WO1997019353A1 - Chemiluminescent assay methods and devices for detecting target analytes - Google Patents
Chemiluminescent assay methods and devices for detecting target analytes Download PDFInfo
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- WO1997019353A1 WO1997019353A1 PCT/US1996/018443 US9618443W WO9719353A1 WO 1997019353 A1 WO1997019353 A1 WO 1997019353A1 US 9618443 W US9618443 W US 9618443W WO 9719353 A1 WO9719353 A1 WO 9719353A1
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- sampling
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- container
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- sample
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- 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
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- 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
Definitions
- the present invention relates generally to chemiluminescent assay methods and devices for detecting target analytes.
- the present invention relates to chemiluminescent assay methods and devices for detecting bacteria on contaminated surfaces.
- Standard culture plate methods for monitoring surfaces for bacterial contamination require a sterile sample collection device (generally a swab or sponge) and suitable culture media which, after inoculation, must be incubated at an appropriate controlled temperature for a minimum of several hours or days.
- a sterile sample collection device generally a swab or sponge
- suitable culture media which, after inoculation, must be incubated at an appropriate controlled temperature for a minimum of several hours or days.
- chemiluminescence detection methods measures adenosine triphosphate (ATP) to indirectly measure bacteria.
- ATP adenosine triphosphate
- ATP detection is a reliable means to detect bacteria because all bacteria contain some ATP Chemical bond energy from ATP is utilized in the chemiluminescent reaction that occurs in the tails of the firefly Photinus .
- the mechanism of this chemiluminescence reaction has been well characterized (DeLuca, M. , et a l ,
- the components of this reaction can be isolated free of ATP and subsequently used to detect ATP in other sources by a reaction that begins with formation of an enzyme bound luciferyl-adenylate complex and free inorganic pyrophosphate and ends with a rapid reaction of this complex with molecular oxygen to produce light, CO 2 and AMP.
- Luciferin-luciferase reactions of the firefly have been used for detecting a threshold level of microorganisms as described in U S Patents 4,385,113 and 5,366,867
- These reported methods suffer a number of deficiencies Lyophilized luciferase-luciferin reagent is unstable at room temperature during long term storage and is unstable after liquid reconstitution over short time intervals. Additionally, after reconstitution, solutions of this reagent display significant emission of light in the absence of ATP This background decreases detection sensitivity and persists for several minutes to an hour.
- This solution to the instability problem lowers sensitivity of the detection method.
- This solution creates a new problem of incomplete transfer of ATP from the collection device to a separate plastic surface that contains the luciferase-luciferin reagent.
- this solution introduces a new variable of time between the transfer and the light emission measurement.
- the first copending application describes a sampling-device holder interface system and a method for performing an assay for a target analyte from a sampling device of the type disclosed in the first copending application.
- the sampling system includes a sampling-device holder interface and a quantifier for converting the output signal to quantifiable data indicative of the amount of the target analytes
- the interface comprises a sampling-device holder and a light detector - means for converting light generated from the sampling device to an output signal corresponding to the amount or intensity of the light generated such as a photomultiplier or photodetector.
- the interface holds a sampling device, which comprises a container and a sampling strip inside the container.
- the sampling strip has a sampling portion for introducing a sample, a reading portion containing a reagent for producing a chemiluminescent reaction with the target analytes, and a transfer portion connecting the sampling and reading portions for transferring the sample from the sampling portion to the reading portion.
- the container has an opening to permit introduction of samples to the sampling portion It also has a light transmissive portion, such as a window or opening, visibly exposing the reading portion.
- the holder includes a housing and a tray.
- the housing has at least first and second walls forming a cavity therebetween. One of the first and second walls has an opening or light transmissive window.
- the tray is received in the cavity and movable between opened and closed positions.
- the tray has a compartment adapted to seat and support the sampling assay device.
- the first opening is in registry with the reading portion when the tray is in the closed position to enable observation of the reading portion through the first opening.
- the light detector is connected to the housing, in registry with the first opening.
- the tray has a second opening (or light transmissive window) extending through the compartment, which opening is in registry with the reading portion of the seated sampling device.
- the second opening is in registry with the first opening to enable observation of the reading portion through both the first and second openings when the tray is in the closed position.
- a second copending application SN. 08/580096, filed December 22, 1995, SAMPLING-ASSAY DEVICE, INTERFACE SYSTEM, AND METHOD, describes a system similar to that of the second copending application, but has a light source opposite the light detector for illuminating a luminescent fluorescent or phosphorescent agent bound to the target analyte, the disclosure of which is also incorporated herein by reference.
- the sampling device in that copending application uses a binding agent immobilized to the reading zone to capture the luminescent agent bound to the target analyte.
- the sampling device disclosed in the first and second copending applications provides a unique means for allowing light detection using known light detection devices. Because the sampling device, however, is, rather flexible, thin, and flat, it can be challenging to remove the same from the tray compartment. The sampling device needs to be removed by prying out with a fingernail or some sharp instrument. One can also turn the interface upside down and drop the sampling device. But in any event, it would be desirable to ease the sampling device removal from the tray, in addition, there is a need to protect the exposed sampling portion from cross contamination. For instance, if the interface or holder is shaken or otherwise turned sideways or upside down, it is possible for the exposed sampling portion to contact the underside of the housing upper wall, which contact could possibly introduce other samples that made contact therewith. The present invention fulfills both of these needs.
- a second object of the invention is to prevent cross-contamination of samples.
- a third object of the invention is to provide enhanced stability for chemiluminescent reagent(s) used in detection of target analytes.
- a further object of the invention is to incorporate a light detection means into an assay device and to allow rapid measurement of target analytes at a sample site.
- one aspect of this invention provides a device for conducting a chemiluminescent assay.
- the device comprises a container, and sampling and reagent portions that are physically associated with the container and with each other.
- the reagent portion is made from absorbent material into which one or more chemiluminescent reagents have been dried.
- the device has a light-permeable portion that permits light generated by a chemiluminescent reaction within the container to exit the container.
- the invention also provides a sampling device for performing an assay for a target analyte with a shield to prevent cross-contamination.
- the sampling device has a container and a sampling strip inside the container.
