Elisa microplate

We used a previously described method (Legorreta-Herrera et al., 2004 (link)). Briefly, 1 µg of Pb ANKA antigen diluted in 100 µl of carbonate buffer was added to each well of the ELISA microplate (Corning, NY, USA) and incubated for 2 h at 37°C. The plate was washed with 0.5% Tween 20 in PBS and blocked with 3% skimmed milk in PBS for 2 h at 37°C. The plate was washed and incubated with 100 µl of (1:20) diluted test plasma for 1 h at 37°C; the plate was washed and precalibrated dilutions of Ab goat anti-mouse specific for IgM, IgG, IgG1, IgG2a, IgG2b, and IgG3 (Zymed, San Francisco California, USA) were added for 1 h at 37°C; the plates were washed and incubated with streptavidin-peroxidase (Sigma-Aldrich) after washing; and the plates were incubated with ortho-phenylenediamine at 0.4 mg/ml in citrate buffer with 0.03% hydrogen peroxide and incubated for 20 min in the dark. The absorbance was measured at 492 nm using a Stat-Fax 2100 microplate reader (Awareness Technology Inc. USA).

In indirect ELISA, the glycosylated samples were diluted using 50 mM Na₂CO₃-NaHCO₃ (CBS) buffer (pH 9.6) to 5 μg/mL and coated in a Corning-Costar ELISA microplate (Corning, NY, USA) with 100 μL per well at 4 °C overnight. Following this, the plates were blocked with 1% (w/v) BSA (200 μL/well) at 37 °C for 2 h. These plates were then incubated with rat antisera (diluted 200,000), rabbit antisera (diluted 320,000), and TM monoclonal antibodies (diluted 1:20,000) against TM for 1 h. In addition, polled shrimp-allergic patients’ sera diluted 10 with PBST at 37 °C for 1.5 h diluted 10-fold with PBST were added and incubated at 37 °C for 1.5 h. These plates were subsequently washed and incubated with HRP-labeled rabbit anti-rat IgG (100 μL, diluted 1:10,000), HRP-labeled goat anti-rabbit IgG (100 μL, diluted 1:10,000), HRP-labeled rabbit anti-mouse IgG (100 μL, diluted to 1:10,000) or HRP-labeled goat anti-human IgE (100 μL, diluted 1:3000) for 1 h at 37 °C. Afterward, the enzyme reaction was initiated upon adding the TMB solution and stopped after adding sulfuric acid. The absorbance was measured at 450 nm using a MultiscanMK3 microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA).