Deviren Gokhan, Gupta Kapil, Paulaitis Michael E, Schneck Jonathan P
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, USA.
J Mol Recognit. 2007 Jan-Feb;20(1):32-8. doi: 10.1002/jmr.805.
The development of high-throughput protein microarrays for rapidly determining antigen-specific T-cell receptor repertoires of diverse T-cell populations can enable comprehensive, broad-based analyses of T-cell responses. Promising applications include medical diagnostics, vaccine development, treatment of autoimmune diseases and detection of potential agents of bioterrorism. In this study, we examined the feasibility of using peptide/major histocompatibility complex (p/MHC) microarrays to selectively capture and enumerate antigen-specific T cells. Results are presented for p/MHC microarrays consisting of a dimeric MHC-immunoglobulin complex, K(b)-Ig, loaded with either a cognate or non-cognate peptide for binding CD8(+) T cells. We quantified the sensitivity of these K(b)-Ig microarrays by measuring a lower detection limit of 0.05% antigen-specific CD8(+) T cells mixed with splenocytes from C57BL/6J mouse. A fivefold increase in this lower detection limit (0.01%) was achieved using a secondary capture anti-Ig antibody to coat the microarray surface. This higher sensitivity is comparable to that obtained using standard state-of-the-art fluorescence activated cell sorting (FACS) instruments. We also found that contacting the T-cell suspension with the K(b)-Ig microarrays under mild shear flow conditions produced more uniform distributions of captured T cells on the individual spots and better spot-to-spot reproducibility across the entire microarray.
用于快速确定不同T细胞群体抗原特异性T细胞受体库的高通量蛋白质微阵列的开发，能够实现对T细胞反应的全面、广泛分析。其有前景的应用包括医学诊断、疫苗开发、自身免疫性疾病治疗以及生物恐怖主义潜在制剂的检测。在本研究中，我们检验了使用肽/主要组织相容性复合体（p/MHC）微阵列选择性捕获和计数抗原特异性T细胞的可行性。展示了由二聚体MHC-免疫球蛋白复合物K(b)-Ig组成的p/MHC微阵列的结果，该复合物负载有用于结合CD8(+) T细胞的同源或非同源肽。我们通过测量与C57BL/6J小鼠脾细胞混合的0.05%抗原特异性CD8(+) T细胞的下限来量化这些K(b)-Ig微阵列的灵敏度。使用二级捕获抗Ig抗体包被微阵列表面，使该下限提高了五倍（达到0.01%）。这种更高的灵敏度与使用标准的先进荧光激活细胞分选（FACS）仪器所获得的灵敏度相当。我们还发现，在温和剪切流条件下使T细胞悬液与K(b)-Ig微阵列接触，能在各个斑点上产生更均匀的捕获T细胞分布，并在整个微阵列上实现更好的斑点间重现性。
