WO1997005209A1 - Procede d'accroissement de la chimioluminescence - Google Patents

Procede d'accroissement de la chimioluminescence Download PDF

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Publication number
WO1997005209A1
WO1997005209A1 PCT/US1996/012300 US9612300W WO9705209A1 WO 1997005209 A1 WO1997005209 A1 WO 1997005209A1 US 9612300 W US9612300 W US 9612300W WO 9705209 A1 WO9705209 A1 WO 9705209A1
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Prior art keywords
luminescent
compound
ofthe
detergent
enhancer
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PCT/US1996/012300
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English (en)
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David E. Kohne
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Kohne David E
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Priority to AU66003/96A priority Critical patent/AU6600396A/en
Publication of WO1997005209A1 publication Critical patent/WO1997005209A1/fr

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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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • Luminescent molecules have been incorporated into a variety of art recognized methods designed to determine the presence, absence and/or concentration of an analyte, a substance whose presence, absence and/or concentration is being determined.
  • art recognized methods include enzyme assays, immunoassays, nucleic acid probe assays, chemical assays, and other chemical probe assays. Many of these are described or mentioned in the books cited above.
  • Luminescent molecules can be used in a variety of ways in these detection and/or quantitation assays.
  • inert luminescent molecules are directly attached to a complex formed between the analyte molecule of interest and a molecular entity designed to specifically detect and complex with the analyte molecule of interest.
  • From one to many (1-100) inert luminescent molecules can be directly associated with the analyte in such a complex.
  • a separation step or its equivalent is required to remove or inactivate any uncomplexed luminescent molecules that are not specifically associated with the analyte molecule of interest.
  • the complexed luminescent molecules remaining in the test vessel are then triggered in some way to emit light, and the light is detected and/or quantitated.
  • Examples of a variety of "direct" label tests are present in the Proceedings of the International Symposiums on Bioluminescence and Chemiluminescence cited above, as well as in the CRC Press volume entitled Luminescence Immunoassay and Molecular Applications, Van Dyke and Van Dyke (eds.), CRC Press, Inc., 1990.
  • Table 1 shows a listing of the prior art regarding the use and enhancement ofthe luminescence of various known luminescent compounds.
  • An alternate method combines the amplification properties of an enzyme with a luminescent signal molecule to produce detection systems with ultrasensitive detection potential.
  • the test process results in the direct association or "labeling" of an analyte-specific detection entity, such as an antibody, with one or more enzyme molecules.
  • a separation step is normally required to remove any enzyme molecules that are not specifically associated with the analyte.
  • a luminescently inert enzyme substrate is then added.
  • the substrate possesses an enzyme-cleavable chemical group which prevents the substrate molecules from emitting light until they are activated by contact with the enzyme. Removal of this specific group by the enzyme produces a luminescent product molecule that can emit light.
  • the product molecule begins to emit light immediately, while with other substrate types, the product molecule requires a subsequent triggering step to initiate emission of light from the product molecule. Examples of such tests are described in the references quoted above.
  • An alternate strategy can be used.
  • An enzyme specifically associated with an analyte-specific recognition complex is reacted with a specific substrate to generate product molecules.
  • product molecules are not capable of luminescing, but interact with other molecules to initiate emission of light.
  • Tanaka and Ishikawa An example of such a test is described by Tanaka and Ishikawa (Analytical Letters 19 (3 and 4) 433, 1986) and is summarized in Table 2.
  • enhancement is used in many contexts in the scientific and patent literature including: enhancement of chemiluminescence; enhancement of bioluminescence; enhanced generation of light from bioluminescent or chemiluminescent molecules; enhancing chemiluminescent or bioluminescent efficiencies; enhanced luminescent quantitation; enhanced chemiluminescent reactions; enhanced chemiluminescent immunoassay; improved enhancers of chemiluminescence; enhanced sensitivity of a chemiluminescent reaction; an enhanced luminometric assay; and others.
