Establishment of an enzyme release assay for cytotoxic T lymphocyte activity.
In the present report, we describe the establishment of a cell line that can be used as the target for measuring the activity of cytotoxic T lymphocytes (CTL) by an enzyme release assay. We transfected P3/NS1-Ag4-1 (NS-1), a myeloma cell line derived from BALB/c mice with Escherichia coli beta-galactosidase (beta-Gal) gene, and isolated a stable transformant designated as NS-1/Z that expressed a high level of the enzyme activity intracellularly. The effector cells showing cytotoxicity against NS-1/Z were induced when the spleen cells of AKR or C3H mice were cultured with mitomycin C-treated BALB/c spleen cells for 4 days. When 2 x 10(4) NS-1/Z cells were incubated with varying numbers of effector cells, beta-Gal activity was released from the target cells depending on the number of effector cells and the time of incubation for up to 8 h. A highly sensitive enzyme assay was performed by using a fluorescent substrate, 4-methylumbelliferyl-beta-D-galactoside. The cytotoxicity was specific for H-2 haplotype of the stimulator cells, and was abolished by treating the effector cells with anti-Lyt 2 plus complement. The sensitivity of the enzyme release assay was comparable to that of 51Cr release assay. These results indicate that NS-1/Z can be used as a target cell line for the non-radioactive measurement of CTL activity.
Ohmori
H
H
Department of Biotechnology, Faculty of Engineering, Okayama University, Japan.
Takai
T
T
Tanigawa
T
T
Honma
Y
Y
D000818:Animals; D002460:Cell Line; D002860:Chromium Radioisotopes; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D002860:Chromium Radioisotopes; D001616:beta-Galactosidase