Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/76—Chemiluminescence; Bioluminescence
  • G01N21/763—Bioluminescence
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/06—Quantitative determination
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase

Definitions

• the present invention relates to new techniques for performing luminometric assay of cellular ATP (adenosine-5 ' - triphosphate) in a liquid sample, and the invention covers a diagnostic means and a diagnostic kit for such luminometric assay and also a method for determining cellular ATP in a liquid sample.
• cellular ATP adenosine-5 ' - triphosphate
• mastitis i.e. inflammation of the cow udder, resulting increased numbers of lumphocytes in the milk. Mastitis testing can be performed at the farm by the
• the light emission may be measured with luminometers using photomultipliers or diods as light detectors or with photographic techniques (e.g. Polaroid ca ⁇ meras) .
• mastitis testing mammalian cells can be speci ⁇ fically determined in the presence of microbial cells by u- sing an extractant lysing mammalian but not microbial cells, e.g. tritonX-100.
• Mastitis testing by luminometric ATP assay has been described in the literature (T. Ols ⁇ on, K. Sand- stedt, 0. Holberg and A. Thore, Biotechnology and Applied Biochemistry, 8, 361-9, 1986).
• Several methods for the extraction of cellular ATP have been compared in various types of cells (A. Lundin and A.
• Extraction with TCA can be' done by adding an equal volume of TCA to the sample directly in the assay cuvette. If the final assay volume is 1 milliliter, the volumes of sample and extractant shall not exceed 10 microli- ter or TCA will cause analytical interference with the fire ⁇ fly reaction. Thus in quantitation of bacterial cells the 1000 cells required for detection must be present in a 10 microliter volume, corresponding to 10 ⁇ cells per milliliter. Increased sensitivity can be obtained by adding more lucife- rase to the reagent or by using a more sensitive light detec ⁇ tor. Each approach may be used to lower the detection limit at least 10 times, but the assay cost will increase making the method less attractive for routine purposes.
• Mammalian cells can often be extracted with lytic agents, e.g. detergents like tritonX-100, which do not cause analytical interference with the firefly reaction. In the presence of EDTA such extracts are stable, since ATP degrading enzymes require divalent metal ions for activity. Microbial or other cells with tough cell walls require more potent extractants like strong acids (e.g. trichloroacetic acid, perchloric acid), certain organic solvents (e.g. dime ⁇ thylsulphoxide) or quarternary ammonium compounds (e.g. dode- cyltrimethylammonium bromide) .
• lytic agents e.g. detergents like tritonX-100, which do not cause analytical interference with the firefly reaction.
• EDTA e.g. ATP degrading enzymes require divalent metal ions for activity.
• Microbial or other cells with tough cell walls require more potent extractants like strong acids (e.g. trichloroacetic acid
• TCA can be removed by extraction with diethyl ether and perchloric acid (another reliable ex ⁇ tractant) can be removed by neutralisation and precipitation of the perchlorate anion using e.g. potas'sium hydroxide (A. Lundin and A. Thore, Comparison of methods for extraction of bacterial adenine nucleotides determined by firefly assay, Appl .Microbiol . 30:713-21, 1975).
• potas'sium hydroxide A. Lundin and A. Thore, Comparison of methods for extraction of bacterial adenine nucleotides determined by firefly assay, Appl .Microbiol . 30:713-21, 1975.
• Dilution of the extract may be acceptable in routine work but would decrease the sensitivity of the assay.
• the present invention is based on four surpirising fin ⁇ dings.
• the first finding is that it is possible to stabilize all reagents needed for biomass estimation by bioluminescence assay of ATP simply by drying them on one or several reagent carriers made of e.g. filter paper. This applies to pretreat ⁇ ment reagents as tritonX-100 and apyrase, to bioluminescence reagents as those based on firefly luciferase and finally to the ATP standard used for calibration of luminometric assays of ATP. Furthermore, it is possible to stabilize sample ATP on a sampling element made of e.g.
• filter paper by including ⁇ DTA or similar chelators of divalent metal ions in dried form on the sampling element and by drying the sample on the sampling element.
• the second finding is that a sampling ele ⁇ ment of fibrous material, e.g. filter paper, can be used for sampling a defined sample volume as well as for performing pretreatments as degradation of non-microbial ATP or extrac- tion of cellular ATP.
• the third finding is that it is possib ⁇ le to perform heat extraction of cellular ATP with simultane ⁇ ous inactivation of ATP degrading enzyme systems on the samp ⁇ ling element and without the dilution inherent in the boiling buffer procedure.
• the fourth finding is that neither sample ATP nor bioluminescence reagent will be permanently bound to the fibrous material but can be dissolved in assay buffer rapidly enough for a convenient assay procedure.