  • An increase in the light intensity of a luminescent system which can be due to: a. an increase in the QY ofthe luminescent molecules. b. a decrease in the decay time of light emission from the luminescent molecules. c. a larger proportional increase in the QY than in the decay tl/2 or a smaller proportional decrease in QY than in the decay tl/2. d. an increase in the number of luminescent molecules which are present at the time of light measurement; this can be due to
  • An increase in the total light emitted from a luminescent system which can be due to: a. an increase in QY. b. an increase in the rate at which an enzyme processes a substrate to produce luminescent product molecules. c. an increase in the number of luminescent molecules present.
  • a decrease in the background light emission of a luminescent system which can be due to: a. an increase in the stability or purity ofthe luminescent molecule, the luminescent substrate, or some other component ofthe luminescent system. b. a decrease in background light emission from the luminescent molecule and/or substrate and/or contaminant and/or some other component ofthe luminescent system. c. a decrease in the luminescent substrate concentration and/or other components which contribute to background light emission.
  • An improvement in the signal/background(S/B) ratio ofthe luminescent system which can be due to: a. an increase in the S/B ratio; such increase would result in an increased detection sensitivity (a lower limit of detection) for the luminescent system. b. an equivalent increase of both signal and background; such increase would result in statistically better S/B values and could significantly contribute to the ease of use ofthe test. c. an improvement in the ability to obtain reproducible background values of light emission and to obtain reproducible light signals on replicate samples containing the same quantity of molecule; i.e. an improvement in the precision of replicate results. Such improvements can increase the reliability ofthe overall test and improve the useful sensitivity of detection by reducing the upper limit for variant signals. TABLE 4
  • Enhancers are present in:
  • Vargula Arthropod luciferin Low -0.3 Luciferase substrate (MW -6.8x10 4 )
  • QY quantum yield
  • luciferase an enzyme which interacts with a luminescent substrate, luciferin, and very efficiently enhances light emission.
  • the QY of this reaction is reported to be -0.9, or almost unity. This is the highest QY known for a bioluminescent system.
  • the enzyme luciferase makes possible this high QY by complexing with the substrate luciferin in such a manner as to create a nearly ideal environment for converting electronic energy to photons.
  • the environment created is a highly hydrophobic environment that essentially excludes water.
  • This basic theme providing a luminescent molecule with a hydrophobic environment by complexing it with a biological macromolecule, has been repeated many times by nature (See examples in Table 4).
  • the same basic strategy of providing a luminescent molecule in aqueous solution with a more hydrophobic environment is the basis for most methods used to increase the QYs of luminescent molecules in aqueous solution. Such methods involve associating the aqueous solution with regions of higher hydrophobicity, with the result that the luminescent molecules present in aqueous solution associate with these regions of higher hydrophobicity and can then produce light more efficiently.
  • Such a higher hydrophobic region can consist of : (a) compounds normally considered to be soluble in water, such as detergents; macromolecular compounds, such as proteins; and commercially available enhancers, such as polymeric quaternary ammonium salts; (b) solid materials, insoluble in water but in contact with water, such as various types of polymers, for example, nylon and other polymeric substances; and (c) other chemical compounds of various types.
  • Tables 5 and 6 provide examples of prior art enhancing substances. These include water soluble and solid phase substances that enhance the QY in an aqueous environment.
  • the change in decay 1 1/2 may not affect the measured light intensity. For instance, a tenfold increase in the decay t l/2 from 10 "3 seconds to IO "2 seconds, would not appreciably affect a light intensity measurement of one second duration, since in both cases the light emission is essentially complete in one second. Similarly, a tenfold decrease in the decay 1 1/2 from IO "3 seconds to IO "4 seconds will result in little, if any, change in the light intensity measurement one second long. Again, this assumes only the decay t l/2 has changed and all other things remain unchanged.
  • the light intensity will change with a decay 1 1/2 change.
  • a twofold increase in the decay t 1/2 from one second to two seconds results in a light intensity decrease of about 1.7 fold.
