A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity.

A simple way of measuring and evaluating lactate dehydrogenase release from lysed tumor cells is described. LDH activity was determined as NADH oxidation or INT reduction over a defined time interval, which was limited by stopping the enzymatic reaction with the inhibitor oxamate. Reaction products were then assayed using a microplate reader. The principle of measuring LDH activity of cellular culture supernatants as a measure of cytotoxicity was successfully applied to a number of murine and human effector-target cell combinations (macrophages, monocytes, NK cells and cytotoxic T cells with P815, A375, K562 and Yac-1 tumor cells) as well as to the determination of TNF activity on L929 cells. Comparison with 51Cr release assays suggests that LDH release assays are an appropriate and possibly preferable means of measuring cellular cytotoxic reactions. This LDH release assay combines the advantages of reliability and simple evaluation characteristic of radioisotope release assays with the convenience of speed and avoidance of radioactivity.

[1]  A. Pacsa,et al.  Measurement of lymphocyte cytotoxicity by assessing endogenous alkaline phosphatase activity of the target cells. , 1981, Journal of immunological methods.

[2]  I. Fidler,et al.  Constitutive production and release of a lymphokine with macrophage-activating factor activity distinct from gamma-interferon by a human T-cell leukemia virus-positive cell line. , 1984, Cancer research.

[3]  T. Lövgren,et al.  Europium-labelled target cells in an assay of natural killer cell activity. I. A novel non-radioactive method based on time-resolved fluorescence. , 1986, Journal of immunological methods.

[4]  R. Schreiber,et al.  Comparison of Five Short‐Term Assays That Measure Nonspecific Cytotoxicity Mediated to Tumor Cells by Activated Macrophages , 1986, Journal of leukocyte biology.

[5]  E. Saksela,et al.  Isolation of human NK cells by density gradient centrifugation. , 1980, Journal of immunological methods.

[6]  R. Arentzen,et al.  Carboxyfluorescein fluorochromasia assays. I. Non-radioactively labeled cell mediated lympholysis. , 1980, Journal of immunological methods.

[7]  T. Decker,et al.  Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages. , 1987, Journal of immunology.

[8]  B. Bonavida,et al.  Studies on the mechanism of natural killer cell-mediated cytotoxicity. VII. functional comparison of human natural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor. , 1987, Journal of immunology.

[9]  C. Korzeniewski,et al.  An enzyme-release assay for natural cytotoxicity. , 1983, Journal of immunological methods.

[10]  G. Gifford,et al.  Rabbit tumor necrosis factor: mechanism of action , 1981, Infection and immunity.

[11]  J. Ortaldo,et al.  Direct comparison of three isotopic release microtoxicity assays as measures of cell-mediated immunity to Gross virus-induced lymphomas in rats. , 1977, Journal of the National Cancer Institute.

[12]  C. Parish,et al.  Automated fluorometric assay for T cell cytotoxicity. , 1983, Journal of immunological methods.

[13]  S. Ferrone,et al.  Detection of human histocompatibility (HLA) antigens with an indirect rosette microassay. , 1979, Journal of immunological methods.