Detection of human serum tumor necrosis factor-α in healthy donors, using a highly sensitive immuno-PCR assay

Abstract

Background: Tumor necrosis factor-α (TNFα) is an important mediator of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNFα, including those in healthy controls, cannot be measured by existing methods. Methods. We developed a sensitive immuno-PCR assay for the detection of TNFα in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNFα sandwiched by antibodies was detected by amplification of the DNA label using PCR. Results: The limit of detection of the assay was 0.001 ng/L, an ~5 x 104-fold improvement compared with a conventional ELISA. The mean serum TNFα concentration (± SD) in healthy donors was 0.021 ± 0.044 ng/L in men (n = 29) and 0.033 ± 0.065 ng/L in women (n = 25). Conclusion. This method may be useful for analyzing the significance of TNFα concentration in various diseases.