  • a twofold decrease in the decay tl/2 from one second to 0.5 seconds results in a light intensity increase of about 1.5 fold.
  • Table 7 presents examples of prior art substances which decrease the halftime of decay of light emission of particular luminescent molecules. As discussed above, an increase in light intensity will be observed only if an appropriate light measurement duration is utilized.
  • Table 8 presents the effect of pH on the emission decay tl/2 and the light emission intensity of AMP ' D anion. Since changing the pH has little effect on the QY of AMP ' D (Kohne, unpublished data), Table 8 also indicates the effect of a changing decay tl/2 on the light emission intensity.
  • a molecule must become electronically excited before it can emit light.
  • a luminescent molecule can be viewed as possessing excess energy.
  • Such molecules in an excited state are unstable, with the degree of instability depending on the type of luminescent molecule involved, and the environment in which the excited molecule exists. Because the luminescent molecules is unstable, the excess energy must be eliminated. Elimination can happen by a variety of different mechanisms or pathways. At least one of these pathways results in the conversion of the excess energy to a photon, which is emitted from the molecule. Generally, only a fraction ofthe luminescent molecules actually emit a photon. The size ofthe fraction depends upon the type of luminescent molecule and its environment.
  • the time required for photon emission from a population of luminescent molecules also depends upon the type of molecule and its environment.
  • the kinetics of photon emission from a population of luminescent molecules are first order in form and are usually expressed in terms ofthe photon or light emission decay tl/2. Under specified conditions the half time of decay of a population of luminescent molecules is characteristic ofthe type of molecule.
  • Chemiluminescent Molecule AMP ' D anion (other 1,2 dioxetanes behave similarly).
  • a primary factor determining the number of luminescent 1,2-dioxetane molecules in solution at any one time is the pH of the solution.
  • the state of ionization of a particular constituent chemical group ofthe 1,2-dioxetane molecule determines whether the molecule is electronically excited and therefore capable of emitting light.
  • the enzymatic product of a dioxetane substrate is an example of an electronically excited luminescent compound.
  • the dioxetane substrate AMPPD serves as a substrate for an enzyme, alkaline phosphatase, which removes the phosphate from the substrate, leaving behind a dephosphorylated phenolic anion AMP ' D (see Figure 1).
  • AMFD which is electronically excited and therefore unstable, is capable of decomposing and emitting a photon with a light emission decay tl/2 of about 3.7 minutes in a specified aqueous solution (0. IM DEA buffer, pH 10).
  • the product of the dephosphorylation is AMP ' D, an anion which is electronically excited and will, in time, emit light.
  • the pH i.e. the H + concentration
  • the pH at which the dephosphorylation of AMPPD occurs determines whether the AMP ' D will remain in the electronically excited anionic state.
  • AMPDH Figure 1
  • essentially all of the AMP ' D formed will immediately interact with a proton and be converted to AMPDH ( Figure 1), which is stable (not electronically excited) and therefore incapable of emitting light.
  • AMPDH Figure 1
  • essentially all ofthe AMFD will remain in the anionic form and begin light emission ( Figure 1).
  • At an intermediate pH only a fraction ofthe AMP ' D will be converted to AMPDH.
  • a very large and predictable increase in light intensity can be obtained by first dephosphory lating the AMP ' D at a low pH where essentially all ofthe AMP ' D is immediately converted to AMPDH; and then adjusting the solution to a pH where a significant portion ofthe AMPDH is converted to AMP ' D. This conversion would occur almost instantly.
  • the largest degree of enhancement will be obtained by adjusting the solution to a high enough pH so that essentially all ofthe AMPDH is converted to AMP ' D.
  • Table 10 presents the fraction of AMP ' D and AMPDH present over a pH range of 5- 12.
  • Dephosphorylating at pH 5 (which can be done with an acid phosphatase) and then adjusting the pH from 5 to 11 results in about a IO 4 increase in the amount of AMP ' D present, and causes a very large increase in light intensity.
