WEHI 164 subclone 13 assay for TNF: sensitivity, specificity, and reliability

Immunological Investigations
M K EskandariD G Remick

Abstract

Tumor necrosis factor alpha (TNF) is a peptide monokine involved in a number of immune reactions. To further understand the role of TNF in disease states it is critical to have an inexpensive, yet sensitive and specific assay. Additionally, the effects of prostaglandin E2 (PGE2), dexamethasone (dex), and cyclosporine A (CsA) on TNF gene expression have been studied, although little is known of the effects these compounds have on TNF containing samples. The aim of this study is to determine the sensitivity and specificity of a highly sensitive cell line to the actions of TNF, and to elucidate parameters which affect the stability of TNF in biological fluids. Dex and PGE2 at concentrations of 10(-5), 10(-7), and 10(-9) M, were shown not to effect the WEHI assay, and neither did CsA (10 ng/ml-1 ug/ml). The cells were not lysed by recombinant murine IL-1 alpha or beta, human recombinant IL-1 alpha or beta, human recombinant IL-2 or human recombinant IL-6 at concentrations ranging from 0.02 pg/ml to 1.0 ug/ml, or murine gamma-IFN from 100 pg/ml to 10 ng/ml. TNF containing samples with 1%-10% fetal calf serum maintained their cytolytic activity even after three freeze-thaw cycles. Serum samples did not lose any cytolytic activity wit...Continue Reading

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