Establishment of an enzyme release assay for cytotoxic T lymphocyte activity

Hitoshi Ohmori, Toshiyuki Takai, Takahiro Tanigawa, Yoichi Honma

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13 Citations (Scopus)


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 β-galactosidase (β-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 × 104 NS-1/Z cells were incubated with varying numbers of effector cells, β-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-β-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.

Original languageEnglish
Pages (from-to)119-124
Number of pages6
JournalJournal of Immunological Methods
Issue number1
Publication statusPublished - 1992


  • 4-MU
  • 4-MUG
  • 4-methylumbel-liferone
  • 4-methylumbelliferyl-β-D-galactoside
  • 5-bromo-4-chloro-3-indolyl-β-D-galactoside
  • CTL
  • Cytotoxic T lymphocyte
  • DNA transfection
  • E/T ratio
  • Enzyme release assay
  • FCS
  • MMC
  • NS-1
  • P3/NSl-Ag4-1
  • X-Gal
  • cytotoxic T lymphocytes
  • effector-to-target ratio
  • fetal calf serum
  • mitomycin C
  • β-Gal
  • β-Galactosidase
  • β-galactosidase


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