Comparison Of Four Anti-Avian IgY Secondary Antibodies Used In Western Blot And Dot-Blot ELISA To Detect Avian Bornavirus Antibodies In Four Different Bird Species

Escandon, Paulina; Heatley, J Jill; Berghman, Luc R; Tizard, Ian; Musser, Jeffrey MB

Recombinant nucleoprotein (N-protein), purified from E. coli, was produced following the method of Hameed et al (2018) . 26 Briefly, the procedure was as follows. Total RNA was extracted from frozen brain tissue of a Yellow-collared macaw (Primolius auricollis) infected with PaBV-4 using Qiagen RNeasy Mini kit (Qiagen, Hilden, Germany). The RNA was used to generate cDNA using reverse transcription kits (High Capacity Reverse Transcription Kit, Applied Biosystems, Foster City, CA, USA) and random hexamers. Subsequent PCR was performed to amplify the N-protein gene with primers Forward 5ʹ-CATG CAT ATG CCA CCC AAG AGA CAA AGA AGC-3ʹ and Reverse 5ʹ-GTAC CTC GAG GTT TGC GAA TCC GGT TAC ACC-3ʹ. The resulting PCR products were cloned, sequenced, and inserted into pET21a vector to generate a His-tagged fusion protein for expression in Escherichia coli (Rosetta, Sigma-Aldrich, St. Louis, MO, USA). Recombinant E. coli was incubated for 12 hrs in Luria broth fortified with ampicillin; the culture was continuously mixed at 150 rpm at room temperature. Recombinant E. coli was transferred to fresh media of Luria broth, ampicillin, and Isopropyl β-D-1-thiogalactopyranoside to induce protein expression and incubated for 6 hrs, while being continuously stirred at 200 rpm at room temperature. The solution was centrifuged at 3500 x g for 30 mins and the supernatant was removed. The bacterial pellet was resuspended in 40 mL of phosphate-buffered saline (PBS) and sonicated for 3 sets of 8 mins to lyse the bacteria. The sonicated solution was then centrifuged at 12,000 x g for 20 mins at 4°C. The supernatant was loaded on a Qiagen Ni-NTA Agarose column, which had been preconditioned with 10 mL of binding buffer (20mM sodium phosphate, 300mM NaCl, pH 7.4, 10mM imidazole); the Qiagen Ni-NTA Agarose column has a high affinity for His-tagged proteins. Ten mL of wash buffer (20mM sodium phosphate, 300mM NaCl, pH 7.4, 20mM imidazole) was loaded on the column and allowed gravity flow. The column was then eluted by gravity flow with 10 mL of elution buffer (20mM sodium phosphate, 300mM NaCl, pH 7.4, 200 mM imidazole) and the elutant was collected in 1mL fractions. The purity of each protein fraction was determined by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis. Fractions containing the N-protein were combined and an Amico Ultra 15 mL centrifugal filter was used to concentrate the N-protein in 1 mL PBS. Finally, the protein concentration was measured using BCA™ Protein Assay Kit (Thermo Scientific™ PIERCE™, Waltham, MA. USA).