Screening of high-affinity scFvs from a ribosome displayed library using BIAcore biosensor.
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 x 10(-11), 1.13 x 10(-10), 5.06 x 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.
Yuan
Qing
Q
Genetic Engineering Research Centre, College of Bioengineering, Chongqing University, People's Republic of China.
Wang
Zhongkang
Z
Nian
Siji
S
Yin
Youping
Y
Chen
Gang
G
Xia
Yuxian
Y
D000327:Adsorption; D000473:Alkanes / Q000737:chemistry; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D015374:Biosensing Techniques / Q000379:methods*; D002417:Cattle; D003911:Dextrans / Q000378:metabolism; D005786:Gene Expression Regulation; D006046:Gold / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007135:Immunoglobulin Variable Region / Q000032:analysis* / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism; D051379:Mice; D012270:Ribosomes / Q000378:metabolism*; D013499:Surface Properties
D000473:Alkanes; D003911:Dextrans; D007135:Immunoglobulin Variable Region; D008070:Lipopolysaccharides; D006046:Gold