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


CONCLUSIONS
This method may be useful for analyzing the significance of TNFalpha concentration in various diseases.
Saito
K
K
Department of Laboratory Diagnosis, Sapporo Medical University, School of Medicine, South-1, West-16, Sapporo 060-0061, Japan.
Kobayashi
D
D
Sasaki
M
M
Araake
H
H
Kida
T
T
Yagihashi
A
A
Yajima
T
T
Kameshima
H
H
Watanabe
N
N
D000328:Adult; D001782:Blood Donors; D005260:Female; D006801:Humans; D007118:Immunoassay; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012016:Reference Values; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D014409:Tumor Necrosis Factor-alpha