A simple nonisotopic in vitro bioassay for LT and TNF employing sodium fluoride-treated L-929 target cells that detects picogram quantities of LT and TNF and is as sensitive as TNF assays done with ELISA methodology.
We have developed a highly sensitive in vitro bioassay for lymphotoxin (LT) and tumor necrosis factor (TNF). This system employs a monolayer of 20,000 L-929 target cells in each well of 96-well microplates that are incubated in dilutions of LT and TNF at 37 degrees C for 20-24 h, after which the viability of the L-929 target cells is assessed by spectrophotometric techniques. We find that incorporation of 2 mM NaF increases the sensitivity of L-929 cells to lysis by 10(3)-10(4) x for LT and 10(4)-10(5) x for TNF. This method allows the regular detection of less than 1-5 pg of bioactive protein. ELISA assays did not detect synthetic TNF and TNF fragments that were not biologically active in this assay. The L-929 bioassay requires no isotopes, is very reproducible, is as sensitive as ELISA techniques, and detects only biologically active LT and TNF.
Jeffes
E W
EW
Department of Dermatology, University of California, Irvine 92717.
Schmitz
K
K
Yamamoto
R
R
Tomich
J M
JM
Beckman
M
M
Nep
R
R
Knauer
M
M
D000818:Animals; D001681:Biological Assay; D002470:Cell Survival / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007739:L Cells; D008233:Lymphotoxin-alpha / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012969:Sodium Fluoride / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D008233:Lymphotoxin-alpha; D014409:Tumor Necrosis Factor-alpha; D012969:Sodium Fluoride