Purification of Immunoglobulins and their Binding to a Bacterial Protein LAG-HRP Conjugate

Abstract

Objective: To purify IgG molecules from several species by SpA-affinity chromatography and to study the interactions of mammalian IgGs with a peroxidase-labelled SpL, SpA and SpG conjugate (SPLAG-HRP) in an enzyme-linked immunosorbent assay (ELISA). Materials and methods: The periodate method described by Nakane and Kawoi was used to prepare the SPLAG-HRP conjugate. The 10% non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of sera and purified immunoglobulins was carried out to characterize molecularly the purified IgGs. The chicken IgY fraction was isolated by the chloroform-polyethylene glycol (PEG) method for its use as a negative control in the ELISA that was used to determine the affinity of different immunoglobulins to a SPLAG-HRP conjugate. Results: The SpA-affinity chromatography and the 10% non-denaturing SDS-PAGE of sera and purified immunoglobulins (IgGs) were useful separation techniques. Most purified IgGs interacted moderately with the SPLAG-HRP including IgGs from horse, dog, skunk, coyote and raccoon. The purified mammalian IgG had a molecular weight (MW) of approximately 150 kDa. Conclusion: The SPLAG-HRP was a versatile heterofunctional reagent useful for the detection of purified immunoglobulins from diverse mammalian species.