Volume 4 • Issue 3 • 1000173 J Chromat Separation Techniq ISSN:2157-7064 JCGST, an open access journal Open Access Justiz Vaillant et al., J Chromat Separation Techniq 2013, 4:3 DOI: 10.4172/2157-7064.1000173 Open Access Separation and Reactivity of Avian Immunoglobulin Y Angel Alberto Justiz Vaillant 1 *, Niurka Ramirez 2 , Armando Cadiz 3 , Belquis Ferrer 4 , Patrick Akpaka 1 and Monica Smikle 5 1 Department of Para-Clinical Sciences, The University of the West Indies, St. Augustine, Trinidad & Tobago 2 Department of Internal Medicine, Freyre de Andrade Hospital, Havana city, Cuba 3 Carlos J, Finlay Vaccine and Serum Institute, Havana, Cuba 4 Department of Immunology, Saturnino Lora Teaching Hospital, Santiago de, Cuba 5 Department of Microbiology, The University of the West Indies, Mona Campus, Jamaica *Corresponding author: Angel Alberto Justiz Vaillant, Department of Para- Clinical Sciences, The University of the West Indies, St. Augustine, Trinidad & Tobago, E-mail: avail4883@gmail.com Received February 22, 2013; Accepted March 23, 2013; Published March 27, 2013 Citation: Justiz Vaillant AA, Ramirez N, Cadiz A, Ferrer B, Akpaka P, et al. (2013) Separation and Reactivity of Avian Immunoglobulin Y. J Chromat Separation Techniq 4: 173. doi:10.4172/2157-7064.1000173 Copyright: © 2013 Justiz Vaillant AA, 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 Immunoglobulin Y (IgY) is the mayor protein present in the avian egg yolk. This antibody fulfls important functions in the protection of the embryo against several challenging stimuli. Separation of IgY from the egg yolk of several birds was carried out by the Polson method. Their capacity to react with immunoglobulin-binding bacterial protein: protein A, L or LA was investigated. The cross-reactivity of an anti-chicken-IgY-HRP conjugate with different avian IgY was tested by ELISA. The results showed that protein L reacts with bantam hem IgY; and proteins A and LA react with ostrich, bantam hen or duck IgYs. These fndings are important for the development of methods of detection and purifcation of avian IgY proteins. Keywords: Protein A; Protein L; Protein LA; Immunoglobulin Y; Avian egg yolk antibodies Introduction Immunoglobulin Y (IgY) is the mayor protein present in the avian egg yolk. Tis antibody fulfls important functions in the protection of the embryo against several challenging stimuli [1]. Egg yolk antibodies are important therapeutically [2,3], but also they have been used as immunological tool in many immunodiagnostic assays [4-7]. Staphylococcal protein A, SpA [8], Peptostreptococcal protein L, SpL [9] and chimeric protein LA, pLA [10] are immunoglobulin- binding bacterial proteins (IBBPs). Tey have the ability of binding to the Fc-fragment of several immunoglobulins (Igs) in a non-specifc manner [11]. Tis interaction is useful in serological diagnosis of infectious diseases, where enzyme-labelled-IBBPs are used as a marker in ELISAs and other procedures for the determination of antibodies, and in the Ig-purifcation. Te binding of IBBPs to Igs of mammalian and avian species because is so important has been extensively documented, specially the interactions between SpA to mammalian Igs [8]. Te binding of pLA to animal Igs has not been extensively documented but it is predicted positive if SpA, which is an integral component of pLA, binds to Igs. However the interaction of SpL to avian immunoglobulins is not well-known and deserves further studies and documentation since this IBBP could also be successfully used for IgY isolation [12], and serological studies. Tis study reports on the separation of IgY from the egg yolk of several avian species using the Polson Method [13] and the IgY interaction with IBBPs, aims to establish binding afnities between them that are unknown or not documented yet, this is important for using IBBP in the purifcation of avian immunoglobulins. Materials and Methods Materials Peroxidase labelled protein A, L or LA, rabbit anti-IgG, protein A antibody purifcation kit, ELISA microtitre plates were obtained from Sigma-Aldrich Co, St. Louis Missouri, USA. All other chemical were of reagent grade, obtained from the same company. Eggs from diferent avian species were collected in Westmorland, Jamaica. Immunoglobulin Y Isolation and Determination by Direct ELISA Te IgY fraction was isolated from the egg yolks of a variety of birds including chicken, bantam hen, guinea hen, quail, goose, duck, pigeon, parakeet, cattle egret, pheasant, and ostrich. Te IgY fraction was isolated by the chloroform-polyethylene glycol (PEG) method [13]. Te eggs were washed with warm water and the egg yolk was separated from the egg white. Te membrane was broken and the egg yolk collected and diluted 1:3 in phosphate bufered saline (PBS), pH 7.4. To 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×g, RT). Te supernatant was decanted and mixed with PEG 6000 (12%, w/v), stirred and incubated for 30 min (RT). Te mixture was then centrifuged as previously described. Te precipitate containing IgY was dissolved in PBS (pH 7.4) at a volume equivalent to 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). Te IgY was removed from the dialysis tubing. IgY concentration was determined by the Bradford method. IgY samples were stored at –20°C. A direct ELISA was used to determine the presence of avian egg yolk IgYs as follows: 96 well microtitre plates were coated overnight at 4°C with 100 µg of duplicates of each IgY sample in carbonate-bicarbonate bufer pH 9.6. Plates were washed 4X with 150 µl PBS-Teen 20 bufer. Ten 50µl of a commercial anti-chicken IgG-HRP diluted 1:30,000 (according to manufacturer’s instructions) in PBS-non-fat milk, was added to each well and incubated for 1h at RT. Te plates were washed 4X with PBS-Tween. 50 µl of 4 mg/ml o-phenylenediamine solution (OPD) was added and the plates were incubated 15 minutes at RT. Te Research Article Journal of Chromatography Separation Techniques J o u r n a l o f C h r o m a t o g r a p h y & S e p a r a t i o n T e c h n i q u es ISSN: 2157-7064