Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Reexamination Certificate
2002-02-08
2004-11-02
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
C435S026000, C435S018000, C435S021000, C435S029000, C435S968000
Reexamination Certificate
active
06811990
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
Not applicable.
REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTING COMPACT DISC APPENDIX
Not applicable.
BACKGROUND OF THE INVENTION
The present invention is generally directed toward luminescent methods and compositions for measuring various biological events, such as cell death, membrane damage, cell proliferation, or enzyme activities. In these methods, something occurring as a result of enzyme activity is able to produce light, which is detected in a luminometer or other instrument capable of detecting light. The invention is more particularly directed to methods of measuring various biological events, such as cytotoxicity, membrane damage, cell proliferation, enzyme activities, or some combination of these events, by coupling the activities of enzymes, which may be supplied by the investigator, or which may have been released from dead or damaged cells, with production or consumption of high-energy molecules such as adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (reduced form) (NADH), and subsequently measuring the concentrations of these high-energy molecules by evaluation of the light produced by a light-producing molecule, such as a luciferase.
Cytotoxicity and Proliferation Assays
Assays for cell death and cell proliferation are very widely performed in many areas of biological and clinical research. They may be used to assess the cytotoxic effects of a drug candidate (such toxicity may be either desirable or undesirable), measure the activity of complement, measure programmed cell death (apoptosis), quantify growth-inhibitory or growth-enhancing effects, detect and characterize environmental toxins, determine the sterility or bioburden of a sample, assess drug sensitivity or resistance of a patient's tumor cells or a culture of an infectious organism, or simply determine cell number. One of the most usefull and efficient applications of cell death and proliferation assays is in high-throughput screening (HTS), a collection of methods currently used by many pharmaceutical and biotechnology companies to determine the properties of large libraries of drug candidates very rapidly. However, the methods of determining cell death and proliferation currently in use all suffer from important limitations. Some of these limitations make the assays impractical for use in HTS, and also limit their utility in traditional research environments.
Assays in current use for cell death, or cytotoxicity assays, fall into several categories. One category is “release” assays, in which a substance released by dying cells is measured. Often the substance is an enzyme, such as lactate dehydrogenase (LDH) or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Traditional enzyme-release assays have exploited the fact that these enzymes create NADH, which can be observed by UV spectroscopy at 340 nm. An alternative is to couple production of NADH to generation of a colored dye, as in the LDH-based CellTiter® assays currently available from Promega. However, these processes are slow and lack sensitivity. For example, the current product from Promega recommends seeding of 5,000-100,000 cell per well, depending on the cell type, and an incubation time with the chromogenic reagents of one hour or more. Other enzymes used in this way include phosphatases, transaminases, and argininosuccinate lyase. These enzymes are typically present in low quantities in most cells, and they do not lend themselves to simple activity assays, making the process of determining cell death cumbersome and insensitive.
Another variety of release assay involves pretreatment of the target cells with a radioactive isotope, generally
51
Cr or
3
H. Upon lysis, the radioactive contents are released and counted in a scintillation counter. Aside from the problems of handling and waste disposal of radioactive materials, these assays also suffer from various artifacts, and are tedious because of the pretreatment and recovery steps required. The same process can also be carried out with fluorescent dyes, such as bis-carboxyethyl-carboxyfluorescein or calcein-AM, but, again, pretreatment is required, and the dyes are spontaneously released at a significant rate by healthy cells.
Another type of release assay is the luminescent assay of ATP released from dead or damaged cells. However, as it is actually used, this is a proliferation assay, and it is discussed further below along with other proliferation assays.
Another category of cytotoxicity assay makes use of dyes which are able to invade dead cells, but not living cells. An example of such a dye is trypan blue. These assays are useful for examining individual cells, but for quantification of overall cytotoxicity they are inefficient because each cell must be counted individually, either by laborious microscopic analysis or by very expensive and time-consuming flow cytometry. Moreover, some modes of death.(such as complement-mediated lysis) are not easily assessed by this method, because the dead cell remains intact for a limited period of time, after which it can no longer be counted because it has disintegrated.
Yet another category of cytotoxicity assays includes those methods directly related to apoptosis. These assays typically look for either protein markers of apoptotic processes or particular effects on DNA that are uniquely associated with apoptosis. The methods are generally slow and tedious, and thus are not suitable for high-throughput screening applications. Another method of studying apoptosis is to look at the ATP:ADP ratios in a cell, which change in a distinct way as the cell enters apoptosis. These assays may be performed by coupled luminescent methods (Bradbury et al. (2000) J. Immunol. Methods 240:79). However, while these methods are useful for qualitative definition of the mode of death, they have no advantages over the ATP-release assay in quantitative determinations of cytotoxicity or proliferation.
Proliferation assays are methods of measuring numbers of live cells. This may be better for some applications than measuring cell death or damage. For example, proliferation assays are able to reveal cytostatic, growth-inhibitory, and growth-enhancing effects which yield no readout in a cytotoxicity assay. Proliferation assays are also in common use as indirect cytotoxicity assays, but there are serious drawbacks with this approach; these are discussed below in connection with the ATP-release assay. Proliferation assays also fall into several categories. Assays of metabolic activity are in widespread use in research laboratories. The commonly used methods make use of tetrazolium salts, which are reduced in living cells to colored formazan dyes. One advantage of these methods is convenience, especially with the newer dyes (MTT and WST-1). The dye is added to the cell culture, and the absorbance of the formazan is read, typically after 0.5-12 hours. However, there are several important disadvantages. Metabolically active cells reduce the dyes at rates much greater than quiescent cells; the readout may therefore be a poor reflection of the cell number. Moreover, the readout is not an instantaneous “snapshot” of the quantity of live cells when the measurement is taken, but rather a complex integral of metabolic activity over the preceding time interval, whose mathematical relationship to the actual live cell number involves the half-life of the dye as well as variations in metabolic activity. Metabolism-based assays are not suitable for measurement of cellular cytotoxicity (for example, the activities of cytotoxic T lymphocytes), or any other assay system in which live cells other than the target cells are present, because these other cells will yield a substantial and often ill-defined background signal. Finally, various artifacts have been associated with the use of these dyes (see for example O'Brien et al. (2000) Eur. J. Biochem. 267:5421-5426; Natarajan et al. (2000) Cancer Detection and Prevention 24:405-414). Although they have not been thoroughly characterized with
Corey Michael J.
Kinders Robert J.
Christensen O'Connor Johnson & Kindness PLLC
Corey Michael J.
Leary Louise N.
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