Evaluation of an immunoblot methodology for the detection of relevant Entamoeba histolytica antigens by antibodies induced in human amebiasis.
The complexity of the clinical spectrum in human amebiasis and the high variability in laboratory methods used to detect Entamoeba histolytica infections have impeded the collection and evaluation of reliable epidemiological data. Thus, more sensitive, specific and standardized methods are needed in order to accurately identify infections with this parasite. An important step in the development of serological diagnostic methods is the identification and isolation of specific parasite antigens which are immunogenic in the host. In this work, we have standardized an electroimmunotransfer blot technique to characterize E. histolytica antigens recognized by antibodies present during human amebic infections. An important aspect was an investigation of technical variations in the preparation of cell lysates including the use of different protease inhibitors and solubilizing agents. The highest yield of protein was achieved by homogenization of trophozoites in the presence of 10 mM p-hydroxymercuribenzoate (pHMB) as a protease inhibitor and by lysis using Triton X-100 and a mixture of protease inhibitors. Recovery of degraded vs non-degraded proteins in the cell extracts was evaluated by gradient polyacrylamide gel electrophoresis. Both quantitative and qualitative differences were noted between the different methods of preparing soluble cell extracts. A more complete set of antigenic components was obtained by homogenization and use of pHMB. Thus parasite extracts prepared by this method were selected for protein transfer. In this, the optimal protein concentration was of 120 micrograms of protein per cm of gel width and efficient transfer of proteins to nitrocellulose sheets was achieved at 100 V for 2 hrs and at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Argüello-García
R
R
Depto. de Genética y Biología Molecular, Centro de Inv. y Estudios Avanzados del IPN, México, D.F.
Sánchez-Guillén
M C
MC
Garduño
G
G
Valadez-Salazar
A
A
Martínez-García
M C
MC
Muñoz
O
O
Ortega-Pierres
M G
MG
D000328:Adult; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology*; D000918:Antibody Specificity; D000953:Antigens, Protozoan / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000737:chemistry / Q000254:growth & development / Q000276:immunology*; D004749:Entamoebiasis / Q000097:blood / Q000276:immunology*; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D007411:Intestinal Diseases, Parasitic / Q000097:blood; D008297:Male; D011480:Protease Inhibitors / Q000494:pharmacology; D015800:Protozoan Proteins / Q000276:immunology* / Q000302:isolation & purification
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D011480:Protease Inhibitors; D015800:Protozoan Proteins