Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Reexamination Certificate
1999-09-15
2004-03-09
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
C435S029000, C435S034000
Reexamination Certificate
active
06703211
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to cellular detection. More specifically, the invention relates to the indirect detection of cells by the detection of ATP in a reaction mixture.
BACKGROUND OF THE INVENTION
The ability to detect very low amounts of cells such as microbes is required in a variety of fields, both where sterilization is necessary and where the presence of microbes is expected as in foods and commercial water sources. By traditional methods, it is generally not possible to detect and quantify very small amounts of microbes directly from a sample. For example, a research scientist may want to know how many cells they have in a sample. A typical way of estimating this is to grow the cells in liquid culture and then to measure the optical density of the culture.
Reducing the amount of microbes to required levels is important in the making of consumer products, such as food and medical products. This is accomplished by autoclaving, irradiation, pasteurization, and filtration, among other techniques. A task of quality control is to ascertain the effectiveness of these techniques. Typically, such is the work of microbiologists, who attempt to grow cultures from swabs or samples of the product to be tested. On the other hand, commercial water sources are expected to contain low concentrations of microbes and microbiologists also grow cultures from these to determine their microbial counts in those sources.
In the food science and water treatment fields, liquid samples are typically analyzed using the Most Probable Number (MPN) method. The Most Probable Number method is basically a statistical analysis based on classical bacteriology culturing techniques, wherein in a range of dilutions of a liquid sample are inoculated into growth medium, and cell growth in the medium is detected. Standard MPN procedures often use a minimum of three dilutions and multiple culture tubes at each level of dilution. A typical worker would consult an MPN table to obtain the statistical multiplier to calculate the most probable number of bacterial cells in the original sample based on the growth distribution in the culture tubes.
Methods such as MPN are useful for calculating the number of living cells in a sample. The living cells in a consumer product are likely to grow during storage or after it is opened. The growth of these cells may adversely affect the consumer.
The time it takes to carry out an analysis by methods such as MPN that rely on the culturing of cells is a major drawback. The analyst must wait for the cultures to grow. Culture growth typically takes from overnight to several days.
All living organisms use adenosine triphosphate (ATP) as an energy source. In the prior art, cells are detected by assaying for the presence of ATP using the ATP-driven chemiluminescent (bioluminescent) luciferase/luciferin couple according to the following reaction.
luciferase
ATP+luciferin+O
2
→AMP+oxyluciferin+PPi+hv.
The light that is emitted from the reaction (hv) is conveniently measurable.
The amount of ATP present in a sample has been used to infer the amount of microbial organisms since the mid-1960s, according to U.S. Pat. No. 5,648,232, citing other work. Chittock et al.,
Analyt. Biochem
., 255:120-126 (1998), discuss the detection of ATP on a food preparation surface as a marker for biological contamination. A firefly luciferase/luciferin system is used to detect the ATP that is present in a sample. In the art discussed in that publication, commercial systems have a detection limit of about 5 pg ATP. Chittock, et al. disclose a method to achieve that detection limit using a less sensitive luminometer and Michaelis kinetics to calculate the time at which the bioluminescence reaches one-half of its maximal value to calculate the initial ATP concentration. Chittock, et al. note that adenylate kinase activity can be driven by excess CTP, which is not a substrate of luciferase, and that adenylate kinase can convert AMP to ATP, citing Brovko et al.,
Anal. Biochem
., 220:410-414 (1994).
U.S. Pat. No. 5,648,232 discloses a more sensitive way to detect microorganisms using a luciferase ATP assay. Rather than to detect only the ATP present at the time of measuring (linear relationship between photons produced and the amount of ATP), those workers detect the presence of adenylate kinase, an enzyme that catalyzes the conversion of ADP to ATP. By providing ADP, a single adenylate kinase molecule can give rise to an amplified amount of light by converting multiple ADP molecules to ATP and AMP. Those workers report that typically 400,000 ADP molecules are converted to ATP by a single adenylate kinase molecule in 10 minutes. The method disclosed in that patent for determining the presence and/or amount of microorganisms and/or their intracellular material present in a sample involves lysing the cells, providing adenosine diphosphate (ADP), luciferase/luciferin and preferably magnesium ions, and then analyzing for light produced from the luciferase/luciferin reaction.
An advantage to the method of U.S. Pat. No. 5,648,232 over mere analysis for the ATP that is present in a sample is that ATP can be present for a number of reasons, but the presence of adenylate kinase means that a living organism is likely present.
A drawback of that method is that solutions of ADP are unstable, resulting in formation of ATP. Thus, in a method of that patent, ADP is preferably kept in solid form until immediately prior to use. Contamination of the ADP, particularly with ATP, is highly undesirable for a cellular assay based on ATP detection. That patent advises purchasing high purity commercial ADP (>99.5% purity), then further purifying the ADP by column chromatography.
International Patent Application Publication No. WO/94/25619 discusses the art of the detection of biological material. Cyclic reactions for the measurement of low levels of NAD(H) or NADP(H) were discussed therein, citing EP-A-0060123, linearly amplifying the detection target, thus requiring a long time for sensitive measurements. WO/94/25619 cites GB-A-2055200 for disclosing a linear amplification of ATP using adenylate kinase to catalyse the reaction of AMP and ATP to form 2 ADP, which is then re-phosphorylated by pyruvate kinase to form ATP. In GB-A-2055200, the ATP is measured using bioluminescence and a luminometer.
The same cycle is discussed therein citing Chittock et al.,
Biochem. Soc. Trans
., 19:160S (1991), wherein adenylate kinase forms two ADP from an AMP and an ATP. Pyruvate kinase then converts the two ADP to two ATP. Chittock et al. close the cycle by permitting luciferase to convert an ATP back to AMP, producing bioluminescence.
WO/94/25619 itself discloses a method of detecting ATP indirectly, by using ADP to convert glucose-6-phosphate to glucose with glucose kinase, then detecting the glucose product, the concentration of which increases exponentially when sufficient AMP is present with adenylate kinase to reform the consumed ADP. They add glucokinase and adenylate kinase and glucose-6-phosphate to determine ATP present.
It would be beneficial if a method were available for fast, highly sensitive detection of the presence of very low amounts of cells. The disclosure that follows provides such a method that also solves the problem posed by the instability of ADP by providing AMP and a high energy phosphate donor that are stable, as alternative precursors to ATP, and uses endogenous cellular enzymes to convert those precursors to ATP, as described below.
BRIEF SUMMARY OF THE INVENTION
The invention contemplates a method for determining the presence and/or amount of cells in an aqueous sample composition. The method contemplates indirectly detecting cellular enzymes that convert AMP into ATP. The process includes providing a high energy phosphate donor, and preferably also providing AMP, and detecting the generated ATP. Such a method comprises the following steps. An aqueous sample solution to be assayed for the presence of cells is admixed with a high energy phosphate donor (other than ADP)
Hall Mary
Pratt Tamara Sue
Shultz John William
Wood Keith V.
Naff David M.
Promega Corporation
Ware Deborah K.
Welsh & Katz Ltd.
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