4 nitrophenyl phosphate disodium salt hexahydrate substrate

The procedure carried out by Laboratory 3 is based on the ELISA described by Szu et al. and uses Vi lot 05 supplied by CBER [18 (link)]. Wells (Nunc Maxisorp) were coated with 100 μL of 2 μg Vi mL⁻¹ diluted in PBS. Plates were covered, incubated overnight at 25 °C, washed 2x with NaCl-Brij and shaken dry. Wells were blocked with BB2 for 2 h at 25 °C, washed 2x with NaCl-Brij and shaken dry. Starting dilutions of test samples were prepared in AD2. Wells of the first row received 200 μL of the starting dilution and all other wells received 100 μL AD2. Top down dilutions were prepared by transfer of 100 μL of the diluted sample to subsequent wells. Plates were covered, incubated overnight at 25 °C, washed 2x with NaCl-Brij and shaken dry. Hundred μL of AP conjugated goat anti-human IgG (Sigma) diluted 1:5000 in AD2 was added to each well and incubated at 37 °C for 4 h. Plates were washed 2x with NaCl-Brij, shaken dry and 100 μL of 4-Nitrophenyl Phosphate disodium salt hexahydrate substrate (Sigma) in Tris/3 mM Mg-buffer (pH 9.8) was added to each well and incubated at RT for 10–30 mins. Plates were read at OD_405nm.

The procedure carried out by Laboratory 7 is a modification of the ELISA by Szu et al. [18 (link)]. The plate surface is coated with directly with Vi PS from S. Typhi (lot 201308, Wuhan Institute of Biological Products Co. Ltd). Wells (Costar, 2592) were coated with 100 μL of 2 μg Vi mL⁻¹ in PBS diluted from 1 mg mL⁻¹ in Milli-Q. Plates were covered, incubated overnight at 4 °C, washed 5x with 1% Brij L23 in 0.8% NaCl (NaCl-Brij 23). Wells were blocked with BB2 for 2 h at 37 °C, washed 5x with NaCl-Brij 23 and shaken dry. Starting dilutions of test samples were prepared in BB2 with 0.1% Brij L23 (AD4). Wells of the first row received 200 μL of the starting dilution and all other wells received 100 μL AD4. Top down dilutions were prepared by transfer of 100 μL of the diluted sample to subsequent wells. Plates were covered, incubated overnight at 25 °C, washed 5x with NaCl-Brij 23 and shaken dry. Hundred μL of AP conjugated goat anti-human IgG (Southern Biotech, 2040-04) diluted 1:2000 in AD4 was added to each well and incubated at 37 °C for 2 h. Plates were washed 5x with NaCl-Brij 23 and shaken dry and 100 μL of 4-Nitrophenyl Phosphate disodium salt hexahydrate substrate (Sigma, 71768) in Tris/3 mM Mg-buffer (pH 9.8) was added to each well and incubated at RT for 90–120 mins, then stopped by 50 μL 3 M NaOH. Plates were read at OD_405nm.