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Detection of human serum tumor necrosis factor-alpha in healthy donors, using a highly sensitive immuno-PCR assay
K Saito1, D Kobayashi, M Sasaki
1Department of Laboratory Diagnosis, Sapporo Medical University, School of Medicine, South-1, West-16, Sapporo 060-0061, Japan.
Background:
Tumor necrosis factor-alpha (TNFalpha) is an important mediator of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNFalpha, including those in healthy controls, cannot be measured by existing methods.
Methods:
We developed a sensitive immuno-PCR assay for the detection of TNFalpha in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNFalpha 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 approximately 5 x 10(4)-fold improvement compared with a conventional ELISA. The mean serum TNFalpha 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 TNFalpha concentration in various diseases.
