Patent ID: 6682900
Filing Date: 2004-01-27
Classification: G01N,Y02A,Y10S

Abstract:
A process for the preparation of Epimastigote Extract (EpEx) antigen from epimastigote forms of T. Cruzi, TulahuÃ©n strain, which process comprises the steps of:a) culturing the parasites thereof at a temperature of 28Â° C. in Schneider's insect culture medium containing 20% fetal calf serum, and after 7-10 days, b) collecting the parasites from the culture supernatant, c) washing the collected parasites 3-5 times with phosphate-buffered saline (PBS) at 100 mM at a pH of 7.3-7.4, d) centrifuging the washed parasites at 12,000-14,000 g for 30-45 minutes at 4-8Â° C., immediately thereafter e) resuspending the parasites in a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer at 10 mM, pH of 7.4-7.5, containing 0.2-0.4 mM leupeptin, 2-3 mM ethylenediaminetetraacetic acid EDA, 1% nonanoyl-N-methylglucamide (lysis buffer), f) submitting the resuspended parasites to 4-6 cycles of freezing and thawing, in liquid nitrogen and in a water-bath at 37Â° C., respectively, g) centrifuging the resultant lysate at 10,000-12,000 g for 5-10 minutes, at 4-8Â° C., h) removing the supernatant containing the EpEx antigenic preparation, and i) storing the supernatant containing the EpEx antigenic preparation at âˆ’70Â° C. until use.