Comparison of cytokine measurements using ELISA, ELISPOT and semi-quantitative RT-PCR
Comparison of cytokine measurements using ELISA, ELISPOT and semi-quantitative RT-PCR
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DOI：
10.1016/s0022-1759(97)00033-1
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发表时间：
1997-05-12
影响因子：
2.2
通讯作者：
Rudin, W
中科院分区：
文献类型：
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作者：
Favre, N;Bordmann, G;Rudin, W
我们研究了三种不同方法(半定量RT-PCR、ELISAT和ELISPOT)用于测量小鼠白细胞细胞因子表达的结果之间的相关性。用三种方法分析细胞因子肿瘤坏死因子-α(TNF-α)、干扰素-γ(干扰素-γ)和白介素4(IL-4)的产生。在一个简单的实验性小鼠体内白细胞刺激模型中，包括一次静脉注射抗CD3抗体，然后在体外短期孵育，用脾细胞对三种细胞因子中的每一种的结果都有很大的不同，这取决于使用的方法。对于肿瘤坏死因子α，在刺激后RNA显著增加，而斑点形成细胞的数量没有增加，并且在血清或细胞培养上清液中未检测到该蛋白。体外培养的脾细胞显示出这三种方法检测干扰素α的相关性很强。在刺激下，IL-4的RNA数量与细胞因子的分泌和斑点形成细胞的数量平行增加。然而，在对照组中观察到大量的斑点形成细胞。我们的结论是，根据研究的特定目的，必须仔细选择不同方法的组合，以检测白细胞对细胞因子表达的激活。
We have investigated the correlation between results obtained by three different methods (semi-quantitative RT-PCR, ELISA and ELISPOT) used to measure cytokine expression by mouse leukocytes. The production of the cytokines tumour necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) and interleukin-4 (IL-4), was analysed with all three methods. In a simple experimental murine in vivo model of leukocyte stimulation, consisting of a single intravenous injection of anti-CD3 antibodies followed by a short incubation in vitro, the results obtained with spleen cells for each of the three cytokines differed greatly, depending on the method used. For TNF alpha, a significant increase in RNA was observed upon stimulation, whereas the number of spot-forming cells did not increase and the protein was not detectable in serum or in cell culture supernatants by ELISA. In vitro cultured splenocytes showed a strong correlation between all three methods for IFN alpha. Upon stimulation, the amount of RNA for IL-4 increased in parallel with the secretion of the cytokine and the number of spot-forming cells. However, high numbers of spot forming cells were observed in controls. We conclude that, depending on the specific aim of an investigation, combinations of different methods have to be chosen carefully in order to detect activation of leukocytes for cytokine expression.