Hydrolases / Q000378:metabolism*
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D010447:Peptide Hydrolases