Aug 12, 2020
Protocol of preparation of a Protein-LAG conjugated to horseradish peroxidase-labeled immunoglobulin Y (IgY-HRP).
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Protocol of preparation of a Protein-LAG conjugated to horseradish peroxidase-labeled immunoglobulin Y (IgY-HRP). . protocols.io https://dx.doi.org/10.17504/protocols.io.bjk8kkzw
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: 40320
Abstract
Peroxidase-labeled anti-IgY conjugated to proteins L, A and G (SpLAG-anti-IgY-HRP) is a new development in the field of biochemistry. This versatile protein binds to more 700 species of animal IgGs including birds, laboratory animals, farm animals, wild animals, pets, and many others. This conjugate can be used in the study of zoonosis and the assessment of the antibody production for the study of the animal immune system. This immunomarker is not available in a recombinant form as their counterpart SpLA-HRP, SpLG-HRP and SpAG-HRP. It can be used in ELISA, immunohistochemistry, Western blot analysis, Dot blot, and RIA [1-4].
References
1. Justiz-Vaillant AA, Akpaka PE, McFarlane-Anderson N, Smikle MF. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.West Indian Med J. 2013;62(1):12-20.
2. 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.
3.Justiz-Vaillant AA, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 2013, 4: 175.
4. Stöbel K, Schönberg A, Staak C. A new non-species dependent ELISA for detection of antibodies to Borrelia burgdorferi s. l. in zoo animals. Int J Med Microbiol. 2002, 291 Suppl 33: 88-99.
Guidelines
All reagents but specially the enzyme and the sodium periodate solution have to be prepared freshly before mixing it with the enzyme.
Materials
MATERIALS
Ammonium SulfateP212121
Anti-Chicken IgY, HRP Conjugate, 300ulPromegaCatalog #G1351
Sodium periodateBio Basic Inc.Catalog #SB0875.SIZE.100g
sodium borohydrideSigma AldrichCatalog #452882
Staphylococcal Protein-ASigma Aldrich
Protein-L from P. Magnus
Streptococcal protein G by Sigma Aldrich
Horseradish peroxidase-labeled IgY (Promega) (500 µl in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml). Then the mixture is incubated in the dark for 2 h.
Mix 500 µg of staphylococcal protein-A (SpA) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate.
Mix 500 µg of streptococcal protein-G (SpG) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate.
Mix 500 µg of Peptostreptococcal protein-L (SpL) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate. These mixtures are incubated for 2 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 each mixture, including to the anti-IgY-HRP.
Then, the preparations are centrifuged (13,000 rpm., 10 minutes at RT). Add to each preparation cold saturated amonium sulphate solution and centrifuge them again (10000rpm, 25 minutes at 4°C).
Mix the 3 bacterial antigen preparations with the anti-IgY-HRP and incubate it in the dark with gentle agitation.
The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
The pellet (SpLAG-anti-IgY-HRP) is re-suspended in 500 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 200 µl of bovine serum albumin, BSA (20 mg/ml).
The SpLAG-anti-IgY-HRP conjugate is then stored at -20°C until further used.