• These fin- dings will be illustrated in "the following description of the invention.
• the principl object of the present invention is to pro ⁇ vide new rapid techniques for facile determination of bio ⁇ mass, i.e. quantitation of microbial or other types of living cells, whereby the practical problems involved in diagnosis or quality control are eliminated or at least significantly reduced.
• the present invention provides for a diagnostic means, such as a stick, for luminometric assay of cellular ATP, which stick comprises a substrate and attached thereto a reagent carrier on which a reagent in dried form to be used for luminescence analysis is arranged.
• a diagnostic means such as a stick
• the reagent carrier can be enclosed in a container, e.g. a tube, so that it can easily be transferred to the buf ⁇ fer in which the luminometric assay is to be performed or so that the buffer can be added to the container and the assay performed in this container.
• reagent carrier is constituted by a fibrous pad.
• a particularly pre ⁇ ferred bioluminescence reagent is based on firefly lucifera- se.
• the diagnostic stick as referred to above also comprises a sampling element attached to said substrate or that one substrate contains the bioluminescence reagent carrier and another substrate the sampling element.
• the sampling element need not always be present as a separate element but may in some analytical situations be inherent in the reagent carrier.
• the sampling element is preferably constituted by a fibrous pad. * -
• firefly luciferase reagent can be stabilized in dried form on a reagent carrier, such as a pad made of porous material, e.g. filter paper.
• a reagent carrier such as a pad made of porous material, e.g. filter paper.
• This arrangement has been found to result in an astonishing high stability, and firefly reagent pads according to the invention made for example of filter paper can be stored at room temperature for several months.
• the bioluminescence reagent is easily redissolved in buffer solution.
• Such a bioluminescence reagent pad can be conveniently handled without risk for con ⁇ tamination if attached to a stick or enclosed in a container.
• a diagnostic stick according to the invention including a sampling element is convenient to use in actual practice, and a defined sample volume can be obtained by soaking the sampling element in the sample subject to assay for a few seconds.
• an appropriate sampling element material e.g. filter paper
• the accuracy of the sample volume is generally within 10 percent, which is acceptable in most analytical situations.
• pretreatment reagents such as tritonX-100 and apyrase, can be stabilized in dried form in the sampling element.
• ATP can be extracted with an extractant not interfering with the firefly reaction
• the extractant present in dried form on or in the sampling element.
• One such situation is mastitis testing performing the extraction of lymphocytes in milk using a sampling element consisting of " . filter paper with tritonX-100 and EDTA in dried form.
• Fur ⁇ thermore according to the present invention it has been sur ⁇ prisingly found that the sample ATP in this and similar si ⁇ tuations can be stabilized by including EDTA or similar che- lators of divalent metal ions and/or by rapidly drying the " sample.
• the sample can be sent to a central laboratory performing the assay of ATP.
• a diagnostic kit for luminometric assay of cellular ATP comprising in combination: T 1) cuvettes containing buffering solution;
• Such a diagnostic kit can further comprise a calibration stick containing a known amount of ATP standard stabilized in dried form.
• the heat treat ⁇ ment under step c) is constituted by microwave heating or steam heating.
• a basic concept of the present invention is to provide for heat extraction without the dilution inherent in the boi ⁇ ling buffer method.
• the requirements on a heat extraction without dilution are: 1) The temperature has to rise almost instantaneously (within a few seconds) to at least 70°C and preferably to 100°C to avoid degradation of ATP by enzyme systems present' —' in the cells or in pretreatment reagents.
• the temperature should be maintained at this elevated temperature during the time needed for complete inactivation of ATP degrading ensyme systems and release of cellular ATP (a few seconds to a few minutes depending on type of cell and the medium in which they are suspended) , but the temperature may never during this time become so high that ATP is des- troyed.
• the preferred temperature for heat extraction without dilution is around the boiling point of water (90-110°C) .
• heat sources for the rapid heating are microwave radiation and steam heating.
• the diagnostic stick with the sampling element is inserted into a small microwave oven for a few seconds.
• microwave ovens can be produced at reasonable prices and need not have physical dimensions lar- ger than a few liters.
• a major advantage of microwave heating is that it is instantaneous and will by its inherent proper ⁇ ties cease when the water in the sample has evaporated. Over- heating resulting in degradation of ATP will thus not appear.
• With a diagnostic stick containing both sampling element and reagent carrier there is no risk that the bioluminescence reagent part of the stick will be heated, since said part -5 does not contain water except in the- special embodiment of the invention where the sampling element is inherent in the reagent carrier (an embodiment that cannot be combined with heat extraction) .
• Steam heating is another preferred technique for provi ⁇ ding rapid heating to an enzyme inactivating temperature of the sample.
