Introduction Determination of Chickens as a Useful Model for Antibody Production to Green...
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Transcript of Introduction Determination of Chickens as a Useful Model for Antibody Production to Green...
Introduction
Determination of Chickens as a Useful Model for Antibody Production to Green Fluorescent Protein and a Tomato Proteinase Inhibitor II Peptide
Tyler Sechrist and Jeffrey P. Thompson, Ph.D.Department of Biological Sciences, York College of Pennsylvania
•In recent years, the use of antibodies as a molecular tool in the lab has exploded. A variety of animals, mostly mammals, are commonly used to produce antibodies specific for a desired target.
•Chickens have been shown to be one of the most useful models for antibody production due to several factors. – they pass their antibodies (IgY) on in the yolk of their egg, eliminating the need for bleeding –IgY in the yolk are very plentiful, typically 2-10 mg/ml of yolk (Bizhanov and Vyshniauskis 2004)
•IgY obtained from chickens are polyclonal, meaning they include all the various antibodies produced against different regions, or epitopes, of the same antigen.
•On the other hand, antibodies produced against one specific eptiope are referred to as monoclonal antibodies, and they cut down on potential cross reactivity with proteins similar to the antigen against which they are produced against.
•Monoclonal antibodies are much more expensive to both produce and purchase, and polyclonal antibodies are sufficient for the majority of biochemical techniques commonly used.
Objectives
Experimental Procedures
GFP produced in and isolated
from E. coli
PIN2 peptide ordered (CEGESDPKRPNA) (Fig. 1)
Crosslinked by primary amine and sulfhydryl
group
Coupled molecules injected subcutaneously with Freund’s complete adjuvant; boosters given at 3 and 6 weeks (Freund’s Incomplete)
Total IgY extracted from eggs yolks 6 weeks post injection as well as pre-
injection eggs
Western Blots run using both pre and post injection IgY as the primary antibody and anti-IgY as secondary (Fig. 3a &b)
GFP linked to UltraLink® beaded
resin (primary amine)(Fig. 4)
Pin2 linked SulfoLink coupling resin
(sulfhydryl)
Antibody Purification
Results
Kal
eido
scop
e Std
Kal
eido
scop
e Std
GFP w
ith
PIN
2G
FP
BSA
with
PIN
2BSA
Tom
ato
Lea
f Ly
sate
Figure 2. Ponceau S analysis of membrane prior to western blot. Staining represents total protein content on membrane.
Kal
eido
scop
e Std
Kal
eido
scop
e Std
GFP w
ith
PIN
2G
FP
BSA w
ith
PIN
2B
SA
Tom
ato
Lea
f Lys
ate
IgY
Figure 3a. Western blot analysis performed using post- injection total IgY as primary antibody. Shaded banding represents IgY specific binding of proteins.
Kal
eido
scop
e Std
Kal
eido
scop
e Std
GFP w
ith
PIN
2G
FP
BSA
with
PIN
2BSA
Tom
ato
Lea
f Ly
sate
Figure 4. Western blot analysis performed using purified anti-GFP IgY as primary antibody.
Conclusions
•Induce production of anti-GFP and anti-PIN2 IgY
• Create IgY towards whole molecule PIN2 using only a surface exposed peptide
•Purify out antigen specific IgY from total IgY using Affinity Chromatography
•Establish chickens as a useful model for antibody production
•IgY specific for GFP and PIN2 was successfully created and extracted
•GFP specific IgY were successfully purified and isolated from the total IgY.
•Antibodies towards two separate antigens can be created simultaneously
•IgY collection was non-invasive, and IgY was plentiful in the yolk (1 yolk contained enough IgY for ≈30 western blots), and chickens average an egg a day!
•Chickens are a very useful model for creation of antibodies for the purposes of scientific research
Figure 3b. Overexposure of the western blot shown in figure 2. Upon overexposure a band is present in the lane containing tomato lysate where a multimer of PIN2 would run.
Literature CitedBizhanov, G. and Vyshniauskis, G. 2004. A Comparison of Three Methods for Extracting IgY from the Egg Yolk of Hens Immunized with Sendai Virus. Veterinary Research Communications 24: 103-113.Polyclonal Antibody image obtained from: www.rndsystems.com/dam_public/6295.gif Egg Yolk image obtained from: https://www.gallusimmunotech.com/Custom-IgY-Purification
Figure 1. 3-D image of PIN2 tetramer with highlighted region representing peptide used.