组成图示
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传感器类型
表面等离子共振(SPR)生物传感器
检测对象
单链Fv抗体片段（scFv，anti-Xac LPS scFv）；样品基质：大肠杆菌TG1/HB2151周质提取物、纯化scFv溶液
检测原理
将Xac脂多糖（LPS）固定于HPA疏水金膜芯片，形成抗原捕获层；未纯化scFv周质提取物经HEPES缓冲液稀释后流过芯片，scFv与LPS发生特异性抗原-抗体结合，使芯片表面质量增加，引起表面等离子共振（SPR）角度偏移，仪器以响应单位（RU）实时记录结合与解离曲线。由于解离速率常数kd不依赖scFv浓度，可据此从ELISA阳性克隆中初筛低kd、高亲和力候选。随后用2–16 nM纯化scFv进行浓度依赖结合实验，通过全局拟合和Scatchard分析计算结合速率ka、解离速率kd和平衡解离常数KD。特异性分析中，scFv胺偶联固定于CM5芯片，不同细菌LPS和BSA流过，仅Xac LPS产生结合信号，从而判定特异性。该过程无标记、无酶催化放大。
检测灵敏度
原文未报告LOD、线性范围、灵敏度斜率或相关系数。
效应效果
该方法对35个ELISA阳性scFv进行BIAcore筛选，31个显示结合（RU约100–300），4个无结合（<20 RU），提示可排除ELISA假阳性。BSA在LPS覆盖芯片上非特异结合增加<50 RU，而在未覆盖表面约1000 RU，说明表面覆盖良好。三个候选scFv的KD为3.51×10^-11、1.13×10^-10、5.06×10^-10 M，竞争ELISA所得3.41×10^-11、1.08×10^-10、5.02×10^-10 M与之相符。scFv B2对Xac LPS高度特异，不与BSA及Xcc、Xoc、Xoo的LPS交叉反应。作者认为BIAcore可实时、无标记、低样品量地评估动力学和亲和力，比ELISA更适合从核糖体展示库筛选高亲和力scFv。
传感器的构成
- 基底/换能器：BIAcore X金膜SPR芯片（HPA/CM5），通过表面等离子共振监测结合质量变化
- 疏水修饰层：HPA长链烷硫醇（long-chain alkanethiol）表面，促进LPS脂质单层吸附
- 捕获/识别层：Xac脂多糖（LPS）固定于HPA金膜表面，作为识别scFv的抗原捕获分子
- 封闭/非特异对照：牛血清白蛋白（BSA），检测非特异结合并封闭暴露疏水区域
- 特异性分析修饰层：CM5羧甲基葡聚糖（carboxymethylated dextran）金膜芯片，用于胺偶联固定scFv
- 固定识别层：scFv A1/B2/C5胺偶联固定于CM5表面，用于检测不同LPS/BSA结合
- 信号读出：SPR角度变化转换为响应单位（RU），计算ka、kd和KD
中文摘要
本文建立了一种利用BIAcore生物传感器从核糖体展示库中筛选高亲和力单链Fv抗体片段（scFv）的实验方案。以柑橘黄单胞杆菌柑橘致病变种（Xac）免疫小鼠脾脏mRNA构建的核糖体展示库为来源，将Xac脂多糖（LPS）固定于HPA疏水传感器芯片表面，使未纯化的scFv周质表达产物流过芯片，并依据解离速率常数（kd）选择亲和力改善的scFv。对35个ELISA阳性scFv进行BIAcore分析后，筛选出kd较低的scFv A1、B2和C5。为验证筛选准确性，将三个scFv在大肠杆菌HB2151中表达并纯化，进一步根据结合速率常数和亲和力常数进行鉴定。结果显示，三者对Xac LPS均具有较高亲和力，平衡解离常数KD分别为3.51×10^-11、1.13×10^-10和5.06×10^-10 M。此外，scFv B2对Xac LPS高度特异，不与牛血清白蛋白及Xac相关细菌的LPS发生交叉反应，表明BIAcore筛选结果可靠。
英文摘要
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.