Hrp rabbit anti human igg secondary antibody

Cell lysates, cell supernatants, purification fractions, rZNS1-His and gel filtration fractions were resolved on 7 % or 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a nitrocellulose membrane (Hybond-ECL, GE Healthcare), analysis by immunoblotting was performed using 0.2 μg/ml ZNS1 monoclonal antibody (mAbia labs, Argentina), 1:1000 glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Sigma-Aldrich) or 0.1 μg/ml anti-His tag monoclonal antibody (mAbia labs, Argentina). Bound mAbs were recognized with a horseradish peroxidase (HRP) goat anti-mouse IgG secondary antibody (Sigma-Aldrich) at a 1:2000 dilution, Alexa Fluor 680 goat anti-mouse IgG secondary antibody (Invitrogen) at a 1:20000 dilution, or with HRP rabbit anti-human IgG secondary antibody (Dako) at a 1:10000 dilution. The signal was visualized with enhanced chemiluminiscence reagent (GE Healthcare) and CL-XPosure Films (Thermo Scientific), or with an Odyssey Infrared Imager (Li-Cor). Densitometric analysis was performed using the NIH ImageJ software.

Microtiter plates (Nunc Maxisorp 96-well ELISA plates) were coated with 100 μl of rZNS1-His (400 ng/well) in coating buffer (0.1 M Na₂HPO₄ buffer pH 9.5) for 18 h at 4 °C. Following incubation in blocking buffer (5 % skimmed milk in TBS) for 1 h at 37 °C, the plates were further incubated with human sera at a 1:100 dilution, or with mouse polyclonal sera at the indicated dilutions, for 1 h at RT in blocking buffer. Following four washing steps in TBS Tween-20 0.05 %, plates were further incubated in HRP rabbit anti-human IgG secondary antibody (Dako) at a 1:4000 dilution, or with HRP goat anti-mouse IgG Fcγ fragment specific secondary antibody (Jackson Immunoresearch) at a 1:2000 dilution, for 1 h at RT in blocking buffer. Finally, plates were washed four times in TBS Tween-20 0.05 % and after incubation with the substrate [0.3 % H₂O₂, 0.1 % 3,3′,5,5′-tetramethylbenzidine (TMB) in 0.1 M citric acid pH 5] for 15–20 min at RT, the reaction was stopped with 0.2 M H₂SO₄. The absorbance at 450 nm was measured with a FilterMax F5 Multi-Mode microplate reader (Molecular Devices).