[Clinical significance of measurement of circulating tumor necrosis factor alpha]

Tumor necrosis factor alpha (TNF alpha) is an important mediator of inflammatory and autoimmune diseases. The 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. 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 by streptavidin to the biotinylated third antibody. TNF alpha sandwiched by antibodies was detected by amplification of the DNA label using PCR. The limit of detection of the assay was 0.001 pg/ml, an approximately 5 x 10(4)-fold improvement compared with conventional ELISA. The mean serum TNF alpha concentration in Duchenne muscular dystrophy patients (27.8 pg/ml) was approximately 1,000 times higher than that in healthy subjects (0.027 pg/ml). The mean value in inflammatory bowel disease patients was approximately 2,000 times that in healthy subjects (mean excess for UC, 1,100 times; for CD, 7,700 times). These findings suggest that measuring the serum concentration using a highly sensitive Immuno-PCR assay may be useful for analyzing the clinical significance of TNF alpha in various diseases.