Use of soluble recombinant TNF receptor to improve detection of TNF secretion in cultures of tumor infiltrating lymphocytes.
The accurate quantitation of picogram amounts of TNF is possible by ELISA and is useful in many areas of biomedical research, including studies of TNF release in vitro by stimulated lymphocytes and macrophages, and of serum levels in patients with cancer and sepsis. However, we show in this report that the detection of recombinant TNF standards by ELISA falls over time with incubation at 37 degrees C, and is further decreased when incubated with tumor infiltrating lymphocytes (TIL), making accurate quantitation difficult. We demonstrate that the soluble dimeric form of the TNF receptor can prevent this decrease, both in the presence and absence of TIL. In contrast, the soluble monomeric TNF receptor was much less effective in preventing this decrease. In addition, the dimeric but not the monomeric TNF receptor was found to inhibit bioactivity of TNF as measured by L929 cytotoxicity. The dimeric TNF receptor does not interfere with the detection of recombinant TNF standards by ELISA, and entirely stabilizes TNF levels incubated over 48 h at 37 degrees C in the presence and absence of TIL. This protection is specific, and the TNF receptor does not stabilize interferon-gamma. The dimeric form of the soluble TNF receptor has proven useful in detecting TNF released by TIL transduced with the TNF cDNA that are currently being used in studies of the gene therapy of cancer with TIL. The dimeric TNF receptor may also prove useful in the accurate quantitation of TNF released by stimulated lymphocytes and macrophages in vitro, and in the quantitation of serum TNF levels in patients.
Hwu
P
P
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Schwarz
S
S
Custer
M
M
Smith
C A
CA
Mulé
J J
JJ
Rosenberg
S A
SA
D001681:Biological Assay; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D066298:In Vitro Techniques; D016246:Lymphocytes, Tumor-Infiltrating / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D012995:Solubility; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha