Peroxidase conjugated anti human iga p0216

Acute-phase titres of fecal IgA anti-RV were determined by ELISA using a modification of method previously described [22] . In brief, microtitre plates of 96 wells (Greiner Bio-One) were coated with guinea pig antibody to RV (SBL), diluted 1:500 in carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. After 2 hours' blocking with phosphate-buffered saline (PBS)-bovine serum albumin 3%, RV5 vaccine (Merck) diluted 1:100 was added and incubated at 37°C for 1 hour. A total of 100 μL of serially diluted stool (1:20, 1:40, 1:80 and 1:160) was added and incubated at 37°C for 1 hour, followed by addition of peroxidase-conjugated anti-human IgA (P0216; DakoCytomation) diluted 1:2000, and incubated at 37°C for 1 hour. The reaction was developed for 10 minutes with tetramethylbenzidine (Thermo Fisher Scientific) and stopped with 2M H 2 SO 4 . Optical density (OD) was determined at 450 nm in each well. The reciprocal of the highest stool dilution with OD 0.100 was considered sample titre. All experiments included negative control (neonate stool) and PBS for background monitoring.

Serum rotavirus-specific IgA titers were determined by enzyme-linked immunosorbent assay (ELISA) using a method previously described. 23 In brief, microtiter plates of 96 wells (Greiner Bio-One, Kremsmünster, Austria) were coated with RV5 vaccine diluted 1:100 in carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. After 2 hours of blocking with phosphatebuffered saline-bovine serum albumin 3%, a total of 100 μL of diluted serum (1:25 and 1:50) was added and incubated at 37°C for 1 hour, followed by addition of peroxidase-conjugated antihuman IgA (P0216; DakoCytomation, Glostrup, Denmark) diluted 1:2000, and incubated at 37°C for 1 hour. The reaction was then developed for 10 minutes with tetramethylbenzidine Q3 (Thermo Fisher Scientific, Waltham, MA) and stopped with 2M H 2 SO 4 . Optical density was determined at 450 nm in each well. Titer was assigned by comparing the absorbance reading of both dilutions with a database of absorbance readings from serum titers (50, 100, 200, 400, 800 and 1:600) of RV5-vaccinated children. Phosphatebuffered saline was used in all experiments for background monitoring.