Abstract
An experimental protocol was developed to screen high-affinity single-chain Fv antibody fragments (scFvs) from a Xanthomonas axonopodis pv. citri (Xac) immunized ribosome display library using BIAcore biosensor. The screening methods involved immobilizing antigen [lipopolysaccharides (LPS) of Xac] on sensor chip HPA and then unpurified expression products of scFvs flowing over the immobilized sensor chip. The affinity-improved scFvs were selected based on dissociation rate constants (k d). Thirty-five enzyme-linked immunosorbent assay-positive scFvs were analyzed by BIAcore, and three of those (scFv A1, B2, and C5) with lower k d were screened. To demonstrate the accuracy of the screening method, the three scFvs were expressed in Escherichia coli HB2151 and purified. The purified scFvs were subsequently further identified according to association rate and affinity constants. The results showed that the three scFvs (A1, B2, and C5) had high affinity for LPS of Xac (3.51 × 10−11, 1.13 × 10−10, 5.06 × 10−10 M, respectively). Furthermore, the scFv B2 was highly specific for LPS of Xac and had no any cross-reactions with bovine serum albumin and LPS from Xac-related bacteria. This provided evidence that the information from the BIAcore screening assay could be accurate.
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Abbreviations
- scFv:
-
single chain antibody Fv fragment
- ELISA:
-
enzyme-linked immunosorbent assay
- BIAcore:
-
biomolecular interaction analysis
- SPR:
-
surface plasmon resonance
- k d :
-
dissociation rate constants
- k a :
-
association rate constants
- K D :
-
equilibrium dissociation constant
- RU:
-
response units
References
Hanes, J., Jermutus, L., Weber-Bornhauser, S., Bosshard, H. R., & Plückthun, A. (1998). Proceedings of the National Academy of Sciences, 95, 14130–14135.
Yau, K. Y. F., Groves, M. A. T., Li, S., Sheedy, C., Lee, H., Tanha, J., et al. (2003). Journal of Immunological Methods, 281, 161–175.
Lee, M. S., Kwon, M. H., Kim, K. H., Shin, H. J., Park, S., & Kim, H. I. (2004). Journal of Immunological Methods, 284, 147–157.
He, M., & Taussig, M. J. (2005). Journal of Immunological Methods, 297, 73–82.
Horn, I. R., Wittinghofer, A., de Bruïne, A. P., & Hoogenboom, H. R. (1999). FEBS Letters, 463, 115–120.
Röthlisberger, D., Pos, K. M., & Plückthun, A. (2004). FEBS Letters, 564, 340–348.
Park, S. G., Jeong, Y. J., Lee, Y. Y., Kim, I. J., Seo, S. K., Kim, E. J., et al. (2005). Antiviral Research, 68, 109–115.
Steukers, M., Schaus, J. M., van Gool, R., Hoyoux, A., Richalet, P., Sexton, D. J., et al. (2006). Journal of Immunological Methods, 310, 126–135.
Katsamba, P. S., Navratilova, I., Calderon-Cacia, M., Fan, L., Thornton, K., Zhu, M., et al. (2006). Analytical Biochemistry, 352, 208–221.
Besenicar, M., Macek, P., Lakey, J. H., & Anderluh, G. (2006). Chemistry and Physics of Lipids, 141, 169–178.
Hawkins, R. E., Russell, S. J., & Winter, G. (1992). Journal of Molecular Biology, 226, 889–896.
Schier, R., Bye, J., Apell, G., McCall, A., Adams, G. P., Malmqvist, M., et al. (1994). Journal of Molecular Biology, 255, 28–43.
Duenas, M., Malmborg, A. C., Casalvilla, R., Ohlin, M., & Borrebaeck, C. A. K. (1996). Molecular Immunology, 33, 279–285.
Pei, R., Cui, X., Yang, X., & Wang, E. (2000). Talanta, 53, 481–488.
Cui, X., Yang, F., Sha, Y., & Yang, X. (2003). Talanta, 60, 53–61.
Leonard, P., Säfsten, P., Hearty, S., McDonnell, B., Finlay, W., & O’Kennedy, R. (2007). Journal of Immunological Methods, 323, 172–179.
Nanduri, V., Bhunia, A. K., Tu, S. I., Paoli, G. C., & Brewster, J. D. (2007). Biosensors & Bioelectronics, 23, 248–252.
Albrecht, M. T., Wang, W., Shamova, O., Lehrer, R. I., & Schiller, N. L. (2002). Respiratory Research, 3, 18–28.
Yuan, Q., Xia, Y., Nian, S., Yin, Y., Cao, Y., & Wang, Z. (2007). Enzyme and Microbial Technology, 41, 383–389.
Malmborg, A. C., Duenas, M., Ohlin, M., Soderlind, E., & Borrebaeck, C. A. K. (1996). Journal of Immunological Methods, 198, 51–57.
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This work was supported by grant for ‘Program for new century excellent Talents in University’, Ministry of Education, P. R China (No. NCET-04-851).
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Yuan, Q., Wang, Z., Nian, S. et al. Screening of High-Affinity scFvs From a Ribosome Displayed Library Using BIAcore Biosensor. Appl Biochem Biotechnol 152, 224–234 (2009). https://doi.org/10.1007/s12010-008-8251-y
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DOI: https://doi.org/10.1007/s12010-008-8251-y