ELISA Optimization and Comparison for Schistosome Antibody Detection

Preliminary titrations with varying antigen concentrations, serum and secondary antibody dilutions were conducted to determine optimal conditions to carry out the enzyme-linked immunosorbent assay (ELISA) using sera from endemic normal people (i.e. heavily exposed to infection but negative for schistosome eggs), negative controls (i.e. British people who had never travelled to schistosome endemic areas) and a pool of sera from the whole population. These titration assays allowed determination of the serum dilution and antigen concentration yielding the best discrimination between negative and positive controls. The Elisa protocol was thus developed. Assays were conducted using MBP-Sh13 and MBP control as is standard [3 (link)]. ELISA plates (Nunc-Immulon, Denmark) were coated with 100 μl/well of 1 μg/ml antigen in 60 mM carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Plates were blocked with 200 μl/well of skimmed milk (5% milk in phosphate buffered saline (PBS)/0.03% Tween 20) for 1 hr and washed three times in PBS/Tween 20, which was used for all washes. 100 μl of serum was added to each well at 1:100 dilution; plates were incubated overnight at 4°C and then washed three times. 100 μl of isotype-specific monoclonal antibody was added at 1:1000 dilution for the detection of IgA (Dako, Denmark, P0216), IgE (Vector Laboratories, UK P0720), IgG1, IgG2, IgG3, IgG4 (The Binding Site, UK, AP006, AP007, AP008 and AP009, respectively) and IgM (Dako, Denmark, P0215). Plates were incubated overnight at 4°C, washed six times and 100 μl of ABTS substrate solution (KPL, Canada) was added. The IgE-specific antibody was biotinylated; so 100 μl/well of streptavadin-horseradish peroxidase (Amersham, UK) was added at 1:6000 dilution to these plates, which were then incubated for 1 hr at 37°C, washed 6 times and developed. The reaction was allowed to take place at 37°C for 30 min for all isotypes, before the absorbance was read at 405 nm. Three negative controls used in the titration assays were included on each ELISA plate and all samples were assayed in duplicate.
For comparative purposes, antibody assays were also conducted with the conventional crude worm antigen (soluble worm antigen preparation, SWAP) prepared following standard protocols [13 (link)]. These assays were conducted following the same protocol as above using 1 μg/ml SWAP to coat the plates (a random subset of 41 samples run using 20 μg/ml SWAP together with negative and positive controls showed that similar results were obtained using 1 μg/ml and 20 μg/ml of SWAP) sera diluted at 1:100, and secondary antibodies diluted at 1:1000 for IgG1 and 1:500 for IgG2, IgG3, IgG4.