Aug 12, 2020
Preparation of horseradish peroxidase (HRP) conjugated to chicken anti-IgY
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Preparation of horseradish peroxidase (HRP) conjugated to chicken anti-IgY . protocols.io https://dx.doi.org/10.17504/protocols.io.bjmvkk66
License: This is an open access protocol 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
Protocol status: Working
We use this protocol and it's working
Created: August 12, 2020
Last Modified: August 12, 2020
Protocol Integer ID: 40341
Abstract
Avian IgY is structurally and genetically different from its mammalian counterpart IgG. This reagent can be used in ELISA, Western blotting and Dot blot to detect specific chicken IgY to infectious agents or immune system proteins [1]. IgY can be isolated by the Polson Method [2] or the water dilution method [3]. Chicken IgY can crossed-react with IgY from diverse bird species [4]. Similarly avian IgY can be isolated from their egg yolk using the same methodology with modifications that was developed for the chicken IgY isolation [5].
Reference
1. Larsson A, Bålöw RM, Lindahl TL, Forsberg PO. Chicken antibodies: taking advantage of evolution--a review. Poult Sci. 1993;72(10):1807-1812. doi:10.3382/ps.0721807
2.Polson A (1990) Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure. Immunol Invest 19: 253-258.
3. Nilsson E, Hanrieder J, Bergquist J, Larsson A. Proteomic characterization of IgY preparations purified with a water dilution method.J Agric Food Chem. 2008;56(24):11638-11642. doi:10.1021/jf802626t
4. Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175
5. : 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
Guidelines
All reagents but specially the enzyme and sodium periodate solution has to be prepared freshly before mixing it with the enzyme. Chicken anti-IgY can be prepared by immunizing an animal species with purified chicken IgY; which can be isolated by a protein purification method and refined by affinity chromatography. The high lipid content of the egg yolk can be separated from the water soluble fraction where is the IgY by the use of chloroform.
Materials
MATERIALS
Ammonium SulfateP212121
Sodium periodateBio Basic Inc.Catalog #SB0875.SIZE.100g
sodium borohydrideSigma AldrichCatalog #452882
AF647 Donkey anti-chicken IgY (IgG)Jackson ImmunoresearchCatalog #703-605-155
Horseradish Peroxidase (HRP) type IVSigma AldrichCatalog #P8375-25KU
Pierce™ Chicken IgY Purification KitThermo FisherCatalog #44922
Horseradish peroxidase (500 µg in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml) followed by incubation in the dark for 2 h.
Mix 500µg of chicken anti-immunoglobulin Y (anti-IgY) developed in a laboratory animal or commercially available with 500 µg of the mix of horseradish peroxidase-sodium periodate that was prepared above.
The mixture is incubated for 3 hours at 4°C with gentle agitation.
Forty µl of freshly prepared NaBH4 solution (5 mg NaBH4 /ml 0.1 mM NaOH) is then added to the preparation.
The preparation is incubated for 90 min at 4°C in the dark with gentle agitation.
Cold 50% saturated ammonium sulphate solution (pH 7.4) is added drop by drop in the ratio 1:1 (v/v).
The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
The pellets is re-suspended in 200 µl of PBS pH=7.4 and dialysed against 1L of PBS for 24 h with 3 buffer changes.
An equal volume of glycerol is added to the dialysate followed by 100 µl of bovine serum albumin, BSA (20 mg/ ml).
The peroxidase conjugated to anti-chicken IgY is then stored at -20°C until further used.