- the sampling strip has a sampling portion for receiving a sample, a reading portion for holding the sample with a compound that can emit light, and a transfer portion connecting the sampling and reading portions for permitting transfer of the cample from the campling portion to the reading portion.
- the container has means to permit introduction of the sample to the sampling portion and a light transmissive portion exposing the reading portion.
- a shield extends from the container adjacent the exposed sampling portion. The shield has a portion extending beyond one end of the container and is movable to and from the sampling portion and can be wrapped around the one end.
- the container comprises a first layer and a second layer sandwiching the sampling strip.
- the shield is attached to or integral with the first layer and has means for -permitting the extending portion to wrap around the one end of the container.
- the wrap around means is preferably a preformed fold or crease, or even a perforation.
- a tab or handle is attached to or formed integrally with the first layer. Alternatively, the tab can also be attached to or integral with the shield.
- the sample introducing means preferably is a first opening formed through the first layer and aligned with the sampling portion.
- the light transmissive layer is preferably a second opening through the second layer and aligned with the reading portion.
- the second layer preferably also includes a light transmissive member to cover at least the second opening.
- the device of the present invention eliminates or reduces much of the complexity associated with prior art assay methods and, as a result, decreases the cost and training requirements for detecting target analytes.
- the sampling strip is composed of an adsorbent material.
- the sampling strip includes a poly-carbonate membrane that is light transmissive.
- the sampling device can further include a sample collecting member, which preferably is adsorbent, in contact with the sampling portion inside the container and aligned with the first opening.
- the sample collecting member receives the sample and transfers the sample to the sampling portion.
- the compound is a reagent, preferably an enzyme in a dried state, contained within the reading portion.
- the reagent When the reagent is mixed with the analyte, it produces chemiluminescent light. More preferably, the reagent is luciferase-luciferin in a dried state.
- the compound which preferably is a luminescent labeling agent
- the sampling strip further includes a collecting portion contiguous with the reading portion. This collection portion is designed to absorb any excess liquid containing the labeling compound not coupled to the binding agent.
- the analyte immobilized in the reading portion carries the labeling agent that glows when exposed to light
- the amount of light produced after exposure to light correlates to the amount of analyte present in the sample.
- the target analyte will pick up the labeling agent.
- Other organisms mixed with the labeling compound and the excess labeling compound are not specific to the binding agent. Thus, they will not be captured in the reading portion, but rather will flow through.
- the target analyte since it is specific to the binding agent, will be captured and remain in the reading portion.
- the sampling device is particularly adapted for use with a sampling-device holding interface, which has a housing and a tray for seating the sampling device.
- the housing has a cavity for accepting the tray with the sampling device.
- the cavity is light-light tight when the tray is closed.
- the housing is connected to a light detector or the like to measure the amount of light generated by the sample.
- the housing can have also a light source for triggering reaction of the luminescent labelling agent.
- the device of this invention includes a film such as self-developing photographic film. This feature facilitates convenient readout and monitoring.
- a convenient method for performing an assay for a target analyte In this method, the sampling portion of the above-mentioned device is made to contact a sample, for example, by wiping the sampling portion over a surface suspected of containing the target analyte. A carrier liquid is then added to the sampling area, which liquid transports target analyte into the reagent portion. The carrier liquid also re-wets chemiluminescence reaction components located in the reagent portion, and thus allows a chemiluminescence reaction to begin.
- the instability problem suffered by prior art methods is overcome by providing the chemiluminescent reagent in a dried state within the reagent portion and/or sampling portion.
- the method is used to rapidly detect bacteria on surfaces such as countertops and equipment used in meat or food production
- high sensitivity is achieved by incorporating components of the luciferase chemiluminescence reaction in a dried form into the reagent portion.
- a bacteriolytic agent e . g . , a detergent
- ATP liberated by lysis of bacteria then participates in a luciferase reaction to produce light.
- Figure 1 is a top perspective exploded view of a device in accordance with this invention.
- Figure 2 is a top perspective exploded view of another device in accordance with this invention depicting the positioning of a fibrous adsorbent sample portion.
- Figure 3 is a top perspective exploded view of another device in accordance with this invention comprising a carrier liquid and a photographic film detection means.
- Figure 4 is an exploded view of a sampling device according to one embodiment of the present invention.
- Figure 5 is an exploded view of a sampling device according to another embodiment of the present invention.
- Figure 6 is a schematic side view of the present sampling device showing the manner in which the shield operates.
- the present inventors discovered that, for a chemiluminescence system, the luciferase enzyme and necessary cofactors could be dried onto a porous adsorbent material and subsequently reconstituted in an assay for bacteria andother analytes. Moreover, the present inventors discovered that such reagents could be combined into a convenient device that included a sampling portion and a reagent portion.
- the process of this invention comprises three steps: 1) contacting the surface, liquid, or other area suspected of containing the analyte by swabbing with the sample portion; 2) applying a carrier liquid to the sample portion to wash target analyte from the sample portion to the reagent portion and, at the same time reconstitute dried reagent, and 3) detecting lignt produced by chemiluminescence in response to the presence of target analyte in a sample It is noted that step 2 (application of a carrier liquid) may not be needed if the target analyte to be tested is in a liquid.
- the sampling portion of the device can collect virtually any type of target analyte not only from physical contact with a solid but also from a fluid applied externally.
- a target analyte as used herein is a molecule such as a protein, cell metabolite or microorganism such as a prokaryotic cell, virus, microplasma or free living eukaryotic cell.
- the target analyte can be for example, introduced to the sampling portion of the device by application of a fluid by means of, for example, an eye dropper or other dispenser, or by brief immersion of the device in a fluid or stream of fluid.
- the target analyte can be introduced by physical contact such as by swabbing a suspected contaminated surface with the device.
- suspect surfaces such as countertops, kitchen utensils and slaughterhouse machinery are tested for bacterial contamination by swabbing the suspect contaminated surface with the sampling portion of the device.
- the sampling portion is comprised of an adsorbent material.
- the adsorbent material may be fibrous, such as glass fiber, cotton, dacron, or paper, and it may be porous, such as porous polyethylene or sintered glass.
- the sampling portion is in proximity to or in physical contact with a reagent portion such that fluid applied to the sample receiving portion will enter the reagent portion.
