PHS 2010-05-103). Mice were infected with O. viverrini by feeding 50 intact, viable metacercariae to each mouse by way of an orogastric tube. Mta1−/− and Mta1+/+ mice were bred in our laboratory as described.23, 28 Seven mice of each genotype, Mta1−/− and Mta1+/+, age- and sex-matched per group, were infected and included in the investigation. Infected mice and control noninfected mice were euthanized

23 days after infection by way of overdose with pentobarbital sodium plus phenytoin sodium (Euthasol, Virbac, Fort Worth, TX). At necropsy, blood for serum was removed by way of cardiac puncture, after which the liver, spleen, kidneys, lungs, and bladder were removed from the mouse. About half of each of the solid ACP-196 purchase organs were stored by snap freezing them in liquid N2, and the remainder were fixed and stored in 4% formalin in phosphate-buffered saline (PBS).

The investigation of O. viverrini infection of these mice was undertaken with the approval of the Institutional Animal Use and Care committee of the George ALK inhibitor Washington University. An indirect enzyme-linked immunosorbent assay (ELISA) was used to measure levels of immunoglobulin G (IgG) to an O. viverrini soluble adult worm preparation produced as described.19 A pool of positive control sera was derived from equal portions of sera from each genotype at 23 days after infection. A pool of negative control sera was sourced from the age- and sex-matched mice without any other apparent infection. PolySorp (Nalge, Nunc International, Rochester, NY) 96-well microtiter plates were coated with 100 μL/well of 5 μg/mL of soluble adult worm antigen, prepared from adult O. viverrini worms in carbonate-bicarbonate buffer (pH

9.6), sealed, and incubated overnight MCE公司 at 4°C. Plates were washed three times with PBS (pH 7.2) and blocked with 100 μL/well of 3% bovine serum albumin (BSA) (Sigma, St. Louis, MO) diluted in PBS (pH 7.2). Control and experimental serum samples were diluted 1:4,000 in PBS (pH 7.2), and 100 μL was added to each well of the microtiter plate in duplicate. The plates were sealed and incubated overnight at 4°C and then washed three times with PBS with 0.05% Tween 20 (pH 7.2). A biotinylated goat anti-mouse IgG antibody (Vector Laboratories Inc., Burlingame, CA) was used at a 1:5,000 dilution in 3% BSA and PBS and applied 100 μL/well and then incubated for 90 minutes at room temperature. After incubation, the plates were washed with PBS with 0.05% Tween 20 and incubated with a 1:1,000 dilution of horseradish peroxidase–conjugated streptavidin (GE Healthcare, Buckinghamshire, UK) in 3% BSA and PBS for 60 minutes at room temperature in the dark. The plates were incubated in the dark at room temperature for 30 minutes with o-phenylenediamine dihydrochloride.