RECEPTOR-MEDIATED LABEL-TRANSFER ASSAY (RELAY) - A NOVEL METHOD FOR THE DETECTION OF PLASMA TUMOR-NECROSIS-FACTOR AT ATTOMOLAR CONCENTRATIONS

We have exploited the extremely high binding specificity of the 55 kDa human tumor necrosis factor (TNF) receptor in an assay designed to de tect TNF with sensitivity limited only by limits in the detection of I -131. Bivalent derivatives of the 55 kDa TNF receptor (referred to her e as the TNF binding protein), in which the extracellular domain is co upled to an IgG heavy chain, ordinarily bind TNF with very high affini ty as a result of the fact that they interact with two separate sites on the trimer surface. The TNF binding protein is radioiodinated to a high specific activity and then added to plasma at a saturating concen tration, so that it binds all active TNF present in the solution. Cova lent adducts between molecules of TNF and molecules of the binding pro tein are then produced by crosslinking with disuccinimidyl suberate (D SS). The complexes are swept out of solution using sepharose beads to which polyclonal anti-TNF antibodies have been affixed. On electrophor esis, the complex presents itself as a band of M(r) = 200 kDa (as dist inct from the uncomplexed binding protein, which has a size of 120 kDa and which in any case is removed by washing). As little as 50 fg of a ctive TNF (600,000 trimers) can be detected in a 5 mi sample of plasma using this approach, corresponding to the detection of TNF at a 200 a M concentration. Notably, no TNF is detectable in normal plasma specim ens, indicating that normal plasma contains active TNF at a concentrat ion beneath 200 aM.