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.