Abstract
The aim of this study was to produce chimeric bacterial protein conjugates, to separate them by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and study their reactivities with human serum immunoglobulins by immunoblot analysis. Universal conjugates: protein LA-peroxidase (SpLA-HRP), protein LG-peroxidase (SpLG-HRP), protein AG-peroxidase (SpAG-HRP), protein LAG-peroxidase (SpLAG-HRP) and others were prepared by the periodate method. The conjugates were polymeric, with high molecular weights (MW) ranging from between 80-90 kDa to greater than 220 kDa. All conjugates have similar numbers of protein bands. However SpLALG-HRP and SpLAG anti-IgY-HRP were less polymeric. Immunoblot blot analysis showed that all conjugates reacted with human serum Igs. These conjugates have a potential use in epidemiological surveys of zoonotic infections affecting avian and mammalian species.The periodate method was effective to producing chimeric conjugates of peroxidase-labelled immunoglobulin-binding proteins and the immunoblot analysis showed that they were funtional and effective in reacting with human serum immunoglobulins.
Highlights
The reactivity of immunoglobulin-binding proteins for immunoglobulins (Igs) of mammalian species is well-known
Yolken and Leister synthesised and used a Staphylococcal protein-A (SpA)-peroxidase conjugate in an ELISA [6] and Nygren and Hansson had reported that the modified periodate method allowed the preparation of conjugated protein A, which when analysed by SDS-polyacrylamide gel electrophoresis showed the presence of polymeric conjugates of large molecular size [7]
Protein LA-peroxidase (SpLA-HRP), protein LG-peroxidase (SpLG-HRP), protein AG-peroxidase (SpAG-HRP) and protein LAGperoxidase (SpLAG-HRP) by the periodate method.Horseradish peroxidase (HRP) labelled SpA, Streptococcal protein-G (SpG) and/or SpL conjugates were prepared using the periodate method described by Nakane and Kawoi [8]
Summary
The reactivity of immunoglobulin-binding proteins for immunoglobulins (Igs) of mammalian species is well-known. These proteins are Staphylococcal protein-A (SpA), Streptococcal protein-G (SpG), Peptostreptococcal protein L (SpL) and a commerciallyprepared recombinant protein LA (SpLA) [1,2,3,4]. Perel'man et al reported that the periodate method was efficient in the preparation of SpA-peroxidase conjugates, while the preparation of bacterial protein conjugates failed when glutaraldehyde was used as a cross-linker [5]. Yolken and Leister synthesised and used a SpA-peroxidase conjugate in an ELISA [6] and Nygren and Hansson had reported that the modified periodate method allowed the preparation of conjugated protein A, which when analysed by SDS-polyacrylamide gel electrophoresis showed the presence of polymeric conjugates of large molecular size [7]. This study aimed to produce chimeric bacterial protein conjugates, to separate them by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and record their reactivities with human serum immunoglobulins by immunoblot analysis
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