Bacterial Proteins and Their Proposed Interactions with Fc or Fab Fragments of Immunoglobulins Angel Alberto Justiz Vaillant * , Sehlule Vuma and Wayne Mohammed Pathology and Microbiology Unit, Department of Para-Clinical Sciences, The University of the West Indies, St. Augustine, Trinidad and Tobago, WI * Corresponding author: Angel Justiz Vaillant MD, PhD, Dept. of Para-Clinical Sciences, The University of the West Indies, St. Augustine, Trinidad & Tobago, Tel: +868-773-5914; E-mail: avail4883@gmail.com; angel.vaillant@sta.uwi.edu Rec date: Oct 22, 2014, Acc date: Nov 05, 2014, Pub date: Nov 11, 2014 Copyright: © 2014 Vaillant AAJ, 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. Abstract The reactivity of Immunoglobulin Binding Proteins (IBP) to Fc and/or Fab fragments of immunoglobulins was summarized in this review. Staphylococcal protein A (SpA), Streptococcal protein G and Peptostreptococcal protein L (SpL) were the IBP reported. SpA reacted with IgG from skunk, coyote, raccoon, mule and donkey. SpG reacted almost with the entire panel of immunoglobulins and SpL binding was restricted to some immunoglobulins including raccoon, ostrich and duck. The various immunological techniques that have been used to test the binding capacity of IBP to Igs were double immunodiffusion, Enzyme-Linked Immunosorbent Assay (ELISA), SpA-affinity chromatography and immunoblot analysis. These protein-protein interactions are important because they can be used in the immunodiagnosis and in the purification of intact Igs or their fragments. Keywords: Immunoglobulin; Streptococcal protein; Bacterial proteins; ELISA; Immunoblot analysis Introduction The binding of Immunoglobulin-Binding Protein (IBP) such as Staphylococcal protein A (SpA) [1], Streptococcal protein G [2] and Peptostreptococcal protein L (SpL) [3,4] to immunoglobulins (Igs) from distinct animal species is known [1-9]. However, the information about their binding capacity to Fc or Fab fragments is scanty. The aim of this review is to report on the reactivity of IBP to immunoglobulin regions from a number of mammalian and avian Ig molecules. The following is what is well-known about IBP: SpA has a molecular weight of 42 KDa. It binds to the Fc fragment of IgG produced by several animal species. The native protein consists of five domains. Of these, four show high structural homology of about 58 aminoacids and they have binding capacity to immunoglobulins [1]. Streptococcal protein G, Type III bacterial Fc receptor, is a small globular protein produced by several streptococcal species and is composed of two or three nearly identical domains, each of 55 amminoacids. SpG is well-known for binding to many species including human, mouse, rat and hamster [2]. SpL comprised of an alpha-helix packed against a 4 stranded beta-sheet. Utilizing immunoblot assays showed that the isolated protein binds to immunoglobulins through L chain interaction [3-5]. Materials and Methods All materials used in the following experiments were obtained from Sigma-Aldrich Co, St. Louis, Missouri, USA. The methods used to study the reactivity or binding capacity of IBP to Igs: Fc, Fab or both regions were double immunodiffusion [6,10], ELISA [6,8,9,11], immunoblot analysis [2,3,6,12] and SpA-affinity chromatography [5-7]. In addition the IgY purification from avian egg yolks was carried out by the method of Polson [5-8,12,13]. Immunoglobulin Y isolation The IgY fraction was isolated from the egg yolks of a variety of birds including chicken, bantam hen, duck, and ostrich. The IgY fraction was isolated by the chloroform-polyethylene glycol (PEG) method [12]. The eggs were washed with warm water and the egg yolk was separated from the egg white. The membrane was broken and the egg yolk collected and diluted 1:3 in Phosphate Buffered Saline (PBS), pH 7.4. To one third (1/3) of the egg yolk mixture an equal volume of chloroform was added, the mixture was then shaken and centrifuged for 30 min, 1000 x g, Room Temperature (RT). The supernatant was decanted and mixed with PEG 6000 (12%, w/v), stirred and incubated for 30 min at RT. The mixture was then centrifuged as described above. The precipitate containing IgY was dissolved in PBS (pH 7.4) at a volume equivalent to one sixth (1/6) of the original volume of the egg yolk and dialyzed against 1L of PBS (pH: 7.4 for 24 h at 4°C). The IgY was removed from the dialysis tubing. IgY concentration was determined by the Bradford method [13]. IgY samples were stored at – 20°C. Purification of immunoglobulins from animal sera and avian eggs A commercially prepared protein-A antibody purification kit, PURE-1A (Sigma-Aldrich Co, St. Louis Missouri) based on SpA- affinity chromatography was used to purify IgG from the sera of skunk, coyote, raccoon, mule, horse, donkey; IgY from ostrich, bantam hen and duck egg yolks and ostrich IgM from the ostrich egg white. The procedure was performed according to the manufacturer’s instructions [7,14]. Binding properties of bacterial immunoglobulin receptors by double immunodiffusion (Ouchterlony) technique The binding of SpA, SpL, and SpG with animal sera, avian IgY, avian egg whites and purified IgG were investigated by double immunodiffusion as previously described [8]. Vaillant et al., Biochem Physiol 2014, 3:4 DOI: 10.4172/2168-9652.1000143 Research Article Open Access Biochem Physiol ISSN:2168-9652 BCP, an open access journal Volume 3 • Issue 4 • 1000143 Biochemistry & Physiology: Open Access B i o c h e m i s t r y & P h y s i o l o g y : O p e n A c c e s s ISSN: 2168-9652