Affinity Separation and Characterization of IgG Subfragments by Fast Protein Liquid Chromatography with HiTrap_r Protein A Column

Abstract

It has been previously reported that the antibody molecules can be cleaved into fragments by the proteolytic enzymes and these fragments provide information to determine an antibody’s structure and function. Antibody fragments offer several advantages over intact antibodies for use in experimental applications and immunochemical techniques. These fragments have smaller sizes and higher chemical and physical resistances. In this study, papain was used for the digestion of human IgG and the resulting fragments were separated by Fast Protein Liquid Chromatography FPLC . For this purpose, GE Healthcare HiTrap_r Protein A FF column was used, the resolution Rs and theoretical plate numbers N of the column were calculated. The unbound and bound fragments were collected by the Frac 920 fraction unit of the FPLC system and the collected fragments were analysed by enzyme-linked immunosorbent assay ELISA and sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS-PAGE .

Keywords

• Antibody fragmentation,Fab fragment,Fc fragment,FPLC,Protein A column

HiTrap_r Protein A Kolon İçeren Hızlı Protein Sıvı Kromatografisi ile IgG Altbirimlerinin Afinite Ayrılması ve Karakterizasyonu

Abstract

Antibadi moleküllerinin proteolitik enzimlerle alt birimlerine parçalanabildiği bilinmektedir. Bu alt birimler, antibadilerin yapısı ve işlevlerinin belirlenmesi çalışmalarında önemli veriler sunmaktadır. Deneysel uygulamalarda ve immünokimyasal teknikler için antibadi alt birimleri, parçalanmamış bütün antibadi moleküllerine göre önemli avantajlar sunmaktadır. Alt birimlerin boyutları daha küçüktür, kimyasal ve fiziksel dirençleri daha yüksektir. Sunulan çalışmada, insan immunoglobulin G parçalanması için papain kullanılmıştır. Elde edilen alt birimler, hızlı protein sıvı kromatografisi FPLC sistemi ile ayrılmıştır. Bu amaç için GE Healthcare HiTrap_r Protein A FF kolon kullanılmış, ayırıcılık Rs ve teorik plaka sayısı N hesaplanmıştır. Kolona tutunan ve tutunmayan alt birimler, FPLC sisteminin Frac 920 fraksiyon toplama birimi ile toplanmış ve enzim-bağlı immünosorbent kit ELISA ve sodyum dodesil sülfat-poliakrilamid jel elektroforez SDS-PAGE yöntemleri ile karakterize edilmiştir

Keywords

• Antibadi parçalanması,Fab fragmanı,Fc fragmanı,FPLC,Protein A kolon

References

1. T. Vo-Dinh, B. Cullum, Biosensors and biochips: advances in biological and medical diagnostics, Fresenius J. Anal. Chem., 366 (2000) 540.
2. L. Uzun, R. Say, A. Denizli, Porous poly(hydroxyethyl methacrylate) based monolith as a new adsorbent for affinity chromatography, React. Functl. Polym., 64 (2005) 93.
3. K. Huse, H. Bohme, G.H. Scholz, Purification of antibodies by affinity chromatography, J. Biochem. Biophys. Methods, 51 (2002) 217.
4. C.L. Morgan, D.J. Newman, C.P. Price, Immunosensors: technology and opportunities in laboratory medicine, Clin. Chem., 42 (1996) 193.
5. D.S. Hage, Immunoassays, Anal. Chem., 67 (1995) 455R.
6. R.S Yalow, The Nobel lectures in immunology. The Nobel Prize for Physiology or Medicine, 1977, awarded to Rosalyn S. Yalow Scand., J. Immunol., 35 (1992) 4.
7. T. Porstmann, S.T. Kiessig, Enzyme immunoassay techniques: an overview., J. Immunol. Methods, 150 (1992) 5.
8. M.L. Yarmush, A.M. Weiss, K.P. Antonsen, D.J. Odde, D.M. Yarmush, Immunoaffinity purification basic principles and operational considerations, Biotechnol.

9. Adv., 10 (1992) 413.
10. S. Özkara, H. Yavuz, A. Denizli, Purification of Immunoglobulin G from human plasma by metal- chelate affinity chromatography, J. Appl. Polym. Sci., 89 (2003) 1567.
11. N. Bereli, S. Akgöl, H. Yavuz, A. Denizli, Antibody purification by Concanavalin A affinity chromatogra- phy, J. Appl. Polym. Sci., 97 (2005) 1202.
12. Q. Luo, X. Mao, L. Kong, X. Huang, H. Zou, High- performance affinity chromatography for characteri- zation of human immunoglobulin G digestion with papain, J. Chromatogr. B, 776 (2002) 139.
13. D. Beale, Molecular fragmentations. Some applica- tions in immunology., Dev Comp. Immunol., 11 (1987) 287.
14. S.J. Boguslawski, Improved procedure for preparation of F(ab’)2 fragments of mouse IgGs by papain digestion, J. Immunol. Meth., 120 (1989) 51.
15. K.M Wilson, A new enzymatic method to obtain high- yield F(ab’)2 suitable for clinical use from mouse IgG1., Mol. Immunol., 28 (1991) 69.
16. R.S. Kerbel, B.E. Elliot, Detection of Fc receptors., Method. Enzymol., 93 (1983) 113.
17. K. Haupt, K. Mosbach, Plastic antibodies: develop- ments and applications, Tibtech November, Vol 16, 1998.
18. R. Cortese, Identification of biologically active peptides using random libraries displayed on phage, Curr. Opin. Biotechnol., 6 (1995) 73.
19. L. Uzun, D. Türkmen, V. Karakoç, H. Yavuz, A. Denizli, Performance of protein A based affinity membranes for antibody purification, J. Biomat. Sci. Polym. Ed., 2011, In Press, doi: 10.1163/092050610X538731.