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

Background: Tumor necrosis factor-alpha (TNF alpha) is an important mediato r of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNF alpha including those in healthy controls, cannot be measured by existing methods. Methods: We developed a sensitive immuno-PCR assay for the detection of TNF alpha in human serum. The DNA label was generated by PCR amplification usi ng biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNF alpha sandwiched by antibodies was detected by amplific ation of the DNA label using PCR. Results: The limit of detection of the assay was 0.001 ng/L, an similar to 5 x 10(4)-fold improvement compared with a conventional ELISA. The mean ser um TNF alpha concentration (+/- SD) in healthy donors was 0.021 +/- 0.044 n g/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 TNF alpha concentration in various diseases. (C) 1999 American Association for Clinical Chemistry.