Yuan Qing, Wang Zhongkang, Nian Siji, Yin Youping, Chen Gang, Xia Yuxian
Genetic Engineering Research Centre, College of Bioengineering, Chongqing University, People's Republic of China.
Appl Biochem Biotechnol. 2009 Feb;152(2):224-34. doi: 10.1007/s12010-008-8251-y. Epub 2008 Jun 24.
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.
已开发了一种实验方案，用于利用BIAcore生物传感器从经柑橘溃疡病菌免疫的核糖体展示文库中筛选高亲和力单链Fv抗体片段（scFvs）。筛选方法包括将抗原[柑橘溃疡病菌的脂多糖（LPS）]固定在传感器芯片HPA上，然后使scFvs的未纯化表达产物流过固定化的传感器芯片。基于解离速率常数（k(d)）选择亲和力提高的scFvs。通过BIAcore分析了35个酶联免疫吸附测定呈阳性的scFvs，并筛选出其中k(d)较低的3个（scFv A1、B2和C5）。为了证明筛选方法的准确性，将这3个scFvs在大肠杆菌HB2151中表达并纯化。随后根据结合常数和亲和力常数对纯化的scFvs进行进一步鉴定。结果表明，这3个scFvs（A1、B2和C5）对柑橘溃疡病菌的LPS具有高亲和力（分别为3.51×10(-11)、1.13×10(-10)、5.06×10(-10) M）。此外，scFv B2对柑橘溃疡病菌的LPS具有高度特异性，与牛血清白蛋白和柑橘溃疡病菌相关细菌的LPS无任何交叉反应。这证明了来自BIAcore筛选测定的信息可能是准确的。