  • Adjusting the pH from 8 to 11.3 results in an increase of about 11-fold in AMP ' D concentration and a 20-fold increase in light intensity; while adjusting the pH from 10 to 13.6 results in a 1.4-foId increase in light intensity.
  • pH has a large effect on light intensity, and the rough magnitude of enhancement due to a pH change can be predicted.
  • any aqueous solution containing AMP ' D and AMPDH can result in an enhancement ofthe light intensity, as discussed above, if raising the pH results in a significant increase in the ratio of AMP ' D anion concentration over AMPDH concentration (and an increase in light emission) for that solution.
  • the observed half time of photon emission is shortened by roughly the factor of enhancement.
  • the half-time of photon emission is the time necessary for emission of one half of the total number of photons that will be released by the solution over time.
  • the half-time of photon emission is also the time necessary for one half of the total population of molecules in the solution (i.e., AMP ' D and AMPDH molecules) to become activated and decay to the ground state.
  • the half-time of light emission for AMP ' D anion at pH 13, a pH where very little AMPDH can be present, is about 3 minutes at 25 °C in aqueous solution in the absence of enhancers of any kind.
  • LUMINESCENT ENHANCEMENT CAN BE DUE TO A FAVORABLE SHIFT IN THE WA VELENGTH OF LIGHT EMITTED FROM THE L UMINESCENT SYSTEM. Any particular luminescent molecule will emit photons with an emission spectrum characteristic ofits type.
  • the data concerning detection and quantitation of photons emitted from luminescent molecules is generally obtained using a photon counting luminometer , such as Optocomp 1 model (MGM instruments, Inc., Hamden, CT), in which the photons are detected by a photomultiplier tube (PMT).
  • PMT photomultiplier tube
  • a PMT has a characteristic spectral response, and detects photons of certain wavelengths
  • a particular luminescent molecule will emit photons with an emission spectrum characteristic ofits type. Any directed change that results in an emission spectrum that better matches the spectral response ofthe PMT can result in an increased light intensity.
  • the present invention relates to a method for obtaining increased luminescent enhancement from luminescent systems. It has been found that the luminescent enhancement obtained from art known luminescent systems can be significantly increased by the inco ⁇ oration of one or more detergent compounds into the system at some time before the light measurement.
  • the present invention provides a method to detect lesser amounts of luminescent molecules in solution or on a solid surface, and allows significant enhancement of luminescence from luminescent molecules over previous art methods.
  • the present invention has broad application in any area where a signal generation system is required, including medical, veterinary, agricultural, and industrial diagnostics and quality control.
  • the invention has application in any area where it is desirable to use a signal generating system in any assay designed to detect and/or quantitate the presence of any analyte, including industrial and pharmaceutical compounds of all types as well as biological compounds and organisms of all types, including proteins, carbohydrates, nucleic acids and lipids, bacteria and viruses.
  • the present invention can be used in concert with other technical advances to detect 10-30 enzyme molecules in less than one hour.
  • These signal generation and detection systems are readily adaptable to a variety of diagnostic assay formats.
  • This capability along with the capability to readily achieve levels of detection as low as 10-30 enzyme molecules, has great utility in many areas, including medical and industrial diagnostics and quality control, and further provides the opportunity to greatly reduce the diversity of signal generation and detection technologies required to meet the needs of clinical and other diagnostic tests.
  • Figure 1 is a schematic illustration ofthe process ofthe formation of a 1,2-dixoetane luminescent molecule from AMPPD substrate, its subsequent decomposition, and emission of a photon.
  • QUANTUM YIELD the ratio of (the total number of photons emitted by a population of luminescent molecules) over (the total number of luminescent molecules present in the population). If a total of 10 photons are emitted from 1000 luminescent molecules the QY is 0.01.
