Biopolym. Cell. 2010; 26(6):492-498.
http://dx.doi.org/10.7124/bc.000179
Molecular and Cell Biotechnologies
The development of strategy for obtaining single-chain recombinant antibodies against cell-surface biomarkers on the example of human CD34
1Nikolaiev Iu. S., 2Gorbatiuk O. B., 1Tsapenko M. V.
  1. State Organization of Genetic and Regenerative Medicine NAMS Ukraine
    67, Vyshhorodska Str., Kyiv, Ukraine, 04114
  2. Taras Shevchenko National University of Kyiv
    64, Volodymyrska Str., Kyiv, Ukraine, 01601

Abstract

Antibodies against cell-surface proteins play an important role in cell detection, separation and determination of differentiation stage. The globular structure of extracellular region of cell-surface antigens is frequently characterized by heavily glycosilation and/or is stabilized by disulphide bonds. In this case the obtaining of antibodies against such proteins is a substantive problem. Aim. The development of strategy for obtaining recombinant antibodies against cell-surface biomarkers. Methods. The research strategy is based on the construction of cDNA library of VH and VL genes of animals, immunized with recombinant antigen, and subsequent cell-based bio-panning for the library enrichment with desired phage clones. High-throughput automated systems were used for the detection and isolation of antigen-specific clones. Results. We have obtained a panel of five antibodies that recognize an antigen on CD34+ cell surface using the methods of immunocytochemistry and flow cytometry. Conclusions. The proposed strategy may be used for obtaining antibodies against cell-surface antigens.
Keywords: CD34, phage display, single-chain recombinant antibodies (ScFv), biopanning
Full text: (PDF, in Russian)

References

[1] Civin C. I., Strauss L. C., Brovall C., Fackler M. J., Schwartz J. F., Shaper J. H. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J. Immunol. 1984; 133(1):156–165.
[2] Berenson R. J., Andrews R. G., Bensinger W. I., Kalamasz D., Knitter G., Buckner C. D., Bernstein I. D. Antigen CD34+ marrow cells engraft lethally irradiated baboons J. Clin. Invest 1988 81, N 3:951–955.
[3] Azzazy H. M., Highsmith W. E., Jr. Phage display technology: clinical applications and recent innovations Clin. Biochem 2002 35, N 6:425–445.
[4] Maniatis T., Fritsch E. F., Sambrook J. Molecular cloning: a laboratory manual New York: Cold Spring Harbor Lab. publ., 1982 545 p.
[5] Pavlova MV, Hil'chuk PV, Pokholenko IaO, Nikolaiev IuS, Kordium VA. Construction and characterization of immune combinatorial cDNA library of mouse variable immunoglobuline genes. Tsitol Genet. 2008;42(2):10-5.
[6] Studier F. W. Protein production by auto-induction in high density shaking cultures Protein Expr. Purif 2005 41, N 1:207–234.
[7] Gilchuk P. V., Okunev O. V., Irodov D. M., Pavlova M. V., Yakovenko O. Ya. Immobilized single chain antibodies for affinity purification of recombinant human IFN-2b Biopolym. Cell 2006 22, N 2:157–161.
[8] Giordano R. J., Cardo-Vila M., Lahdenranta J., Pasqualini R., Arap W. Biopanning and rapid analysis of selective interactive ligands Nat. Med 2001 7, N 11:1249–1253.
[9] Gilchuk P. V., Okunev O. V., Irodov D. M., Pavlova M. V. Overproduction of single-chain antibodies against human IFN-2b in Escherichia coli and its obtaining in biologically active form. Ukr Biokhim Zh. 2006;78(1):163–171.
[10] Lipes B. D., Chen Y. H., Ma H., Staats H. F., Kenan D. J., Gunn M. D. An entirely cell-based system to generate singlechain antibodies against cell surface receptors J. Mol. Biol 2008 79, N 2:261–272.
[11] Huie M. A., Cheung M. C., Muench M. O., Becerril B., Kan Y. W., Marks J. D. Antibodies to human fetal erythroid cells from a nonimmune phage antibody library Proc. Natl Acad. Sci. USA 2001 98, N 5:2682–2687.
[12] de Bruin R., Spelt K., Mol J., Koes R., Quattrocchio F. Selection of high-affinity phage antibodies from phage display libraries Nat. Biotechnol 1999 17, N 4:397–399.