Immulon 2 plates

The full sample cohort (N=371) of serum samples from the gold, diamond, and emerald mining populations was utilized for enzyme-linked immunosorbent assay to validate the protein array data. Immulon2 plates (ThermoScientific) were coated with peptide cocktail including all three peptides (Table 1) (0.25 μg/ml in PBS) and incubated at 4°C overnight. The plates were washed with wash buffer (2% FBS in PBS) and diluted serum samples (1:500 and 1:2500 in PBS) were added in triplicate. A triplicate 8-point standard curve was generated with 2-fold serial dilutions of rabbit anti-serum and negative controls included two dilutions of pre-immune serum (1:500 and 1:2500). Plates were incubated for 2 hours at room temperature, washed, and peroxidase-conjugated recombinant protein G (Thermo Scientific) (0.1 μg/ml) was added. Anti-GSTA1 relative titer was determined by colorimetric analysis with TMB substrate solution (Sigma) at 450nm. The limit of detection was set per plate as the standard deviation of the blanks × 3. Any reading below the limit of detection was set to the limit of detection/√2.

Immulon 2 plates (ThermoLabsystems, Franklin, MA) were coated overnight at 4°C with 5 μg/ml PC-BSA (50 μl/well). Plates were washed, blocked with 3% BSA + PBS at 37°C for 2 hours, washed, and 100μl/well of eight twofold dilutions of sera in PBS +1% BSA were added starting at 1/400 for the IgG and IgM ELISAs and 1/50 for the IgG subclasses. Plates were incubated for 1 hour at 37°C and washed. Goat anti-mouse AP conjugated antibodies (100μl/well) were added for 1 hour at 37°C. Plates were washed, phosphatase substrate tablets (Sigma-Aldrich) dissolved in p-Nitrophenyl Phosphate, Disodium Salt (PNPP) buffer and added to wells. Absorbance was read at 405nm on a μQuant spectrophotometer using KCJunior software (Bio-Tek Instruments, Winooski, VT).