Hrp conjugated rabbit anti chicken igy

Protein concentration was determined using the QuickStart Bradford Assay (BioRad, UK), following the manufacturer's protocol. Recombinant proteins were transferred to polyvinylidene difluoride (PVDF) membrane using the TransBlot Turbo system (Biorad, UK) and analysed by Western blot with anti-GST (Santa Cruz Biotech, USA) or anti-MBP antibody (New England Biolabs, UK) at 1:10,000 dilutions. Bound antibodies were detected using appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies at 1:10,000 dilutions. To assess if the cloned antigens were immunogenic following natural Campylobacter infection, pooled serum from Campylobacter-infected non-vaccinated White Leghorn birds collected three weeks post-infection was used at a 1:100 dilution. Bound serum IgY was detected with an HRP-conjugated rabbit-anti chicken IgY (Sigma-Aldrich, UK) at a 1:3000 dilution. In order to analyse the subcellular localisation of SodB a Western blot of subcellular fractions (Section 2.9) was probed with sera from GST–SodB vaccinated birds as above. Blots were developed using Clarity ECL (BioRad, UK) and autoradiography (Amersham Hyperfilm ECL, GE Lifesciences, UK).

A dot-blot assay was performed to determine the specificity of the purified anti-S IgY antibodies. The PVDF membrane was activated by soaking in methanol for 15 s, then washed with distilled water. Three different concentrations (500, 100, and 50 ng) of each recombinant antigen (S, S1, nucleocapsid protein [NP], and receptor binding domain (RBD)) was separately dot-blotted onto PVDF membrane. The membrane was incubated in 20 mL of blocking buffer for 1 h at room temperature. After being washed three times with TBS-T, the PVDF membrane was immersed in primary antibody, anti-S IgY antibodies of the MERS-COV (at 1:200 dilution), in blocking buffer with gentle agitation for 1 h at room temperature. The membrane was next incubated with HRP-conjugated rabbit anti-chicken IgY (Sigma, USA) as a secondary antibody (at dilution 1:10,000) in blocking buffer with gentle agitation for 1 h at room temperature. After being washed, the membrane was incubated with HRP colorimetric substrate (Immun-Blot Opti-4CN colorimetric Kit, Cat. No. 1708235) to 30 min at room temperature. The reaction was stopped with distilled water, and the image was captured after dot color development.