• Steam heating can be provided by injecting water 5 into an electrically heated chamber with a hole through which the steam passes.
• the part of the diagnostic stick to be hea- ted is- ositioned in front of this hole.
• the bioluminescence - ⁇ reagent In using a combined stick the bioluminescence - ⁇ reagent must, of course, not be heated. In this case it may thus be more practical to use a 0 diagnostic stick wherein the bioluminescence reagent is well separated from the sampling part of the stick, or even two separate sticks rather than a combined stick.
• the use of steam heating is advantageous in that it is based on very low cost components and may accordingly be the most competitive technique for many applications.
• Fig. la illustrates an embodiment with a combined diagnostic stick
• Fig. lb illustrates an embodiment using separate sampling and firefly reagent sticks.
• a cuvette generally designa ⁇ ted 1 containing a buffer solution 3.
• a combined diagnostic stick generally designated 5 including a firefly reagent pad 7 and a sampling pad 9 arranged separately on stick 5.
• Fig. lb shows a corresponding cuvette 1 containing buf ⁇ fer solution 3.
• Fig. lb illustrates separate sampling and firefly reagent sticks, the sampling stick 11 containing at one end thereof a sampling pad 13, and the rea- gent stick 15 containing at one end thereof a firefly reagent pad 17.
• Example 1 Kerman and analytical procedure for estimating bacte ⁇ rial cells in absence of other types of cells. The components of the kit are as follows:
• C s t andarcj The value of C s t andarcj is easily calculated by assuming that the known amount of ATP standard applied on the calibra ⁇ tion pad is in the same volume as soaked up by the sampling 0 pad. This volume can be estimated by weighing a few sampling pads before and after sampling.
• a bacteriuria kit prototype was developed and consisted of the following components: 1) A Luminometer 1250 (LKB-Wallac, Turku, Finland) and a steam extraction device. The latter was a household egg boi ⁇ ler with a tube on top to guide the steam and an arrangement for holding the sampling stick.
• the calibration step may be avoided in a routine proce ⁇ dure with carefully standardized instrumentation and rea- gents, since the ATP Monitoring Reagent is extremely stable when dried on pads.
• a combined stick may be used.
• Example 3 Kerman and analytical procedure for mastitis testing
• a mastitis testing prototype kit was developed and con ⁇ sisted of the following components:
• a milk sampler consisting of a stick with a filter pa ⁇ per (Schleicher & Schiill No. 2668/2 (0 approx. 5 mm)) attach ⁇ ed to a plastic stick with a fracturing point.
• the filter paper had been prepared by applying 20 microliters of a solu ⁇ tion containing 0.6% tritonX-100 and 250 mM of EDTA (Merck, Darmstadt, FRG) followed by drying over night at room tempe ⁇ rature.
• the plastic stick was contained in a plastic container excluding humidity and con- taining moisture absorbing material (silica gel) .
• a modified Polaroid camera home-built allowing light emission from inserted cuvettes to be measured.
• the light intensity could be estimated from the diameter of the light area on the film.
• a series of various grey fil- ters inserted between the cuvette and the film could be used to obtain a pattern of light areas from which the light in ⁇ tensity could be estimated.
• the analytical procedure when performed at the farm was as follows: 1) Allow a jet of milk to soak the filter paper of the milk sampler stick.
• the ATP assay can also be performed in the laboratory using conventional instrumentation, e.g. automatic luminome- ters as Luminometer 1251 (LKB-Wallac, Turku, Finland). In this case the special version of the kit including the plas- tic container should be used. After applying the milk the plastic container is sealed and transported to the laborato ⁇ ry. During the transport ATP is stabile due to the presence of EDTA in the sampling pad and the drying effect of the si ⁇ lica gel. In the laboratory the milk sampler is put in a cu- vette with buffer and the automatic luminometer performs the additions of firefly reagent and ATP standard. The automatic luminometer can be connected to a computer for rapid calcula ⁇ tion of results.
• automatic luminome- ters as Luminometer 1251 (LKB-Wallac, Turku, Finland).
• the special version of the kit including the plas- tic container should be used.
• the plastic container After applying the milk the plastic container is sealed and transported to the labora

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• 1987
  • 1987-09-23 SE SE8703675A patent/SE8703675L/ not_active Application Discontinuation
• 1988
  • 1988-09-13 ZA ZA886830A patent/ZA886830B/xx unknown
  • 1988-09-14 WO PCT/SE1988/000478 patent/WO1989002929A1/en unknown
  • 1988-09-14 EP EP88850303A patent/EP0309429A3/en not_active Withdrawn
  • 1988-09-14 AU AU24849/88A patent/AU2484988A/en not_active Abandoned

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