  • LIGHT EMISSION DECAY HALF TIME OF LUMINESCENT MOLECULES (decay tl/2): the time required for emission of one-half of the total light which will be released. The light emission kinetics are first order in form. This is also the time required for one-half of the excited unstable chemiluminescent molecules to decompose to a non-excited stable state. STABILIZATION OF LUMINESCENT MOLECULES: the ability to make unstable luminescent molecules more stable so that fewer of these molecules will be lost or decay with time.
  • Detergents or surface active agents or amphipathic compounds, are characterized as having an amphipathic nature. Therefore as used herein, the term "detergent” is synonymous with and encompasses all surface active agents and amphipathic compounds. A part of each detergent molecule is solvent-loving, while a different part of such a detergent is solvent-hating. When water is the solvent, one part of the detergent molecule is water-attracting; while another portion of same molecule is water-repelling. Detergents are most commonly characterized on the basis ofthe nature of their hydrophillic or water soluble groups. On this basis there are four separate classes of detergents or surface active agents:
  • detergents include compounds that are not normally regarded as detergents, but which have detergent-like, that is, amphipathic properties. These compounds can also be used in the practice of the invention.
  • Example 4 presents results obtained with a commonly used buffer compound, CHES [2-(N- cyclohexylamino)-ethanesulfonic acid]. This buffer compound has a pK of 9.3 at 25 °C in aqueous solution.
  • LUMINESCENT OR LIGHT INTENSITY a measure ofthe rate of photon emission from luminescent systems.
  • BDMQ Poly[vinylbenzyl(benzyldimethyl-ammonium chloride)]; a polymeric (Sapphire I) enhancer
  • TBQ Poly[vinylbenzyl(tributyl-ammonium chloride)]; a polymeric enhancer
  • AMPPD Disodium 3-(4-methoxyspiro ⁇ l,2-dioxetane-3 ,2"- tricyclo[3.3.1.1 3 ' 7 ] decan ⁇ -4-yl) phenyl phosphate
  • CDP Disodium 3-chloro-5-(4-methoxyspiro ⁇ l,2-dioxetane-3,2'-(5'chloro)- tricyclo[3.3.1.1 3,? ] decan ⁇ -4-yl)- 1 -phenyl phosphate
  • MDP Disodium 3-methoxy-5-(4-methoxyspiro ⁇ l,2-dioxetane-3,2'-(5'chloro)- tricyclo[3.3.1.1 3,7 ] decan ⁇ -4-yl)- 1 -phenyl phosphate
  • CDP-STAR Disodium 2-chloro-5-(4-methoxyspiro ⁇ 1 ,2-dioxetane-3,2'-(5'chloro)- tricyclo[3.3.1.1 3,7 ] decan ⁇ -4-yl) phenyl phosphate
  • CTAB cetyltrimethylammonium bromide (a cationic detergent)
  • TX-100 Triton X-l 00 or octylphenolpoly(ethylene-glycolether) n (a neutral detergent)
  • the invention relates to a method for enhancing luminescence from a luminescent system in a solution or on a solid surface by contacting the luminescent compound with a luminescence-enhancing combination of one or more detergent and one or more enhancer compound. More particularly, the method of enhancement of luminescence of this invention is practiced by bringing together under the proper conditions, usually in a polar solution, such as an aqueous solution, or on a solid surface, a mixture comprising: (a) an effective concentration of a detergent compound ;
  • both a detergent and an enhancer compound be utilized to increase the luminescent intensity of the luminescent system relative to that obtained in the absence of either the detergent or the enhancer compound.
  • surfactants and enhancers as individual luminescence enhancers
  • the increase in the luminescent intensity is substantially greater than the sum ofthe individual effects that can be achieved from the use of either the surfactant or the enhancer alone.
  • 1,2-dioxetane luminescent signal obtained from art known chemiluminescent systems containing 1,2- dioxetane luminescent molecules, such as those described in EP patent application 85/109759.2, as well as U.S. Patent No. 5,145,772.
