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 detect TNF with sensitivity limited only by limits in the detection of 131I. Bivalent derivatives of the 55 kDa TNF receptor (referred to here as the TNF binding protein), in which the extracellular domain is coupled to an IgG heavy chain, ordinarily bind TNF with very high affinity 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 concentration, so that it binds all active TNF present in the solution. Covalent adducts between molecules of TNF and molecules of the binding protein are then produced by crosslinking with disuccinimidyl suberate (DSS). The complexes are swept out of solution using sepharose beads to which polyclonal anti-TNF antibodies have been affixed. On electrophoresis, the complex presents itself as a band of M(r) = 200 kDa (as distinct 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 active TNF (600,000 trimers) can be detected in a 5 ml sample of plasma using this approach, corresponding to the detection of TNF at a 200 aM concentration. Notably, no TNF is detectable in normal plasma specimens, indicating that normal plasma contains active TNF at a concentration beneath 200 aM.
Poltorak
A
A
Howard Hughes Medical Institute, Department of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas 75235-9050.
Peppel
K
K
Beutler
B
B
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007158:Immunologic Techniques; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D011817:Rabbits; D018124:Receptors, Tumor Necrosis Factor / Q000276:immunology*; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000276:immunology
D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha