Isolation, kinetic analysis, and structural characterization of an antibody targeting the Bacillus anthracis major spore surface protein BclA
Isolation, kinetic analysis, and structural characterization of an antibody targeting the Bacillus anthracis major spore surface protein BclA
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DOI：
10.1002/prot.22971
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发表时间：
2011-04-01
影响因子：
2.9
通讯作者：
Liu, Chun-Qiang
中科院分区：
文献类型：
--
作者：
Nuttall, Stewart D.;Wilkins, Michelle L.;Liu, Chun-Qiang
实验室或现场炭疽检测的一种方法是用高亲和力、高特异性结合试剂检测炭疽芽胞。从一组单抗中，我们选择了一个与炭疽杆菌外孢杆菌蛋白BCLA的截短版本tBclA具有高亲和力的候选株(A4D11)。在Biacore生物传感器上使用标准滴定和动力学滴定进行的动力学分析表明，重组tBclA的抗体亲和力在300 pm范围内，A4D11抗体也被重排为scFv构型，而亲和力没有损失。然而，对炭疽杆菌和相关芽孢杆菌的检测显示，完整孢子的结合有限，物种之间存在显著的交叉反应。通过对scFv-tBclA络合物的三维晶体结构的测定，这些结果得到了合理的解释。A4D11结合tBclA三聚体的侧面，接触通常包装的抗原的一个表面，以对抗外孢子体结构中的相邻三聚体；这种孢子间界面在芽孢杆菌物种之间高度保守。我们的结果表明，产生高亲和力抗体来区分高度保守的近缘物种的孢子结构是困难的，但也暗示了未来针对这一困难目标进行基于结构的抗体设计的可能性。
One method of laboratory- or field-based testing for anthrax is detection of Bacillus anthracis spores by high-affinity, high specificity binding reagents. From a pool of monoclonal antibodies, we selected one such candidate (A4D11) with high affinity for tBclA, a truncated version of the B. anthracis exosporium protein BclA. Kinetic analysis utilising both standard and kinetic titration on a Biacore biosensor indicated antibody affinities in the 300 pM range for recombinant tBclA, and the A4D11 antibody was also re-formatted into scFv configuration with no loss of affinity. However, assays against B. anthracis and related Bacilli species showed limited binding of intact spores as well as significant cross-reactivity between species. These results were rationalized by determination of the three-dimensional crystallographic structure of the scFv-tBclA complex. A4D11 binds the side of the tBclA trimer, contacting a face of the antigen normally packed against adjacent trimers within the exosporium structure; this inter-spore interface is highly conserved between Bacilli species. Our results indicate the difficulty of generating a high-affinity antibody to differentiate between the highly conserved spore structures of closely related species, but suggest the possibility of future structure-based antibody design for this difficult target.