Rs232c

Antibody capture ELISA with electroeluted proteins and synthetic peptides was performed using 96-well flat bottom ELISA plates (Nunc Maxisorp, Denmark). Briefly, 1 µg/well of electroeluted antigens, EF1-α, α-tubulin and GP63, as well as the crude antigen, LAg were coated in 0.02 M phosphate buffer, pH 7.4 (100 µl/well) and kept overnight at 37 °C. In another set of ELISA 10 µg/well of synthetic peptides P1, P2 and P3 along with antigen LAg were used. Plates were blocked with 1% of bovine serum albumin (BSA) (Sigma, USA) in 200 µl/well of 0.02 M phosphate buffer saline (PBS, pH 7.4) for 2 h at 37 °C. Subsequently, 100 µl/well of urine samples (1:10 dilution in blocking) followed by HRP-conjugated anti-human IgG (Bangalore GeNei, India) (1:4000 dilution) were applied and incubated at 37°C for 1 h. ELISA plates were washed three times with PBS containing Tween-20 in each step. Finally, 0.05% (w/v) o-phenylenediamine dihydrochloride (OPD) (Sigma, USA) as substrate was added in 50 mM phosphate-citrate buffer (100 µl/well) with 0.05% H₂O₂ (Merck, Germany). Subsequently, 2 N sulfuric acid (50 µl/well) was used to stop the reaction and the optical density was determined at 492 nm in an ELISA reader (RS232C, Thermo Scientific, MA, USA).

Indirect ELISA to capture antibodies was performed on 96-well flat bottom plates (Maxisorp Nunc; Thermo Fisher Scientific, MA, USA). In brief, wells were coated with 1 μg/well concentration of purified and electroeluted proteins with phosphate buffer (100 μl/well) and incubated overnight in 4 °C. The next day, antigen-coated wells were blocked with 1% BSA in PBS (200 μl/well) for 2 h at 37 °C. Subsequently, serum samples (1:2000) followed by peroxide conjugated antihuman IgG (1:4000) were applied to the wells in PBS buffer (100 μl/well) and incubated for 1 h at 37 °C. Plates were washed in each step with PBS and Tween 20 to remove any non-specific binding. Finally, wells were incubated in the substrate, o-phenylenediamine dihydrochloride (OPD), and H₂O₂ in the phosphate-citrate buffer (50 μl/well). The biological reaction was stopped with sulfuric acid and optical density values were acquired by using an ELISA plate reader (RS232C; Thermo Fisher Scientific, MA, USA) at a wavelength of 492 nm.