PRODUCTION OF ANTIBODY IgY ANTI-c-Myc IN CHICKEN EGGS
Abstract
Bioinformatics analysis has been performed on c-Myc peptide sequences to predict its epitopes in order to design antigen for IgY anti-c-Myc production in chickens. Peptide sequences for human c-Myc protein obtained from the NCBI database were analyzed with ProPred-1 softwares. Manual screening has been conducted on the prediction results.One candidate of c-Myc epitope, EQKLISEEDL, have been selected and prepared as an antigen, by adding one molecule of Cystein to the peptide, and conjugated it with Keyhole limpet hemocyanin (KLH). Twelve hens, aged 22-24 weeks, were immunized a week after their first eggs were produced. Antigen diluted in aquabidest and emulsified with Freund’s Complete Adjuvant. The suspension was injected into the area of the chicken breast, subcutaneously or intramuscularly, with a dose of 0.1 mg for the first immunization. Booster injection performed on days 10, 20, and 30 with the same volume of emulsion, but using Incomplete Freund’s Adjuvant and the amount of antigen half of the first immunization. Extraction of IgY from egg has been carried out using kits from Gallus Immunotech. IgY levels quantified using a spectrophotometer, absorbance measured at 280 nm. The results showed that chicken antibody production gave rise to a sigmoid curve pattern, just like in mammals. The IgY content of the egg was stable at high levels (> 5 mg / ml yolk) after the third immunization. In the intramuscular group, the immune response seems to work earlier than in the subcutaneous group, but eventually the subcutaneous group reaches the same level, and even higher. In general, from an egg produced after the second booster it can be extracted at least 50 mg of IgY. Higher yields could be obtained when IgY was extracted from egg 2 to 3 days after the third booster.
Keywords: epitope prediction, c-Myc, IgY antibody, ProPred-1
References
Andersen, H.P., M. Nielsen, and O. Lund. 2006. Prediction of residues in discontinuous B-cell epitopes using protein 3D structures. Protein Sci 15 (11): 2558–2567.
Angeletti, R.H. 1999. Design of Useful Peptide Antigens. J Biomol Tech. No.10: 2-10.
Anonymous. 2011. Pharmaceuticals & Biotech Industry Global Report - 2011. IMAPS.
Gassmann, M., P. Thommes, T. Weiser, and U. Hubscher. 1990. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. The FASEB Journal Vol. 4: 2528-2532.
Hanly, W.C., J.E. Artwohl, and B.T. Bennett. 1995. Review of Polyclonal Antibody Production Procedures in Mammals and Poultry. ILAR Journal V37(3) 1995.
Harkins, K.R. 2001. Antibody Purification Methods. In: Howard, G.C., and D.R. Bethell (editors). Basic Methods In Antibody Production And Characterization. CRC Press LLC, USA. 141-168.
He, C., H. Hu, R. Braren, S-Y. Fong, A. Trumpp, T.R. Carlson, and R. A. Wang. 2008. c-myc in the hematopoietic lineage is crucial for its angiogenic function in the mouse embryo. Development 135: 2467 - 2477.
Kulkarni-Kale, U., S. Bhosle, A.S. Kolaskar. 2005. CEP: a conformational epitope prediction server. Nucleic Acids Res (33 Web Server):W168-171.
Polson, A. 1990. Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure. Immunol Invest 19: 253–258.
Ponomarenko, J.V., and M.H.V. van Regenmortel. 2009. B-Cell Epitope Prediction. In: Gu, J., and P.E. Bourne. Structural Bioinformatics Second Edition. John Wiley & Sons, Inc. 849-79.
Rubinstein, N.D., I. Mayrose, E. Martz, and T. Pupko. 2009. Epitopia: a web-server for predicting B-cell epitopes. BMC Bioinformatics, 10:287.
Saha, S., and G.P. Raghava. 2006. Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins 65(1): 40-48.
Sweredoski, M.J., and P. Baldi. 2009. COBEpro: a novel system for predicting continuous B-cell epitopes. Protein Engineering Design & Selection, 22(3):113-120.
Singh, H., and G.P.S. Raghava. 2001. ProPred: Prediction of HLA-DR binding site. Bioinformatics Application Notes. Vol. 17 No. 12: 1236-1237.