Separation of equine IgG subclasses (IgGa, IgGb and IgG(T)) using their differential binding characteristics for staphylococcal protein A and streptococcal protein G.

The research explores a method for separating subclasses of equine IgG (a type of immunoglobulin) using bacterial proteins A and G. It reveals that these proteins can purify specific subclasses from the horse serum, giving separate peaks in chromatography for further analysis.

Background

• Immunoglobulins, also known as antibodies, are important proteins produced by the immune system to combat pathogens. IgG or Immunoglobulin G is a class of these antibodies.
• In horses, IgG has four subtypes: IgGa, IgGb, IgGc, and IgG(T), differentiated by their movement in electrophoresis, a technique used to separate molecules.
• The research focuses on IgGa and IgGb because previously there were no reported methods for their separate preparation.

Use of Bacterial Proteins A and G

• Proteins A and G, derived from bacterial cell walls, have known affinities for IgGs. They have been utilized in the past to purify IgG subtypes from animal serum.
• In this study, these proteins were used in combination with other techniques based on the physical and chemical properties of the proteins to purify equine IgG subtypes.

Findings

• IgGa and IgGb were purified by separating the ‘fall-through’ peak from ion-exchange chromatography using FPLC (Fast Protein Liquid Chromatography) protein A and protein G affinity chromatography. Ion-exchange chromatography is a process that separates ions and polar molecules based on their charge.
• This separation process resulted in two distinct peaks, C and D. Peak C consisted of IgGb and peak D consisted of IgGa, which showed slightly faster movement than peak C in IEP (Isoelectric Point) analysis. The isoelectric point is the pH value at which a particular molecule carries no net electrical charge.
• Additionally, the technique revealed potentially two different components within IgG(T). One had a low affinity for protein G whereas the other showed greater affinity.

Impact

• The findings build upon the understanding of equine IgG classes, specifically IgGa and IgGb, providing a clear method for their separation and preparation for further studies.
• The revelation about two distinct components within the IgG(T) subtype adds a new dimension to our understanding of equine immune response and these IgG subclasses.