PATIENTS AND METHODS

Stored sera from 378 patients with PBC from The Toronto Western Hospital, Toronto, Ontario, were screened. IgA EmA was detected with the use of indirect immunofluorescence on human umbilical cord by using the method described by Ladinser et al; the serum was measured at a 1:5 dilution.

Titres of IgA tTG were measured with the use of an ELISA method devised by Dieterich et al, which was modified to account for differences in scientific supplies. In brief, high affinity 96-well microtitre plates (Costar Corporation, USA) were coated with 1/600 U/well of tTG from guinea pig (Sigma-Aldrich Canada Ltd, Canada) in 100 mL 50 mM tris-hydrochloride, 150 mM sodium chloride and 5 mM calcium chloride (pH 7.5) overnight at 4°C. After washing three times with 50 mM tris-hydrochloride, 150 mM sodium chloride, 10 mM EDTA and 0.1% Tween 20 (pH 7.4), the plate was blocked for 10 min at 37°C by using this solution. Samples were then applied to the plates at 1:5 dilution using the same solution as a diluent. The plate was incubated at room temperature for 1 h and then washed three times again. Peroxidase-conjugated rabbit antihuman IgA (Dako Corporation, USA) was applied at 1:3000 dilution for 1 h at room temperature, and the plate was again washed three times. O-phenylenediamine-hydrochloride (Sigma-Aldrich Canada Ltd) 0.4 mg/mL in citric acid 0.05 mM and dibasic sodium phosphate 0.1 mM with hydrogen peroxide 0.06% was applied to the plate and incubated for 1 h in the dark at room temperature before reading using 450 nm wavelength light. A sample from a patient with biopsy-proven, untreated CD was tested in doubling dilutions until the linear portion of the standard curve was obtained. This sample was designated to have 400 arbitrary units (AU)/mL during the initial validation of the assay and was used for the standard curve of each plate. The titre, in AU, of each test sample was then calculated from this curve. Samples with optical densities above the upper limit of the standard curve were repeated at increasing dilutions until final titres were calculated. During the initial validation of the assay, a reference range of up to 140 AU/mL was calculated from titres from gastrointestinal disease control subjects with normal small bowel biopsies. Buy quality medications online: find generic serevent every time you need treatment.

Samples with titres of less than 5 AU/mL were tested for IgA deficiency using NOR-Partigen Total IgA kit (Behring Diagnostics Inc, USA).