- the reagent portion also is comprised of an adsorbent material.
- This material may be fibrous, such as glass fiber, cotton, dacron, or paper and the like, and it may be porous, such as porous polyethylene or sintered glass and the like.
- the porous material is generally flat and can be in various shapes, including rectangular, or narrow in the middle and wider at each end. The shape provides for diffusion and mixing of reagents and, advantageously, allows a maximum exposure of surface area to the light detection means.
- Those skilled in the art will recognize many useful materials, such as those used in chromatographic-type assays currently available.
- the reagent portion often will be only part of a porous adsorbent strips shown in the Figures.
- the reagent portion can contain one or more reagents for the chemiluminscent reaction, generally in dried form. Additionally, it can contain other reagents useful for the assay including, for example, the detergent or other bacteriolytic reagent necessary to extract ATP from bacteria.
- the reagents can be mixed together in the reaction portion, or placed sequentially so that the diffusing fluid contacts the reagents sequentially.
- the chemiluminescent reaction may occur within the reagent portion or may occur downstream of the reagent portion (where the chromatically moving reagents will be), once the reaction has become sufficient to generate detectable light.
- the location on the adsorbent material for detecting the reaction will depend upon such factors as the type and amounts of reagents, type of adsorbent material, etc. A satisfactory location on the adsorbent for detecting the reaction will be easily determined.
- a portion of the device is at least partially transparent to the light emitted by the chemiluminescent reaction. This can be achieved, for example, by using transparent plastic for the device although other means, such as windows or sonic welded transparent portions are suitable. Particularly suitable is the use of a transparent covering over a surface of the reagent portion of a strip so that light can be detected at a location distant from a sample application area.
- chemiluminescent chemistries can be used in the devices and methods of this invention.
- Acceptable chemiluminescence chemistries include, among others, the reaction of hydrogen peroxide with horseradisn peroxidase labelled antibodies and luminol, enhanced horseradish peroxidase, reactions that include the use of diacylhydrazides, acridinium salts, dioxitanes, and bioluminescent reactions involving cofactors such as reduced nicotine adenine dinucleotide in the case of marine bacteria.
- a particularly preferred chemiluminescent chemistry is the firefly ATP assay which utilizes luciferase and at least one cofactor to generate light from ATP that is present in a sample.
- At least one chemiluminescent reaction reagent is present in the reagent portion adsorbent material in a dry state.
- a reagent may be conveniently applied as a wet water solution and dried during manufacture or it may be applied in a dry form, such as a powder or suspension in a organic solvent or slurry. Other methods are known in the art and the preferred one is determined by characteristics of the reaction components.
- Acceptable carrier liquids include, among others, buffer, buffer with detergent, water, blood and urine Buffer solutions of TRIS, HEPES buffers at pH 7 0 to 9.0, and most preferably HEPES buffer at 7 8 with EDTA arc preferred EDTA is a preferred ingredient because ATP degrading enzymes require divalent metal cations for activity and EDTA chelates these.
- detergent is present in the sample or reagent portion, or elsewhere in the porous adsorbent material that contains the reagent portion.
- the detergent dissolves in fluid that is added to the device and it serve to open cells and liberate cell components.
- An optional further use of the carrier liquid is to release one or more substances from microorganisms in the sample portion
- the carrier liquid lyses any collected bacteria in the sampling portion and releases ATP from the bacteria into the solution for transport into the reagent portion.
- Detergent can be included in the carrier liquid for this purpose.
- the carrier liquid is present in a reservoir that is present in the assay device.
- the reservoir can be positioned on the device adjacent to a flexible area (e.g., hinge, or integral hinge).
- the flexible area permits the reservoir to be positioned proximate the sampling portion.
- the portion of the device containing the reservoir can be contorted or folded over so that the reservoir is positioned proximate (e.g., directly on top of) the sampling portion.
- the reservoir then can be broken (e.g. by finger pressure) so that its contents are released directly on the sample portion
- detergents or combination of detergents include, nonionic detergents such as Triton X-100, Nonidet P40, n-Undecyl Beta-D glucopyranoside, Zwitterioinc detergents such as n-hexadecyl-N,N-dimethyl-3-ammonio-1- propanesulfonate, and cationic detergents chloride, cetyldimethyl-ethylammonium bromide, dodecyltrimethyl-ammonium bromide, and cetyltrimethylammonium bromide.
- concentration of detergent solution varies for each type of detergent and can range from 0.1% to 6%, and preferably from 0.5% to 2.0%.
- the container itself should be constructed of liquid impermeable material, such as a plastic.
- the container can be made, for example, by molding a single piece of plastic into a shape that can house the adsorbent material of the sampling and reagent portions.
- the container can be constructed from multiple elements that are sealed to provide a liquid impermeable seal.
- the container optionally may have an opening to enhance the diffusive or chromatographic flow of liquid along the adsorbent material therein. Alternatively, an air pocket within the container can be provided for such purpose.
- the step of detecting chemiluminescent light emitted from the container can be accomplished by a number of means known to those skilled in the art.
- Chemiluminescent light can be detected electronically by, for example, a photomultiplier, photo diode, photo fet or charge coupled device.
- the most preferred electronic light detector is a photomultiplier because of its sensitivity.
- Chemiluminescent light also can be detected chemically, for example by the use of a film.
- Particularly preferred is high speed photographic film such as Polaroid #612 which has a speed equivalent to ASA 20,000.
- Polaroid #612 which has a speed equivalent to ASA 20,000.
- the intensity of light emitted from the chemiluminescent reaction obeys an inverse square relationship to distance following Lambert's Law. Therefore, if a light detector is used, detection sensitivity is optimized by placing the detector as close as possible to the reaction components or the place where the reaction occurs.
- the device is may be inserted into a complementary fitting dark chamber wherein a light detector means is proximal to the test area and distal to the sampling area of the device.
- a light detector means is proximal to the test area and distal to the sampling area of the device.
- Figure 1 depicts a basic form of the sample-test device 102 in which a sample portion and reagent portion are combined as one part.
- the first element 104 comprises an impervious material such as a plastic film having an opening 106.
- the second element 107 is an adsorbent material that has dried reagent in it.
