ELISA. For glycolipids, polysorp microtitration plates (Nunc, Denmark) were coated with 0.3% (IgG4 and IgE) or 0.05% (IgG) of the ceramide containing lipid fraction derived from 4 grams of worm, dissolved in methanol. Each well was coated either with 25 μl of lipid in methanol or with methanol only as a control, and the plates were air-dried overnight at room temperature. For proteins, maxisorp microtitration plates (Nunc, Denmark) were coated overnight at room temperature with 100 μl 5 μg/ml (for IgG4 and IgE) and
ELISA. Concentrations of IL-6, IL-8, FGF2, thymus- and activation-regulated chemokine (TARC) and VEGF in cell culture supernatants were measured by enzyme- linked immunosorbent assay (ELISA) (all purchased from PeproTech, London) according to the manufacturer’s instructions. The standard curves for human IL- 8, VEGF, IL-6 and FGF2 ranged from 16-2000 pg/ml, 16-1000 pg/ml, 32-2000 pg/ml and 63-4000 pg/ml respectively. The individual assays were reported to show no cross-reactivity. A summary of an ELISA protocol is outlined here: A 96-well plate was incubated overnight with the capture antibody at 0.5 µg/ml in PBS (pH 7.20 in sterile water), and subsequently blocked with PBS containing 1 % BSA (R&D Systems). After washing the plate with PBS buffer containing 0.05 % Tween-20 (R&D Systems), appropriate dilutions of samples and standards were added, incubated for 2 hours at room temperature, washed, and incubated with a detection antibody for 2 hours at room temperature. After washing, streptavidin-HRP conjugate (1:200; R&D Systems) was added. Finally, ABTS substrate (100 μl) (R&D Systems) was added. The reaction was stopped with 2 N H2SO4 (R&D Systems), 50 μl per well. The plate was read on an ELISA plate- reader at an optical density (OD) of 450 nm with wavelength correction set at 650 nm.
ELISA. Human insulin growth factor binding protein (IGFBP) 3 and human insulin growth factor binding protein 5 (R&D Systems; Minneapolis, MN) were used according to the manufacturer’s directions. Briefly, sample media was incubated in either IGFBP3 or IGFBP5 antibody-coated microplates for 2 hours, then washed and incubated with appropriate antibody conjugated to horseradish peroxidase for 1 hour. After washing, plates were incubated with color reagent (hydrogen peroxide–chromogen mix) for 30 minutes. Optical density of each well was determined using a microplate reader set to 450 nm. The concentrations were calculated according to the standards supplied with the kit by creating a four parameter logistic curve-fit. Samples were run in triplicate, and experiments were repeated 3 times with reproducible results.
ELISA. Many immunological tests for schistosomiasis infection focus on the identification of circulating cathodic antigen (CCA) and/or circulating anodic antigen (CAA). Both substances are antigens in the adult worm gut and are released into the host blood stream (Xxxxxxx et al., 1991). However, they are relatively quickly eliminated so that their presence in mummified remains is indicative of infection at the time of death. Because the concentration of antigens in a tissue sample is largely dependent upon the number of schistosomes in the individual (Xxxxx et al., 1995; Xxx Xxxxxxxx et al., 1995a), antigen concentrations will serve as a biomarker of infection intensity. CCA levels in human serum have been shown to correlate well with egg counts in repeated samplings (Xxx Xxxxxxxx et al. 1995). This suggests CCA levels provide an accurate reflection of current worm burden (Xxxxxx et al., 2001). As circulating antigen levels show less day to day variability, they provide a more quantitatively stable measure of infection intensity than egg count (Xxxxxx et al., 1998). However, the level of antigens detected in mummified tissues is largely dependent upon the vascularization of the tissues. Hence, measurement of CCA from well vascularized tissue samples would suggest higher intensity infections than less vascularized tissue samples from individuals with similar intensity of infection. Comparison of infection intensity based on antigen concentration between individuals would therefore be dependent upon the ability to obtain tissue samples of known anatomical source and similar vascularization. An enzyme-linked immunosorbent assay (ELISA) was used to detect biotinylated
