Staphylococcal protein A binding to canine IgG and IgM

Abstract

Staphylococcal protein A (SpA) binds with high affinity to immunoglobulins from many species, making it a usefel reagent for immunoassays and immunoglobulin purification procedures. However, its use is limited by poor reactivity with some immunoglobulin subclasses including human IgG 3 and murine IgG 1. Reports of SpA's reactivity with canine immunoglobulins have been inconsistent. Because the most recent reports indicated a much greater reactivity of purified SpA with canine immunoglobulins than was suggested by our observations, we quantitatively reevaluated the binding of canine IgG and IgM to Cowan I strain SpA under the conditions of use in our laboratory. IgG and IgM were purified from pooled normal canine plasma by affinity chromatography with heavy chain specific polyclonal anti-IgG and anti-IgM antibodies. The purified IgG and IgM were assessed for SpA reactivity by affinity chromatography using a SpA-agarose column. The relative proportions of total chromatographed IgG or IgM in the flow-through (SpA-nonbindable) and eluate (SpA-bindable) fractions were determined by absorbance at 280 nm. The IgG and IgM in each immunoglobulin fraction were also nonspecifically adsorbed to microtitration plates and tested for reactivity with 125I-SpA using a solid phase immunoradiometric assay (IRMA). Approximately 18% of the affinity purified canine IgG and 33% of the affinity purified canine IgM did not bind to the SpA affinity column and were also unreactive with 125I-SpA using the IRMA. A second approach using a different polyclonal antibody to canine IgG yielded similar results: about 21% of the purified IgG was unreactive with SpA. Incomplete reactivity of SpA with canine IgG and IgM limits the usefulness of SpA in canine immunologic procedures.