- An area distal from the sampling portion 108 is a detection area 110. Light is preferably detected from detection area 110 although, if desired, light can also be detected from other parts of the device.
- the sampling portion is contacted with an area suspected of containing the target analyte.
- a carrier liquid (if necessary) is added to the sampling portion and carries with it any analyte into the second element, and mixes the analyte with the chemiluminescent reagent within the second element 107.
- the reagent diffuses into the detection area 110.
- dried luciferase reagent can be present throughout the second element 107 between sampling portion region 108 and detection area 110.
- the third element 112 is comprised of an impervious material such as plastic film and is light permeable at least in region 114 directly below the detection area 110.
- the light permeable region 114 can be positioned close to a light detector.
- the adsorbent material of the second element 107 is sandwiched betweenplastic layers
- Figure 2 depicts another device in accordance with this invention 202 that comprises separate sampling portion and reagent portion adsorbent materials.
- the first element 204 comprises a liquid impervious material such as a plastic film having an opening 206.
- the sampling portion 208 is comprised of fibrous adsorbent material.
- the sampling portion 208 is positioned directly under opening 206 between the top impervious layer 204 and one end 210 of a porous absorbent material 211.
- Reagent portion 212 is wider at each end and narrow in its middle (although many other configurations are possible) .
- Element 214 comprises a liquid impervious material which is light permeable, at least in the area where chemiluminescent light will be emitted from 211.
- one or more holes may be present in elements 204 and 214 near end 216 in order to facilitate the passage of fluid away from sampling portion 208.
- Figure 3 depicts another device in accordance with this invention that is useful for detecting the presence of bacteria.
- the device comprises separate sampling portion and reagent portion adsorbent materials as shown in Figure 2, but additionally comprises a carrier liquid (located in reservoir 308) and instant photographic film light detection means.
- the first element 310 comprises a liquid impervious material such as a plastic film having an opening 304 and a reservoir 308 with a surface 312 attached at flexible area (e.g., hinge) 310.
- Sampling portion 306 is comprised of fibrous adsorbent material, and is positioned directly under opening 304 between the top impervious layer 310 and one end 314 of porous absorbent material 316.
- Reagent portion 318 is wider at each end and narrow in its middle (although many other configurations are possible) .
- Another clement 320 comprises a liquid impervious material which is at least partially light permeable. Although not shown in the figure, one or more holes may be present in element 310 and 320 in order to facilitate the passage of fluid away from sampling portion 306.
- the device of Figure 3 also comprises negative film layer 322, compressing bar 324, developing gel container 326, and positive print film 328 with attached tab 330. These components allow detection of chemiluminescent light by instant photographic means.
- finger pressure is placed on backing 302 in an area behind opening 304 through which exposed adsorbent of sample portion 306 is used, for example, to wipe the surface of a test area.
- the portion of the device comprising carrier liquid reservoir 308 is folded over at flexible area 310. Pressure is applied to reservoir surface 312 which causes the reservoir to break and release the carrier liquid from the reservoir and into sample portion 306.
- the carrier liquid comprises a bacteriolytic agent that releases ATP from any bacteria present in the sampling portion 306.
- the ATP diffuses through sampling portion 306 and into absorbent material 316 at point 314.
- the solution diffuses through the body of the adsorbent strip where it rehydrates dried chemiluminescent reagent. Any ATP present in the carrier liquid reacts with the rehydrated chemiluminescent reagent present in portion 318 and light is emitted.
- Figure 4 illustrates a first embodiment of a sampling assay device 400 according to the present invention, which device is particularly adapted for use with the interface described in the second copending application.
- Figure 5 illustrates a second embodiment of a sampling assay device 400 according to the present invention, which device is particularly adapted for use with the interface described in the third copending application
- the device 400, 400' includes a housing 410 for containing a sampling strip 440, 410', which has a sampling portion 442, 442' for receiving a sample, a reading portion 444, 444' for emitting light, and a transfer portion 446, 446' for transferring the sample to the reading portion
- the housing 410 is defined by a top layer 420 and a bottom layer 430, each having an opening 422, 432 in alignment respectively with the sampling portion 442, 442' and the reading portion 444, 444' .
- Each of the top and bottom layers 420, 430 is preferably composed of a thin liquid impervious material, such as a plastic film.
- the sampling strip 440, 440' is sandwiched and retained between the top and bottom layers 420, 430, which can be sealingly joined together, for example, by heat, adhesive, ultrasonic welds, or any physical means that retain layers together while containing the sampling strip therein.
- a single film sheet can also be folded to form the upper and lower layers.
- a housing with three pre-sealed sides can also be used.
- the bottom layer 430 pref erably inc ludes a sealing film 450 positioned underneath as shown in Figures 4 and
- This film 450 is preferably light transmissive
- the film 450 could be structured so that only the region directly below the bottom layer opening 432 is clear.
- Figures 4 and 5 show the film 450 positioned below the bottom layer, it can also be positioned above the bottom layer, below the sampling strip 440, 440'.
- the sealing film 450 can be omitted altogether if the bottom layer, at least the portion below and in registry with the reading portion 444, 444', is formed of a clear material to provide a window for the reading portion In this regard, the entire bottom layer could be made of a clear material if desired. Even just the opening 432 could be covered with a light transmissive material, from either above or below the bottom layer 430.
- the sampling portion 440, 440' is positioned directly under the top-layer opening 422, sandwiched between the top layer 420 and the bottom layer 430.
- an optional sample collecting member 460 is sandwiched between the top layer 420 and the sampling portion 442, 442', with the collecting member exposed to the top layer opening 422.
- the sampling portion is thus accessible through the envelope 410.
- the collecting member is preferably adsorbent, which is preferably composed of fibrous material, such as glass fiber, cotton, dacron, or paper, and it may be porous, such as porous polyethylene or sintered glass.
- the sampling strip 440 is also composed of a similar adsorbent material, which may be fibrous, such as glass fiber, cotton, dacron, or paper and the like, and it may be porous, such as porous polyethylene or sintered glass and the like.
- adsorbent material such as glass fiber, cotton, dacron, or paper and the like, and it may be porous, such as porous polyethylene or sintered glass and the like.
