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Clinical Chemistry 45: 665-669, 1999;
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(Clinical Chemistry. 1999;45:665-669.)
© 1999 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Detection of Human Serum Tumor Necrosis Factor-{alpha} in Healthy Donors, Using a Highly Sensitive Immuno-PCR Assay

Kaori Saito, Daisuke Kobayashi, Masateru Sasaki, Hiroshi Araake, Takashi Kida, Atsuhito Yagihashi, Tomomi Yajima, Hidekazu Kameshima and Naoki Watanabea

Department of Laboratory Diagnosis, Sapporo Medical University, School of Medicine, South-1, West-16, Sapporo 060-0061, Japan.
a Author for correspondence. Fax 81-11-622-7502; e-mail watanabn{at}sapmed.ac.jp

Background: Tumor necrosis factor-{alpha} (TNF{alpha}) is an important mediator 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 using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNF{alpha} 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 TNF{alpha} 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 TNF{alpha} concentration in various diseases.© 1999 American Association for Clinical Chemistry




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