Purification of Immunoglobulins and their Binding to a Bacterial Protein LAG-HRP ConjugateAngel Justiz-Vaillant1*, Wayne Mohammed1, Sehlule Vuma1, Arvind Kurhade1 and Geeta Kurhade2
- *Corresponding Author:
- Angel Justiz-Vaillant
Department of Para-Clinical Sciences
Faculty of Medical Sciences, The University of the West Indies
St Augustine Campus, Trinidad and Tobago, West Indies
E-mail: [email protected]
Received date: December 14, 2015; Accepted date: January 18, 2016; Published date: January 20, 2016
Citation: Vaillant AJ, Mohammed W, Vuma S, Kurhade A, Kurhade G (2016) Purification of Immunoglobulins and their Binding to a Bacterial Protein LAG-HRP Conjugate. J Veterinar Sci Technol 7:288. doi:10.4172/2157-7579.1000288
Copyright: © 2016 Justiz-Vaillant A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.