- the sampling strip 440' preferably is made of a similar material as on the first embodiment, particularly a material that permits lateral liquid flow, such as a bibulous, adsorbent, or hydrophilic membrane. At least the reading portion, however, is light transmissive so that it transmits light. A poly-carbonate membrane suits this requirement.
- the transfer portion 446' and the sampling portion 442' can be made of glass fiber for example. An ordinary skilled artisan will recognize many useful materials that permit lateral liquid flow and are light transmissive.
- the sampling strip 440' also has a collecting portion 448 for absorbing excess liquid and sample components not immobilized in the reading portion 444' .
- the collecting portion preferably is made of an adsorbent or absorbent material such as paper, cellulose filter paper, etc. If the entire sampling strip is formed of the light transmissive material, such as a polycarbonate membrane, the collection portion 448 preferably includes an absorbent layer, such as cellulose paper to wick away excess liquid away from the reading portion.
- sampling and reading portions 442, 442' and 444, 444' are preferably wider than the transfer portion 446 connecting these wider portions, although many other configurations are possible.
- the sampling and reading portions can come in various shapes, including circular (as shown), rectangular, or triangular. The shape can be maximized for particular needs.
- the sample collecting member 460 if used, is preferably in physical contact with the sampling portion 442, 442' to maximize liquid transfer
- the reading portion 444 contains one or more chemiluminescent reagents, preferably in a dried form to produce a chemiluminescent reaction with the target analyte. Additionally, the reading portion can contain other reagents useful for the assay including, for example, the detergent or other bacteriolytic agent for extracting ATP from bacteria.
- chemiluminescent chemistries can be used with the present sampling device
- Acceptable chemiluminescence chemistries include, among others, the reaction of hydrogen peroxide with horseradish peroxidase labelled antibodies and luminol, enhanced horseradish peroxidase, reactions that include the use of diacylhydrazides, acridiinum salts, dioxitanes, and bioluminescent reactions involving cofactors, such as reduced nicotine adenine dinucleotide in the case of marine bacteria.
- a particularly preferred chemiluminescent chemistry is the firefly ATP assay, which utilizes luciferase and at least one cofactor to generate light from ATP present in the sample.
- Reagents may be conveniently applied as a solution and then dried or they may be applied in a dry form, such as a powder or suspension in an organic solvent or slurry other methods are known in the art and the preferred one can be determined by characteristics of the reaction components desired.
- the transfer portion 446' further has a labeling portion 447 adjacent or near by the reading portion
- This labeling portion 447 contains a luminescent (fluorescent or phosphorescent) labeling agent, such as chelated euiopium or europium compound (phosphorescent) or phycobiliproteins (fluorescent) for labeling the analyte.
- a luminescent labeling agent such as chelated euiopium or europium compound (phosphorescent) or phycobiliproteins (fluorescent) for labeling the analyte.
- the labeling portion 447 contains a conjugate (typically two molecules held together by one or more bonds) of a luminescent reporter molecule (agent) and a first binding agent that binds the target analyte.
- the luminescent agent can be either fluorescent such as, for example, fluorescein or a phycobiliprotein, or phospnorescent, such as a chelated europium or europium compounds.
- the "reporter molecule” for purposes of this invention can include larger associations of molecules and atoms such as liposomes filled with fluors or enzymes, and particles such as gold or polystyrene coated with luminescent molecules.
- the first binding agent binds to the target analyte in a solution.
- a preferred first binding agent is an antibody although other substances that can bind a particular antigen such as, for example, protein A (antibody antigen), lectin (glycolipid or glycoprotein antigen), streptavidin (antibody antigen) , avidin (biotin target analyte) , and hormone or trophic factor (cell surface receptor) .
- the first binding agent and luminescent reporter molecule are conjugated by any of a number of techniques known to an ordinary skilled artisan.
- the conjugate formed from a first binding agent and a luminescent reporter molecule is placed into the labeling portion 447 by preparing a solution of the conjugate by spotting the solution onto labeling portion 447. The solution is then dried.
- a carrier protein such as bovine serum album or milk
- Detergent such as TWEEN-20 or TRITON X-100 can also be included to prevent non-specific binding of the conjugate to the labeling portion.
- the labeling agent does not spontaneously react with the target analyte, but rather attaches thereto and glows when it is exposed to light. It thus needs to be triggered. This advantageously enables selective light measurement to be taken at anytime between 2 to 60 minutes after sampling.
- the sample in liquid such as carrier liquid or buffer
- the labeling agent which has a specific binding agent complementary to the target analyte.
- the labeling agent will attach only to the target analyte.
- the carrier liquid now with the tagged or labeled target analyte (and any excess labeling agent not tagged with the target analyte) continues into the reading portion.
- the reading portion 444' contains a second binding agent, which could be same as the first, such as an antibody or antigen complementary to the target analyte to capture the labeled analyte within the reading portion. This time, however, the second binding agent is bound to the base, the light transmissive member, so that it does not flow out of the reading portion. Thus, the labeled analyte remains immobilized within the reading zone.
- a second binding agent which could be same as the first, such as an antibody or antigen complementary to the target analyte to capture the labeled analyte within the reading portion.
- the second binding agent functions to bind to the target analyte and prevent or significantly slowing its movement.
- the second binding agent is preferably an antibody but any substance that can bind the target analyte, is suitable.
- the primary difference between the second binding agent and the first binding agent is that the second binding agent is immobilized to, for example, the polycarbonate member such that the target analyte that flows over or through the polycarbonate can react with the second binding agent to become immobilized there.
- Immobilizing the second binding agent can be achieved by a number of methods known to the ordinary skilled artisan.
- the second binding agent can be indirectly immobilized through an intermediary material such as latex articles or derivatized glass particles, which are commercially available.
- an antibody can coat latex and derivatized particles by non-specific absorption, followed by washing and blocking with a second protein in excess, such as bovine serum albumin.
- the coated latex or glass beads can then be immobilized within the polycarbonate membrane by applying the latox or glass suspension to the polycarbonate membrane in a solution form.
- the collecting portion 448 absorbs excess liquid containing the labeling agent that has not been coupled to the second binding agent in the reading portion.
- Acceptable carrier liquids include, among others, a buffer solution or a buffer solution with detergent. Buffer solutions neighboring in the pH range of 5-10, and more preferably 6-8 (neighboring neutral pH) and compatible with the labeling agent can be used, such as TRIS, HEPES.
