Aug 12, 2020
Chicken immunization with KLH-gp120 fragment (254-274) conjugate.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Chicken immunization with KLH-gp120 fragment (254-274) conjugate.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjnjkmcn
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: 40363
Abstract
Chicken immunization with peptides is inefective if only just the peptides are being inoculated. However, to make the immune response effective the fragment 254-274 of HIV-1 was conjugated with a carrier protein (KLH) that produced a critical immune response, assessed by ELISA, Immunoblot analysis and dot blot [1-4]. The Polson method (1990) can be used effectively to separate the IgY antibody from the egg yolk of immunized chickens [5].
References
1. Schade R, Pfister C, Italatsch R, Henklien P (1991) Polyclonal antibodies from chicken egg yolk-an alternative to the production of mammalian IgG type antibodies in rabbits. ATLA 19: 403-419.
2. Justiz Vaillant AA, Akpaka PE, Smikle M, McFarlane-Anderson N (2012) In vitro Inhibition of Staphylococcus aureus Isolates by Anti-Anti-Idiotypic Antibodies to Staphylococcal Protein (SpA). J Vaccines Vaccin 3: 127-131.
3. Collins AM, Roberton DM, Hosking CS, Flannery GR (1991) Oral immunization with xenogeneic antibodies stimulates the production of systemic and mucosal anti-idiotypic antibodies. Immunology 73: 388-393.
4. Ho DD, Kaplan JC, Rackauskas IE, Gurney ME (1988) Second conserved domain of gp120 is important for HIV infectivity and antibody neutralization. Science 239: 1021-1023.
5. Polson A (1990) Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure. Immunol Invest 19: 253-258.
Materials
MATERIALS
5 x 2ml No-Waste™ Freund's Incomplete Adjuvant (FIA)G-BiosciencesCatalog #786-099
2ml No-Waste™ Freund's Complete Adjuvant (FCA)G-BiosciencesCatalog #786-709
Fragment 254-274 of Gp120 of HIV (peptide)
Two healthy layer chickens (brown Leghorn), aged approximately 6 months, are injected intramuscularly at multiple sites on the breast with the peptide-keyhole limpet hemocyanin (KLH) conjugate.
The chickens are immunized on day 0, with 0.2 µmol/ml of the fragment 254-274 of HIV gp120-conjugated to KLH (immunogen) in 0.5 ml complete Freund’s adjuvant (Sigma-Aldrich Co, St. Louis Missouri).
On days 15, 60, and 90 chickens are immunized with 0.2 µmol/ml of the immunogen in 0.5 ml incomplete Freund’s adjuvant (Sigma-Aldrich Co, St. Louis Missouri).
The eggs are collected post-immunization.The immunoglobulin Y is separated using the Polson method (1990).