96 well flat bottom elisa plates

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).

Flat-bottom 96-well ELISA plates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were coated with 100 ng per well of the antigens from L. intracellularis. Plates were washed with PBS 0.05% Tween 20 (PBST) and blocked with 3% skimmed milk in PBS. After washing, diluted serum from the pigs was added (100 μL/well) and incubated for 2 h at 37 °C. Plates were washed, and the 1/10,000 diluted goat anti-pig IgG-HRP polyclonal antibody (Abcam, Boston, MA, USA) was added. Plates were washed and revealed with a substrate solution of o-phenylenediamine dihydrochloride (OPD) 0.4 mg/mL (Sigma, Burlington, MA, USA). The absorbance at 450 nm was read using the Synergy™ HTX Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). Titer was determined using pre-immune serum absorbance value as cut-off multiplied by two, using serum sample serial dilutions (1:500 to 1:64,000), assigning titer value as indicative of the last dilution in which the antibody was detected.