- Detergent which is preferably present in the carrier liquid, dissolves in liquid that is added to the sampling device and serves to improve flow as surfactants at one concentration, and if necessary, to solubilize the cell wall or organism for release of antigen or the element to be detected.
- suitable detergents or combination of detergents are known to those skilled in the art and include, nonionic detergents such as TRITON X-100 and NONIDET P40.
- the concentration of detergent solution varies for each type of detergent and can range from 0 01% to 6%, and preferably from 0.5% to 1.0%.
- carrier liquid or buffer is preferably introduced into the sampling portion and preferably includes an agent for facilitating the detection.
- the agent releases ATP from any bacteria present in the sampling portion
- Acceptable carrier liquids include, among others, a buffer solution or a buffer solution with detergent.
- Buffer solutions of TRIS, HEPES buffers at pH 7.0 to 9.0, and most preferably HEPES buffer at 7.8 with EDTA are preferred when used with firefly luciferase from Photinus pyral i s (first embodiment) .
- EDTA is a preferred ingredient because ATP degrading enzymes require divalent metal cations for activity and EDTA chelates these.
- the pH of the chemiluminescent reaction determines the ratio of the 562 nm peak to the 616 nm peak emission associated with firefly luciferasefrom photinuspyralis. At pH5.4, the
- 616 nm peak is at a maximum and there is no 562 nm peak.
- the emission spectra change with pH due to the protonation of the oxyluciferin molecule (the light emitter) .
- Oxyluciferin as a dianion emits a yellow-green light (562 nm) and as a monoanion it emits a red light (616 nm) .
- detergent also can be present in the sampling or reading portion or included with the carrier, which detergent dissolves in liquid that is added to the sampling device and serves to open cells and liberate cell components.
- suitable detergents or combination of detergents include, nonionic detergents such as Triton X-100, Nonidet P40, n-Undecyl Beta-D glucopyranoside, zwitterionic detergents such as n-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, and cationic detergents such as alkyltrimethylammonium bromides, benzalkonium chloride, cetyldimethyl-etiylammonium bromide, dodecyltrimethylammonium bromide and cetyltrimethylammonium bromide.
- nonionic detergents such as Triton X-100, Nonidet P40, n-Undecyl Beta-D glucopyranoside
- concentration of detergent solution varies for each type of detergent and can range from 0.1% to 6%, and preferably from 0.5% to 2.0%.
- acceptable carrier liquids include, among others, a buffer solution or a buffer solution with detergent.
- Buffer solutions neighboring in the pH range of 5-10, and more preferably 6-8 (neighboring neutral pH) and compatible with the labeling agent can be used, such as TRIS, HEPES
- detergent which is preferably present in the carrier liquid, dissolves in liquid that is added to the sampling device and serves to improve flow as surfactants at one concentration, and if necessary, to solubilize the cell wall or organism for release of antigen or the element to be detected.
- suitable detergents or combination of detergents include, nonionic detergents such as Triton X-100 and Noni ⁇ et P40.
- the concentration of detergent solution varies for each type of detergent and can range from 0 01% to 6%, and preferably from 0.5% to 1.0%.
- a chield 470 is attached to or made integral with the top layer 420.
- the shield preferably extends beyond the proximal end 424 of the sampling device 400 and preferably has a preformed crease or fold 472 in line with the proximal end so that a portion 474 extending beyond the proximal end 424 can easily be tucked un ⁇ erneath the bottom layer 430 or the sealing film 450, as shown in Figure 6.
- the sampling device preferably comes packaged in a sealed envelope with the shield in the folded condition as shown in solid This will maintain the shield 470 in place when seated in the tray, for example, even when the tray is opened
- the shield 470 also is preferably composed of a thin liquid impervious material, such as a plastic film and it can be attached A the top layer by any conventional means, such as heat (melding), adhesive, ultrasonic welds, etc.
- the attachment area 476 forms a hinge 477, which is defined by the attachment area demarcation line to allow the shield to move or pivot thereabout. If the hinge is made of a relatively rigid material, the hinge should be made so that it preferably does not have any positional retention. Sometimes, however, it may be useful to bias the shield toward the closed position A as shown in Figure 6, where the shield is substantially parallel to the housing 410. This may be achieved by playing desired creases or folds, even along the hinge 477.
- a tab or handle 480 is attached or formed integral with the shield to ease removal of the sampling device 400, 400' from a tray for example.
- the tab also can be formed of the same material as the shield.
- the tab can be attached to the shield 470 or even to the top layer 420. It is, however, preferable to attach the tab with the shield, more preferably to the attachment area 476 as shown.
- the tab and the shield are preferably attached together in a single step, for instance by ultrasonically welding these members to the top layer 420 before the sampling strip 440, 440' is sandwiched between the top and bottom layers.
- the tab is preferably biased in the upright position, away from the upper layer or the shield as shown in Figures 4-6.
- the sampling device 400, 400' preferably comes packaged in another sealed container or envelope (not shown) . Once the sampling device 400, 400' is taken out, sampling can begin.
- the shield 470 is moved back from the position A toward the distal end 426 of the sampling device, toward the position B shown in Figure 6 to expose the sample collecting member 460.
- the collecting member is then exposed to the target area, for instance, by contacting or swabbing the suspected surface, liquid, or other area suspected of containing the analyte with the member 460.
- the sample to be tested can be directly introduced to the sample collecting member 460.
- carrier liquid is introduced to the member, if needed, to wet the target analyte and move any analyte present to the reading portion.
- the carrier liquid may not be necessary but is preferred.
- the degree of luminescence light produced in response to the presence of target analyte in tne sample is detected, using for example the interface disclosed in the second and third copending applications, where the degree of light detected correlates to the amount of analyte present.
- the target analyte can be introduced by physical contact such as by swabbing a suspected contaminated surface with the device or by introducing the sample in liquid form by, for example, an eye dropper or other dispenser, or by brief immersion of the device in the liquid to be tested.
- finger pressure is preferably placed on the bottom layer 430, behind the opening 422 to more fully expose the sample collecting member 460 to the sample.
