Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1998-02-17
1999-05-11
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 4, 435 11, 435 13, 435968, 435975, 435973, 435 71, 435 72, 435 721, 435 723, 435 724, C12Q1/02;1/00
Patent
active
059027339
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a method to determine the action of effectors on target cells in particular the activity of natural killer cells (NK cells) as well as a suitable reagent kit for this.
NK cells play an important role in the immune system of mammals and are defined by their function as immune cells which exhibit a spontaneous non-MHC-restricted cytotoxic activity towards numerous target cells such as tumour cells, virus-infected cells or allogenic target cells. The morphological majority of these effectors of natural cell-mediated immunity are large granulated lymphocytes and are defined immunophenotypically as CD3 negative lymphocytes which express CD56 and/or CD16. A small number of T lymphocytes (CD3.sup.+ CD56.sup.+) also exhibit NK activity.
A reduced NK activity was found in patients with AIDS, "AIDS related Brenner B. et al. 1989, J. Leuk. Biol. 46: 75-83, Quan, C. et al. 1990, J. Acq. Immune Deficiency Syndromes 3: 669-676!, patients with Down's Immunol. 139: 3306-3313, Klimas, N. et al. 1990, J. Clin. Microbiol. 28: 1403-1410, Landay, A. et al. 1991, Lancet 338: 707-713!. In addition the 1989, Adv. Immunol. 47: 187-376!. Patients in an advanced stage of cancer Immunol. 127: 1817, Takasugi, M. et al. 1977, Cancer Res. 37: 413!.
The previous most common assay for the determination of the cytotoxic al. 1968, Immunology 14:18!. However, this method has several important disadvantages for a broad application in routine diagnostics and namely the necessity of a specially equipped isotope laboratory, the use of a radioactive isotope with a relatively short half-life, high costs, a relatively high spontaneous release of .sup.51 Cr and long labelling and incubation periods.
Other methods for the quantification of cytotoxicity have been described and comprise the measurement of released substances such as e.g. determination of the enzyme activity of released cytoplasmic enzymes
In addition flow cytometric methods have been described for the determination of NK effector activity which enable the cytotoxicity to be Methods 86: 7-15, Zarzone, D. et al. 1986, J. Immunol. Methods 94: 247-255, Papa, S. et al. 1988, J. Immunol. Methods 107: 73-78!. However, a disadvantage of these methods is that they cannot unequivocally differentiate between target and effector cells. Therefore flow cytometric methods have been developed in which the membranes of the target cells are labelled with fluorescent dyes to differentiate them from effector cells K. et al. 1990; J. Immunol. Methods 135: 81-89!. determination of NK activity using the lipophilic membrane dye 3,3'-dioctadecyloxacarbocyanine perchlorate (DiOC.sub.18). The B. et a. 1976, J. Natl. Cancer Inst. (U.S.) 56: 627! kept in cell culture was used as target cells.
A disadvantage of the previously described methods is the necessity to culture the target cells in a cell culture laboratory before actually carrying out the assay.
Therefore an object of the present invention was to develop a method in which the disadvantages of the state of the art are at least partially eliminated. In particular it was intended to develop a method for the cryopreservation of cells in which the cells can be stored at -70.degree. C. without loss of vitality and can thus be used directly without prior culture in a diagnostic test e.g. the flow cytometric NK test.
This object is achieved by a method for the determination of the action of effectors on target cells which is characterized in that thawed cryopreserved mammalian cells are used without prior culture as target cells which have a vitality of at least 85% after storage at -70.degree. C. over a period of 4 weeks and which are obtainable by preparing a suspension of the target cells in a medium, adding a cryopreservative to a final concentration of 2-10% (v/v) and freezing the cells with a temperature decrease of no more than 2.degree. C. per minute. The freezing is preferably carried out down to a temperature of at least -50.degree. C. particularly preferably of at least -65.degree. C. and most preferably of about -70.degr
REFERENCES:
patent: 4983515 (1991-01-01), Maley et al.
H.T. Holden et al., "Standardization of the Chromium-51 Release, Cell-Mediated Cytotoxicity Assay: Cryopreservation of Mouse Effector and Targeted Cells," Journal of the National Cancer Institute, 58(3):611-622 (Mar. 1, 1977).
Ehemann Volker
Hirt Werner
Wink Alexander
Leary Louise N.
Orpegen Pharma GmbH
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