Characterization of an in situ IFN-γ ELISA assay which is able to detect specific peptide responses from freshly isolated splenocytes induced by DNA minigene immunization
Characterization of an in situ IFN-γ ELISA assay which is able to detect specific peptide responses from freshly isolated splenocytes induced by DNA minigene immunization
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DOI：
10.1016/s0022-1759(00)00138-1
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发表时间：
2000-04-03
影响因子：
2.2
通讯作者：
Sette, A
中科院分区：
文献类型：
--
作者：
McKinney, DM;Skvoretz, R;Sette, A
已开发并优化了用于新鲜分离和肽再刺激的脾细胞的模拟IFN-γ ELISA测定。该测定基于ELISPOT测定，但利用可溶性发色团，使其易于适应高通量分析。我们表明，在这两个主要的和再刺激试验，这种技术是更敏感的比传统的上清液ELISA或Cr-51释放试验，在原位ELISA中观察到的反应是无法检测到在这些其他的测定。在每个细胞的基础上，原位ELISA的灵敏度约为1个IFN-γ分泌细胞/10（4）个平板细胞。使用原位IFN-γ ELISA来描述IFN-γ I对pMin.1的DNA疫苗接种应答的动力学。对于新鲜分离的脾细胞，在免疫后10至12天观察到所有测试肽的峰值响应，其中早在7天就观察到对某些肽的响应。当添加6天体外肽再刺激步骤时，在7天体内免疫后观察到所有测试的肽的应答。该数据表明，DNA疫苗的单次肌内施用可以诱导T细胞应答，其可以在新鲜分离的脾细胞中检测到。(C)2000 Elsevier Science B. V.保留所有权利。
An in sim IFN-gamma ELISA assay has been developed and optimized for both freshly isolated and peptide-restimulated splenocytes. This assay is based on the ELISPOT assay, but utilizes a soluble chromagen, making it readily adaptable to high-throughput analysis. We show that in both the primary and restimulation assays this technique is more sensitive than either a traditional supenatant ELISA or the Cr-51-release assay, in that responses are observed in the in situ ELISA that are not detectable in these other assays. On a per-cell basis, the sensitivity of the in situ ELISA is approximately one IFN-gamma secreting cell/10(4) plated cells. The in situ IFN-gamma ELISA was utilized to describe the kinetics of the IFN-gamma I response to DNA vaccination with pMin.1. For freshly isolated splenocytes, the peak response for all the peptides tested was observed from 10 to 12 days after immunization, with responses seen to some peptides as early as 7 days. When a 6-day in vitro peptide restimulation step was added, responses were seen for all the peptides tested after 7 days of in vivo immunization. This data demonstrates that a single intramuscular administration of a DNA vaccine can induce T-cell responses that can be detected in freshly isolated splenocytes. (C) 2000 Elsevier Science B.V. All rights reserved.