Hrp conjugated rabbit anti human igg antibody

The culture supernatant sample or purified S protein was coated onto each well of the microtiter plate and left overnight at 4°C. After the wells were blocked with 1% BSA for 1 h, dilutions of human anti-S1 antibody (Abcam) were added to the wells and incubated for 3 h at room temperature. After being washed, HRP-conjugated rabbit anti-human IgG antibody (Abcam) was added and incubated for 1 h at room temperature. The bound antibodies were detected using TMB substrate (Sigma-Aldrich). Absorbance was read at 450 nm using a Multimode Plate Reader (Promega, Madison, WI, USA).

In vitro prepared IgG-ddIgA1 complexes (0.2μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking, the membrane was incubated with HRP-conjugated mouse anti-human IgA antibody (AbD Serotec, Kidlington, UK) and HRP-conjugated rabbit anti-human IgG antibody (Abcam, Cambridge, UK) at 4 °C overnight. Binding was detected by western lightning plus ECL reagent (PerkinElmer, Waltham, MA, USA).

WCL containing 10 μg of total protein was separated by SDS-PAGE (10% acrylamide gels) and then transferred onto a PVDF membrane (Cat# IPVH00010, Millipore, USA) or stained with Coomassie brilliant blue. After blocking with 5% skim milk for 2 h, the membrane was incubated with a P144 specific monoclonal antibody or a control mouse IgG at a concentration of 1 μg/ml. After washing, the membrane was incubated with HRP conjugated rabbit anti-human IgG antibody (Cat# ab6759, Abcam, UK) or HRP conjugated goat anti-mouse IgG antibody (Cat # 115–035-003, Jackson Immuno Research, USA) diluted 1:5000 in TBST (Tris-buffered saline, pH 8.0, 0.05% Tween 20) containing 5% skim milk. After wash, the bands were developed with an ultra-sensitive ECL substrate (Cat# K-12045-D10, Advansta, USA). The area corresponding to the specific WB bands were excised from the gel stained with Coomassie blue and analyzed using the mass spectrometry.