  • chemiluminescent systems containing 1,2- dioxetane luminescent molecules such as those described in EP patent application 85/109759.2, as well as U.S. Patent No. 5,145,772.
  • AMPPD alkaline phosphatase 1,2-dioxetane substrates marketed, all by Tropix, Inc., Bedford, MA. Their trade names are AMPPD, CDP, CDP * , MDP, and CSPD.
  • Each of these 1,2-dioxetane substrates has a different chemical structure.
  • 1,2-dioxetane substrates for other enzymes are also available from Tropix. The chemical names of these compounds are found in the Definitions and Abbreviations Section
  • dioxetane luminescent molecules can be used in the practice ofthe invention.
  • AMPD 1,2-dioxetane luminescent molecules
  • CSPD 1,2-dioxetane luminescent molecules
  • MDP 1,2-dioxetane luminescent molecules
  • CDP CDP* anions
  • Figure 1 illustrates the process of the formation of a 1,2-dioxetane luminescent molecule from AMPPD substrate and its subsequent decomposition and photon emission.
  • the art-known enhancers used in the description of this invention with regard to enhancement ofthe 1,2-dioxetane luminescent signal from known 1,2-dioxetane luminescent molecules are such as those described in U.S. Patent No. 5,145,772, which is inco ⁇ orated herein by reference.
  • Two of these enhancers are sold by Tropix, Inc., Bedford, MA, under the trade names Sapphire 1 and Sapphire 2.
  • Sapphire 1 is designated as BDMQ
  • Sapphire 2 is designated as TBQ. Both of these compounds are water soluble poly(vinylbenzylquaternary ammonium salts).
  • Tropix also available from Tropix is a mixture of TBQ or BDMQ with fluorescein, thus providing enhancement from the polymeric enhancer TBQ or BDMQ and energy transfer enhancement from the fluorescein in one solution.
  • Other compounds can also be used as enhancer substances, including those listed in the abovementioned U.S. patent.
  • the enhancers of luminescence of 1,2-dioxetane compounds suitable for use in the practice of this invention are naturally-occurring and synthetic compounds, generally macromolecular in nature. There are no known limitations on the size and/or the degree of polymerization of the enhancer compounds in order to practice the invention.
  • Water soluble polymeric 1,2 dioxetane luminescence enhancers are also described in detail in U.S. Patent No. 5,145,772.
  • Representative examples of these water-soluble enhancers include water soluble globular proteins having hydrophobic regions: mammalian serum albumins such as bovine serum albumin (BSA) and human serum albumin (HSA), or water soluble polymeric quaternary ammonium salts. Polymeric quaternary ammonium, phosphonium, or sulfonium salts, and mixtures thereof, are preferred.
  • Enhancer compounds can be used in combination with detergent compounds in the practice ofthe invention.
  • Polymeric enhancer compounds for 1,2-dioxetane luminescent compounds include but are not restricted to TBQ, BDMQ and other quaternary ammonium, phosphonium, and sulfonium compounds. These include seven other enhancing compounds obtained from Tropix Inc., which are not currently available commercially, in addition to TBQ and BDMQ, the art known enhancers mentioned above. Each of these seven other compounds represents a different chemical structure, and each has been used in the practice ofthe invention. Although the specific chemical formulae of these seven compounds is not known in the art, the general chemical nature of these additional enhancer compounds is described in U.S. Patent No. 5,145,772. The utility of using any particular type of enhancer compound in the practice of any particular mode ofthe invention can readily be determined empirically by one of skill in the art.
  • the amount of enhancer compound, or mixture thereof, employed in combination with one or more surfactant compounds when practicing this invention to achieve an increased enhancement of luminescence is an effective or luminescence-enhancing amount, and can vary within wide limits, depending on the particular enhancer substance chosen, the amount and type of chemiluminescent compound present, and the type and concentration of detergent used in combination with the enhancer. In general, amounts of enhancer substance ranging from about 0.001% (w/v) to about 5% (w/v) can be employed to practice one or the other modes of the invention. The factors affecting the amount of enhancer used include the specific enhancer type, the specific detergent type and compound, and the specific mode of practice ofthe invention.
