One hundred microlitre volumes were used in each well for the ELISA, unless stated otherwise, with samples loaded in duplicate and incubation stages carried out in a humid chamber at 37 C. Samples and standards were diluted in PBST (005 mol lÿ1 phosphate, 015 mol lÿ1 NaCl, 001% Tween 20, pH 74), which was supplemented with 1% bovine serum albumin (BSA) for the dilution of BS1-IgG-biotin and streptavidin-alkaline phosphatase. In between each stage, plates (Dyrex Technologies, Billinghurst, UK) were washed three times with PBST.