- the applied carrier liquid diffuses through the sampling strip, where it, upon reaching the reading portion, rehydrates the dried chemiluminescent reagent contained therein. Any ATP present in the carrier liquid reacts with the rehydrated chemiluminescence reagent present in the reading portion to spontaneously emit light.
- the applied carrier liquid carrying the sample diffuses through the transfer portion and passes through the labeling portion, which contains luminescent agent attached to a binding agent that is complementary with the target analyte.
- the liquid releases the labeling agent, which attaches only to the target analyte.
- the binding agent captures the target analyte, which carries the labeling agent, while the non-complementary elements, including excess labeling agent, (excess) pass through the reading zone
- the collection portion 448 helps to wick out the excess from the reading portion 444'.
- the chemiluminescent reaction in the first embodiment provides a spontaneous light emission upon mixing the chemiluminescent reagent with the target analyte
- the luminescent reaction in the second embodiment does not produce light until it is first triggered with light exposure.
- the labeling agent associated with the captured target analyte glows or emits light when exposed to light.
- the amount of analyte present can be calculated based on the amount of light exposed and collected thereafter. Accordingly, the reading phase can advantageously take place at a later time, about between 2
- luminescence light produced in response to the presence of target analyte in the sample can be triggered and read, as described in the third copending application.
- the shield 470 is placed over the sampling portion, with the extending portion 474 folded below the bottom layer 430 or the sealing film 450 if used, as shown by the position A in Figure 6. This prevents the sampling portion from accidentally receiving any additional sample or contamination.
- the sampling device 400 can then be positioned in the interface Because the shield is implemented, any accidental cross-contamination between samples within the interface is eliminated. After the reading is completed, the sampling device can easily be removed by conveniently grabbing the tab and lifting the sampling device out of tray.
- Luciferase-luciferin (Analytical Luminescence Systems) is reconstituted with a solution of 5% alpha-D-Glucopyranosyl alpha-D-glucopyranoside in 0.05M Dithiothreitol. Fifteen microliters of the Luciferase-luciferin solution are pipetted at approximately 15mm from the distal end of the filter paper strip and dried in vacuo.
- An adhesive coated plastic is cut into a 12.5mm ⁇ 40mm strip.
- a 8.0mm diameter hole is punched out at the proximal end of the strip.
- the perimeter of the hole is centrally located approximately 3 5mm from the proximal end of the strip.
- An adnesive coated translucent plastic strip is cut into a 12.5mm ⁇ 40mm strip. With the adhesive side of the translucent plastic strip facing up, the filter paper strip is positioned wherein the edge of the distal end of the filter paper is 2.5mm from the distal end of the translucent plastic strip and the proximal portion of the filter paper is 2.5mm from the proximal end of the translucent strip.
- a plug of dacron fibers approximately 10mm in diameter is centered close to but not extending beyond the edge of the proximal portion of the filtei paper strip.
- the adhesive coated plastic strip with adhesive side facing downward is positioned directly over the translucent strip and oriented so that the punched hole is directly over the dacron plug Pressure is applied to the device to seal the adhesive backings together.
- An individual Polaroid film packet (#612 ISO 20,000) is cut down into a 12 5mm ⁇ 20mm rectangle and glued to the proximal undersurface of the device, becoming an integral part of the device forming a dark chamber wherein the photographic film is adjacent to the test portion of the filter paper.
- the device is held between the fingers with dacron side facing the surface to be monitored for bacteria. Finger pressure is applied on the back side of the device directly behind the sampling area, and the dacron sampling portion is used to wipe a defined area
- an extraction buffer that contain 0.05M HEPES buffer pH 7.8 and 0.5% Cetyldimethyl-ethylammonium bromide is added to the punched out hole containing the dacron sampling area.
- the extracted ATP if present, diffuses across the porous filter, the luciferase-luciferin reagent dissolves, and light is emitted as the solution flows across the distal portion of the filter paper.
- a film is exposed to light emitted over a given period of time at the test area. After 2 minutes, the film is developed and peeled back from the device to reveal a white spot if positive, or no white spot if negative.
- luciferase and luciferin were separately applied to the paper strip.
- Three ug Firefly Luciferase dissolved in 8 microliters of 0.05M HEPES buffer pH 7.8, 0.025M dithiothreitol, 0 05% Triton X-100 as described above were applied and dried onto the distal ends of 10mm by 35mm cellulose filter paper strips (Whatman #1)
- Three ug of sodium D- luciferin (Sigma Chemical Company, St. Louis, Mo.) in water were applied and dried onto the proximal ends of each strip.
