Protein g peroxidase conjugate

Cf2Th cells (canine fetal thymus cells, Cat. No. 90110521, European Collection of Authenticated Cell Cultures (ECACC), UK) were co-cultured with BFV100-infected Cf2Th cells in a proportion of 10 to 1 and grown in DMEM, supplemented with 10% fetal bovine serum in the 5% CO₂ atmosphere. Three days after infection, when the cytopathic effect appeared, cells were lysed using a CHAPS buffer (0.5 M EDTA, 1 M Tris HCL pH 8.8, 100 mM NaCl, 0.5 M CHAPS, 0.5 M sodium deoxycholate; Sigma, Poznan, Poland). Uninfected Cf2Th cells were grown under the same condition. Of the total cell lysates, 10 µg of infected and uninfected control cells were separated by SDS-PAGE and served as the antigen for western blotting analyses (WB) [4 (link)]. Wild ruminant sera were used at 1:100 dilutions (v/v in 3% bovine albumin, 0.01% Tween 20, PBS) and Protein G–peroxidase conjugate (Sigma, Poznan, Poland) at 1:10,000 dilution. As positive and negative controls, the pools of serum samples from five BFV naturally infected cows and five uninfected animals, diagnosed by GST-ELISA and PCR tests [32 ], were used at 1:100 dilutions. ECL Plus reagents (GE Healthcare, Warsaw, Poland) were used for the detection of specifically bound antibodies.

20 µg of purified recombinant EFVeca Gag protein was separated by SDS-PAGE and served as antigen for western blotting analyses (WB) [32 (link)]. Horse serum samples and controls were used at 1:100 dilutions (v/v in 2% bovine albumin, 0.01% Tween 20, PBS) and Protein G-peroxidase conjugate (Sigma-Aldrich, St. Louis, MO, USA) at 1:10,000 dilution. 4-chloro-naphtol (Sigma-Aldrich, St. Louis, MO, USA) was used as the substrate for peroxidase. Immunoblotting was established and optimized using 29 horse sera from semi-nested PCR-positive horses and 40 serum samples from PCR-negative individuals.

A GST-capture ELISA was used to examine the antibody response to the BFVbta antigen in the sera of the experimentally infected calves, as described in [6 (link)]. In brief, all of the serum samples were tested in duplicate for GST and GST-Gag fusion proteins as antigens. Prior to their application to the plates, all the serum samples were additionally incubated with the lysate of a GST-expressing E. coli culture (2 μg/μL) so that they would pre-absorb GST-binding antibodies. Protein G peroxidase conjugate (Sigma-Aldrich, St. Louis, MO, USA, 1:5000) was used as a secondary antibody, and tetramethylbenzidine (TMB, Sigma-Aldrich, St. Louis, MO, USA) (0.1 mg per 1 mL of acetate buffer) was added as a substrate. The specific reactivity against the BFVbta antigen (net absorbance value) was calculated for each serum sample by subtracting the absorbance measured for the GST from the absorbance noted for the GST-Gag protein (this is presented as an average value from two replicates).