Separation of equine IgG subclasses (IgGa, IgGb and IgG(T)) using their differential binding characteristics for...

Equine IgG possesses four well-defined subisotypes, designated IgGa, IgGb, IgGc and IgG(T) on the basis of their increasing anodal mobility in electrophoresis. However, the preparation of IgGa and IgGb reference proteins has not previously been reported. Certain bacterial cell wall proteins, termed protein A and protein G, have been used for purification of IgG subisotypes from the serum of domestic animals which, combined with other techniques utilising differences in the physico-chemical properties of the proteins, has allowed the purification of Ig isotypes. This paper describes purification of the subisotypes of equine IgG. Purification of IgGa and IgGb was achieved by the separation of a 'fall-through' peak from ion-exchange chromatography consisting of IgGa and IgGb into two fractions (peaks C and D) by FPLC protein A and protein G affinity chromatography. Peak C consisted of IgGb and peak D consisted of IgGa exhibiting slightly faster cathodal migration than peak C in IEP analysis. Affinity chromatography using protein A and G columns also indicated that there may be two different components of IgG(T); one with a low affinity for protein G and the other having a greater affinity for protein G.