Production of an Anti-Glucocorticoid Receptor Antibody from Chicken IgY

Stephanie Luke Department of Biological Sciences, York College of Pennsylvania

INTRODUCTION

The glucocorticoid receptor (GR) is a steroid receptor that regulates genes responsible for maintaining cell homeostasis.

Once activated, the glucocorticoid receptor translocates from the cytoplasm into the nucleus where it can activate or inhibit gene transcription.

Although antibodies may be purchased through a variety of manufactures, production of antibodies at York College is a great educational tool for students and provides practical uses for research.

Chickens are an ideal host for antibody production. The phylogenetic distance between humans and chickens allows for an increased immune response to human proteins. Unlike mammalian hosts, chicken antibodies (IgY) are deposited into their eggs; eliminating the need to bleed the animal for antibody collection.

There are many kits commercially available for extracting total IgY from egg yolks; however, utilizing a protocol that does not require a kit is more practical when producing large quantities or a variety of antibodies.

OBJECTIVES

1) To create a chicken anti-glucocorticoid receptor antibody using a peptide sequence with a protein conjugate.

2) To assess the use of a polyethyleneglycol precipitation isolation technique for total IgY, compared to the Gallus Immunotech IgY EggsPress Purification Kit.

METHODS

Week 1: Primary250µl GR/KLH + 250µl Freund’s

Complete Adjuvant

Week 2: Booster250µl GR/KLH + 250µl Freund’s

Incomplete Adjuvant

Week 3: Booster 250µl GR/KLH + 250µl Freund’s

Incomplete Adjuvant

Week 4: Collect Eggs

Inoculations

Antigen Preparation1

2

3 Total IgY Extraction

EggsPress Kit

Separate egg yolk from white ( 1 egg)

Add 5X volume of Reagent A to yolk; incubate 2 hours at 4°C; centrifuge

Collect supernatant, add equal volume of Reagent B and incubate for 1 hour; centrifuge

Resuspend pellet in PBS; equal to original egg yolk volume Filter sterilize

Separate egg yolk from white (3 eggs)

Add 2X volume of PBS to yolk

Add 3.5 % PEG per new volume, precipitate for 10 min and centrifuge

Collect supernatant, add 8.5 % PEG per new volume, precipitate 10 min and centrifuge

Resuspend pellet in 10 mL PBS; add 12 % PEG per new volume, precipitate 10 min and centrifuge

Dissolve pellet in 2mL PBS, dialyze, and filter sterilize

GR peptide sequence: CEF EFP KTH SDV SSE Conjugate hapten peptide sequence to Keyhole Limpet

Hemocyanin (KLH) carrier protein

PEG Precipitation

RESULTS

CONCLUSIONS

1) Total IgY bound to proteins within the nuclear and cytoplasmic extracts of the H4IIE hepatoma cells. Banding patterns seen were not exactly as expected.

2) Polyethyleneglycol precipitation isolation for total IgY extraction can be used as an alternative to the Gallus Immunotech IgY EggsPress Purification Kit.

Figure 1. Absorbance spectra of total IgY from EggsPress extraction kit. Absorbance was taken at 280nm with an extension coefficient of 13.30. One egg yolk yielded a final total IgY concentration of 5.53 mg/ml.

Figure 3. Absorbance spectra of total IgY from PEG precipitation extraction. Absorbance was taken at 280nm with an extension coefficient of 13.30. Three egg yolks yielded a final total IgY concentration of 28.94 mg/ml.

FUTURE STUDIES

Affinity purify the total IgY to isolate and verify presence of a specific anti-glucocorticoid receptor antibody.

Use purified antibody and fluorescent tag to visualize translocation of GR from cytoplasm to nucleus, and to compare presence of GR in normal versus malignant cells.

ACKNOWLEDGEMENTS

I would like to thank Dr. Thompson for his guidance, Dr. Kaltreider for his help (and protein extracts), and Ebony for all her hard work and wonderful eggs (she’s a hen).

WORKS CITED

IgY EggsPress Purification Kit. Available from: http://www.gallusimmunotech.com/IgY-Purification-Kit. Accessed 2011 November 4.Necela, B.M. and Cidlowski, J.A. 2004. Mechanisms of glucocorticoid receptor action in noninflammatory and inflammatory cells. Proceedings of the American Thoracic Society 1: 239-246 Pauly, D., Chacana, P., Calzado, E., Brembs, B. and Schade, R. 2011. IgY technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. Journal of Visualized Experiments[serial online] 50: 1-5. Schade, R., Staak, C., Hendriksen, C., Erhard, M., Hugl, H., Koch, G., Larsson, A., Pollmann, W., Regenmortel, M., Rijke, E., Spielmann, H., Steinbusch, H. and Straughan, D. 1996. The production of avian (egg yolk) anitbodies: IgY ATLA 24: 925-934.

Figure 2. Western blot of H4IIE hepatoma extracts against total IgY. Expected size of the GR was 98 kD. Bands were present around the 100 kD and 50 kD markers.

Figure 3. Western blot of H4IIE hepatoma extracts against total IgY. Expected size of the GR was 98 kD. Bands were present around the 100 kD and 50 kD markers.

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