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Screening of High-Affinity scFvs From a Ribosome Displayed Library Using BIAcore Biosensor

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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

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Acknowledgments

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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Correspondence to Yuxian Xia.

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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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