Standardization of procedures of Plasmodium falciparum antigen preparation for serologic tests.
The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20 degrees C up to 10 days or at -70 degrees C for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.
Instituto de Medicina Tropical, Faculdade de Medicina, Universidade de São Paulo, Brasil. sands@usp.br
S L
SL
Avila
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Tozetto-Mendoza
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VG
Arruk
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Ferreira
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood*; D000953:Antigens, Protozoan / Q000737:chemistry* / Q000302:isolation & purification; D001782:Blood Donors; D015153:Blotting, Western / Q000592:standards; D004591:Electrophoresis, Polyacrylamide Gel / Q000592:standards; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D006801:Humans; D016778:Malaria, Falciparum / Q000276:immunology; D010963:Plasmodium falciparum / Q000276:immunology* / Q000302:isolation & purification; D012698:Serologic Tests / Q000379:methods* / Q000592:standards*
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan