Elution of snapper, Pagrus auratus (Bloch and Schneider) Ig from a protein A affinity chromatography column yields contamination from the binding ligand

Staphylococcal protein A (SpA) affinity chromatography was used to single step purify immunoglobulins (Ig) from snapper (Pagrus auratus) serum. Elution of Ig was successful, with analysis of the eluent using SDS-PAGE under fully reducing conditions and PAGE under native conditions revealing that the product was of high purity. Polyclonal antisera to SpA purified snapper Ig was produced in rabbits. Probing of reduced purified Ig with the rabbit-anti-snapper Ig antisera in a Western blot demonstrated that the antisera were most reactive with the heavy (H) chains but also with an unknown protein of approximately 65 kDa in molecular weight. Non-immune rabbit serum (pre-bleed) was not reactive with the H chains of the reduced Ig however, reaction with the unknown protein (65 kDa) occurred. Further, probing of purified Ig in a Western blot with a suite of control sera and antisera demonstrated that the unknown protein was reactive with all mammalian sera and antisera tested. SpA affinity chromatography without prior application of snapper serum was performed. Eluent was probed in a Western blot by rabbit-anti-snapper Ig antisera and non-immune rabbit sera (pre-bleed) once more and were also reactive. In addition, elution buffer pH (3-5) had no effect on the elution of the unknown protein. In contrast, only H and light (L) chains of reduced snapper serum were positive when probed with the antisera. It was concluded that due to the non-specific binding in the Western blot the unknown protein was SpA contamination.