Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy
Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy
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DOI：
10.1002/jmr.682
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发表时间：
2004-11-01
影响因子：
2.7
通讯作者：
Hodges, RS
中科院分区：
文献类型：
--
作者：
Cachia, PJ;Kao, DJ;Hodges, RS
建立了一种利用EK-Coli异二聚体-Coli多肽捕获系统的表面等离子体共振谱测定抗体亲和力和交叉反应性的方法。该系统允许在生物传感器芯片表面可逆捕获合成肽配体，其优点是可以使用单个生物传感器芯片分析多个抗体-抗原相互作用。该方法已在合成肽抗铜绿假单胞菌(PA)疫苗的研制中得到应用。将4株PA的菌毛蛋白受体结合区对应的人工合成的多肽配体连接到异源二聚体结合区的大肠杆菌链上，通过与固定化K-Coli链的二聚反应分别捕获在生物传感器芯片上。将针对菌毛蛋白表位的兔多克隆免疫球蛋白注射到传感器芯片表面，用于抗原-抗体相互作用的动力学分析。计算了动力学速率常数k(ON)和k(OFF)，以及平衡缔合和解离常数Ka和Kd。抗体亲和力在1.14×10~(-9)~1.60×10~(-5)M之间。结果表明，免疫过程中使用的载体蛋白和佐剂对抗体亲和力和交叉反应有很大影响。使用弗氏佐剂系统与锁孔帽状血蓝蛋白偶联的PA株K菌毛蛋白表位的抗体比使用佐剂与破伤风类毒素偶联的同一表位的抗体具有更广泛的交叉反应。本文描述的方法有助于详细描述多克隆抗体与一组合成肽配体的相互作用，目的是在疫苗开发中获得高亲和力和交叉反应的抗体。版权所有(C)2004 John Wiley Sons，Ltd.
A method has been developed for measurement of antibody affinity and cross-reactivity by surface plasmon resonance spectroscopy using the EK-coli heterodimeric colied-coli peptide capture system. This system allows for reversible capture of synthetic peptide ligands on a biosensor chip surface, with the advantage that multiple antibody-antigen interactions can be analyzed using a single biosensor chip. This method has proven useful in the development of a synthetic peptide anti-Pseudomonas aeruginosa (PA) vaccine. Synthetic peptide ligands corresponding to the receptor binding domains of pilin from four strains of PA were conjugated to the E-coli strand of the heterodimeric colied-coli domain and individually captured on the biosensor chip through dimerization with the immobilized K-coli strand. Polyclonal rabbit IgG raised against pilin epitopes was injected over the sensor chip surface for kinetic analysis of the antigen-anti body interaction. The kinetic rate constants, k(on) and k(off), and equilibrium association and dissociation constants, KA and KD, were calculated. Antibody affinities ranged from 1.14 x 10(-9) to 1.60 x 10(-5) M. The results suggest that the carrier protein and adjuvant used during immunization make a dramatic difference in antibody affinity and cross-reactivity. Antibodies raised against the PA strain K pilin epitope conjugated to keyhole limpet haemocyanin using Freund's adjuvant system were more broadly cross-reactive than antibodies raised against the same epitope conjugated to tetanus toxoid using Adjuvax adjuvant. The method described here is useful for detailed characterization of the interaction of polyclonal antibodies with a panel of synthetic peptide ligands with the objective of obtaining high affinity and cross-reactive antibodies in vaccine development. Copyright (C) 2004 John Wiley Sons, Ltd.