The Nuclear Factor Kappa B (NFκB) pathway is an important signalling pathway in the immune system. Single gene defects in the NFκB pathway are described in a number of immunodeficiency diseases. These conditions provide a unique opportunity to investigate the mechanisms of NFκB function and how genetic mutations that disrupt this function lead to human disease. Here we describe a robust method for quantifying small differences in the functional activity of the NFκB pathway.

Peripheral blood mononuclear cells from healthy donors were stimulated over several days, with a combination of anti-IgM antibody and multimeric CD40 ligand. Nuclear proteins were thereafter extracted and tested for the ability of activated transcription factors, to bind known NFκB DNA binding motifs.

Repeatability experiments showed that DNA binding Activity can be quantified with an average inter and intra assay coefficient of variation of less than 10% (RelB and p52) and less than 15% (p50 and RelA). In healthy individuals there is a significant increase in the DNA binding activity of NFκB transcription factors in response to stimulation, although the magnitude of this response varies across individuals. The kinetics of the DNA binding activity also differs between the canonical and non-canonical transcription factors. P50 and RelA DNA binding activity responds within hours of stimulation, whilst RelB and p52 response was delayed to more than a day after stimulation.

Activation of NFκB signalling in response to B cell specific stimulation, can be precisely measured to distinguish individuals with differences in the functional activity of this pathway. This test may prove to be an important biomarker for investigating the functional impact of genetic variants on NFκB signalling.

核因子 Kappa B (NFκB) 通路是免疫系统中的重要信号通路。许多免疫缺陷疾病都描述了 NFκB 通路中的单基因缺陷。这些条件为研究 NFκB 功能的机制以及破坏该功能的基因突变如何导致人类疾病提供了独特的机会。在这里，我们描述了一种强大的方法，用于量化 NFκB 通路功能活性的微小差异。

用抗 IgM 抗体和多聚体 CD40 配体的组合刺激来自健康供体的外周血单核细胞数天。此后提取核蛋白并测试激活的转录因子结合已知 NFκB DNA 结合基序的能力。

重复性实验表明，DNA 结合活性可以用小于 10%（RelB 和 p52）和小于 15%（p50 和 RelA）的平均分析间和分析内变异系数进行量化。在健康个体中，NFκB 转录因子的 DNA 结合活性对刺激作出反应显着增加，尽管这种反应的程度因人而异。DNA结合活性的动力学在规范和非规范转录因子之间也不同。P50 和 RelA DNA 结合活性在刺激后数小时内响应，而 RelB 和 p52 响应延迟到刺激后一天以上。

可以精确测量响应 B 细胞特异性刺激的 NFκB 信号激活，以区分具有该途径功能活性差异的个体。该测试可能被证明是研究遗传变异对 NFκB 信号传导的功能影响的重要生物标志物。