- the amount of relative light units emitted were integrated for 10 seconds and background values were subtracted.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96940503A EP0882230A4 (en) | 1995-11-17 | 1996-11-18 | Chemiluminescent assay methods and devices for detecting target analytes |
AU77367/96A AU7736796A (en) | 1995-11-17 | 1996-11-18 | Chemiluminescent assay methods and devices for detecting target analytes |
JP9516901A JP2000500568A (en) | 1995-11-17 | 1996-11-18 | Chemiluminescence analysis method and analyzer used for detection of an analyte |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/560,094 | 1995-11-17 | ||
US08/560,094 US5783399A (en) | 1995-11-17 | 1995-11-17 | Chemiluminescent assay methods and devices for detecting target analytes |
US57762495A | 1995-12-22 | 1995-12-22 | |
US08/577,624 | 1995-12-22 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997019353A1 true WO1997019353A1 (en) | 1997-05-29 |
Family
ID=27072247
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/018443 WO1997019353A1 (en) | 1995-11-17 | 1996-11-18 | Chemiluminescent assay methods and devices for detecting target analytes |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0882230A4 (en) |
JP (1) | JP2000500568A (en) |
AU (1) | AU7736796A (en) |
WO (1) | WO1997019353A1 (en) |
Cited By (11)
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FR2781056A1 (en) * | 1998-07-08 | 2000-01-14 | Draeger Sicherheitstech Gmbh | TEST STRIP FOR THE DETECTION OF SUBSTANCES, DETECTION METHOD AND USES |
WO2001081618A1 (en) * | 2000-04-26 | 2001-11-01 | Kikkoman Corporation | Method of measuring biological luminescence |
GB2422663A (en) * | 2005-01-27 | 2006-08-02 | Martyn Johnson-Townley | Device for surface assay |
WO2020006530A3 (en) * | 2018-06-29 | 2020-04-30 | International Paper Company | Chemiluminescent wetness indicator for absorbent products |
US11008326B2 (en) | 2018-06-29 | 2021-05-18 | International Paper Company | Synthesis of coelenterazine |
US11078200B2 (en) | 2018-06-29 | 2021-08-03 | International Paper Company | Synthesis of coelenterazine |
WO2021176101A1 (en) * | 2020-03-06 | 2021-09-10 | Biosynth Ag | Luciferase-based methods for detecting bacterial and fungal cells and assessing susceptibility of bacterial cells to antibiotics |
US20210405012A1 (en) * | 2018-12-14 | 2021-12-30 | Luminultra Technologies Ltd. | Portable system for analysing microbial population in a fluid |
US11365140B2 (en) | 2017-10-31 | 2022-06-21 | Luminultra Technologies Ltd. | Decision support system and method for water treatment |
US11505787B2 (en) | 2014-10-16 | 2022-11-22 | International Paper Company | Chemiluminescent wetness indicator for absorbent products |
US11619628B2 (en) | 2017-02-03 | 2023-04-04 | Osaka University | Device and determination system using same |
Families Citing this family (3)
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JP5001611B2 (en) | 2006-09-13 | 2012-08-15 | 新日本製鐵株式会社 | Method for producing high magnetic flux density grain-oriented silicon steel sheet |
JP5332178B2 (en) * | 2007-11-02 | 2013-11-06 | 東洋紡株式会社 | Method for detecting multicolor luciferase |
WO2024019165A1 (en) * | 2022-07-22 | 2024-01-25 | キッコーマン株式会社 | Method and kit for detecting microorganism or cell, or detecting microorganism related substance or cell related substance |
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US4863689A (en) * | 1987-04-23 | 1989-09-05 | University Of Victoria | Apparatus for the detection of chemiluminescence |
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US4806311A (en) * | 1985-08-28 | 1989-02-21 | Miles Inc. | Multizone analytical element having labeled reagent concentration zone |
US4916056A (en) * | 1986-02-18 | 1990-04-10 | Abbott Laboratories | Solid-phase analytical device and method for using same |
US5075078A (en) * | 1989-10-05 | 1991-12-24 | Abbott Laboratories | Self-performing immunochromatographic device |
US5141850A (en) * | 1990-02-07 | 1992-08-25 | Hygeia Sciences, Inc. | Porous strip form assay device method |
US5354692A (en) * | 1992-09-08 | 1994-10-11 | Pacific Biotech, Inc. | Analyte detection device including a hydrophobic barrier for improved fluid flow |
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1996
- 1996-11-18 WO PCT/US1996/018443 patent/WO1997019353A1/en not_active Application Discontinuation
- 1996-11-18 EP EP96940503A patent/EP0882230A4/en not_active Withdrawn
- 1996-11-18 AU AU77367/96A patent/AU7736796A/en not_active Abandoned
- 1996-11-18 JP JP9516901A patent/JP2000500568A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US4863689A (en) * | 1987-04-23 | 1989-09-05 | University Of Victoria | Apparatus for the detection of chemiluminescence |
Non-Patent Citations (1)
Title |
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See also references of EP0882230A4 * |
Cited By (17)
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DE19830405A1 (en) * | 1998-07-08 | 2000-01-27 | Draeger Sicherheitstech Gmbh | Immunoassay test strip for detecting drugs, low molecular pollutants, etc. |
DE19830405C2 (en) * | 1998-07-08 | 2000-09-21 | Draeger Sicherheitstech Gmbh | Test strips for immunochemical substance detection |
US6228658B1 (en) | 1998-07-08 | 2001-05-08 | Drager Sicherheitstechnik Gmbh | Method of using a test strip for the immunochemical detection of substances |
FR2781056A1 (en) * | 1998-07-08 | 2000-01-14 | Draeger Sicherheitstech Gmbh | TEST STRIP FOR THE DETECTION OF SUBSTANCES, DETECTION METHOD AND USES |
WO2001081618A1 (en) * | 2000-04-26 | 2001-11-01 | Kikkoman Corporation | Method of measuring biological luminescence |
GB2422663A (en) * | 2005-01-27 | 2006-08-02 | Martyn Johnson-Townley | Device for surface assay |
US11505787B2 (en) | 2014-10-16 | 2022-11-22 | International Paper Company | Chemiluminescent wetness indicator for absorbent products |
US11619628B2 (en) | 2017-02-03 | 2023-04-04 | Osaka University | Device and determination system using same |
US11365140B2 (en) | 2017-10-31 | 2022-06-21 | Luminultra Technologies Ltd. | Decision support system and method for water treatment |
US11078200B2 (en) | 2018-06-29 | 2021-08-03 | International Paper Company | Synthesis of coelenterazine |
US11008326B2 (en) | 2018-06-29 | 2021-05-18 | International Paper Company | Synthesis of coelenterazine |
WO2020006530A3 (en) * | 2018-06-29 | 2020-04-30 | International Paper Company | Chemiluminescent wetness indicator for absorbent products |
US11926624B2 (en) | 2018-06-29 | 2024-03-12 | International Paper Company | Synthesis of coelenterazine synthesis intermediate |
US11939332B2 (en) | 2018-06-29 | 2024-03-26 | International Paper Company | Synthesis of coelenterazine |
US20210405012A1 (en) * | 2018-12-14 | 2021-12-30 | Luminultra Technologies Ltd. | Portable system for analysing microbial population in a fluid |
EP3894841A4 (en) * | 2018-12-14 | 2022-09-28 | Luminultra Technologies Ltd. | Portable system for analysing microbial population in a fluid |
WO2021176101A1 (en) * | 2020-03-06 | 2021-09-10 | Biosynth Ag | Luciferase-based methods for detecting bacterial and fungal cells and assessing susceptibility of bacterial cells to antibiotics |
Also Published As
Publication number | Publication date |
---|---|
EP0882230A1 (en) | 1998-12-09 |
JP2000500568A (en) | 2000-01-18 |
EP0882230A4 (en) | 2001-05-02 |
AU7736796A (en) | 1997-06-11 |
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