Hrp conjugated rabbit anti mouse ig

Ten μl aliquots of individual toxin conjugates at a concentration of 100 μg/ml were manually dotted onto nitrocellulose membrane (Pall, India). Sterile PBS (pH 7.4) served as negative control. The NC membrane was air dried, blocked by placing into 5% skim milk in PBS (pH 7.4), incubated at 45°C for 30 min, and washed with three changes of 1× PBST (PBS + 0.5% Tween 20). The membrane was then probed with OTA1 mAb for 30 min at room temperature. After the incubation, the membrane was washed with PBST as described above and then incubated for 30 min at room temperature with HRP conjugated rabbit anti-mouse Igs (Dako, Denmark). After 30 min, the membrane was washed with PBST three times, immersed in the substrate solution (TMB + 0.4% H₂O₂) for color development for 1 min, washed with distilled water, and air dried.

Ten μl aliquots of individual toxin conjugates at a concentration of 100 μg/ml were manually dotted onto nitrocellulose membrane (Pall, India). Sterile PBS (pH 7.4) served as negative control. The NC membrane was air dried, blocked by placing into 5% skim milk in PBS (pH 7.4), incubated at 45°C for 30 min, and washed with three changes of 1× PBST (PBS + 0.5% Tween 20). The membrane was then probed with OTA1 mAb for 30 min at room temperature. After the incubation, the membrane was washed with PBST as described above and then incubated for 30 min at room temperature with HRP conjugated rabbit anti-mouse Igs (Dako, Denmark). After 30 min, the membrane was washed with PBST three times, immersed in the substrate solution (TMB + 0.4% H₂O₂) for color development for 1 min, washed with distilled water, and air dried.

The nitrocellulose (NC) membrane strips were divided into squares 1.0 cm × 1.0 cm with a hard lead pencil, and 20 μL aliquots of a range of rabbit polyclonal IgG (1–5 mg/ml) against OTA-BSA diluted in PBS were dotted on separate squares. The strips were allowed to dry and then the remaining unbound sites were blocked with a solution of 5% skim milk in PBS. After washing with PBST, the strips were cut into the squares and placed in 24 well microtitre plate. Two hundred μL to 1 ml of suspected sample containing OTA was added into each well and the plate was incubated at 37°C for 1 h. Each assay always had OTA positive and OTA negative control antigens. The NC squares were washed and then incubated at 37°C for 1 h with 500 μL of the 0.5 μg/ml mouse monoclonal IgG against OTA. After washing with PBST, the plates were incubated at 37°C with 1:5000 diluted HRP conjugated rabbit anti-mouse Igs (Dako, Denmark). After 45 min, the membrane was washed with PBST three times, immersed in the substrate solution (DAB + 0.4% H₂O₂) for color development for 1 min, washed with distilled water and air dried.