  • concentration of enhancer to use with a particular detergent compound and a particular mode of practice of the invention can readily be determined empirically by one of skill in the art using the guidelines provided herein.
  • concentration of enhancer compound generally ranges from about 0.005% to about 1%, will be employed.
  • detergents can be used in the practice of one or another mode ofthe invention. Individual detergents as well as mixtures of detergents can be used in combination with one or more ofthe polymeric enhancers disclosed above in the practice ofthe invention. Such detergents include anionic, cationic, non-ionic and zwitterionic detergents. The utility of using any particular type of detergent or specific detergent compound in the practice of any particular embodiment ofthe invention can readily be determined by one of skill in the art.
  • a representative list of anionic, cationic, zwitterionic, and neutral detergents useful in the practice of this invention includes sodium hexyl sulfate; sodium octyl sulfate; sodium decyl sulfate; sodium dodecyl sulfate; lithium dodecyl sulfate; sodium tetradecyl sulfate; sodium diisobutyl sulfonylsuccinate; sodium lauryl sarcosinate; sodium dodecanesulfonic acid; sodium dodecylbenzene- sulfonic acid; dextran sulfate; sodium oleate; sodium deoxycholate; piperazine-N-N'- bis(2-ethane-sulfonic acid (PIPES); 2-(N-cyclo-hexylamino) ethane sulfonic acid (CHES); dodecyl phenol; sodium taurodeoxycholate; sodium chol
  • the amount of detergent compound, or mixture thereof, employed together with the enhancer to achieve an increase in chemiluminescent intensity when practicing this invention is an effective or luminescence-enhancing amount and can vary within wide limits, depending on the particular detergent compound chosen, the amount and type of chemiluminescent compounds and type and concentration of enhancer compounds present, as well as the particular embodiment of practice ofthe invention. In general, however, an amount of detergent compound, or combination thereof, ranging from about 0.0001% (w/v) to about 25% (w/v) and preferably from about 0.001% to about 10%, is employed.
  • concentration of detergent to use with a particular enhancer compound and a particular mode of practice ofthe invention can readily be determined empirically by one of skill in the art.
  • chemiluminescent compounds including anionic, cationic, zwitterionic and non-ionic detergent compounds; positively, negatively and uncharged enhancer compounds; and negatively, positively and uncharged luminescent molecules can be used.
  • the chemiluminescent-enhancing amount of each detergent and enhancer compound to be used can be determined empirically by one skilled in the art using the teachings herein and the general knowledge ofthe art as guidelines.
  • the "proper conditions" for contacting together effective concentrations of the luminescent, enhancer and detergent compounds in the practice of this invention include the volume, pH, type of buffer, type of solvent, the presence of other non-enhancer, non- detergent additives, and the temperature of the solid surface or solution in which the invention is practiced, as well as the type of solid surface utilized, if any. These can vary widely. Many factors can affect the selection ofthe proper conditions, including the type of enhancer, the type of luminescent molecule, the type of detergent, the desired sensitivity of detection of luminescent molecules, the type of substrate, the type of enzyme used, the type of analyte being tested for, the format selected, and the chosen mode of practice of the invention. The proper conditions can be determined empirically by one skilled in the art, and many aspects ofthe proper conditions will be readily apparent to one with skill in the art.
  • non-detergent, non-enhancer additives include the following.
  • Inorganic and organic salts such as sodium chloride, sodium acetate, magnesium chloride, zinc chloride, and sodium ethylene diamine tetraacetate
  • Organic and inorganic acids such as phosphoric acid, sulfuric acid, hydrochloric acid, and acetic acid, are often inco ⁇ orated into buffer solutions used in luminescent systems for various reasons, and aside from the effect on the pH ofthe solution, have little effect on the properties ofthe luminescent compounds.
  • organic and inorganic acids can be used to lower the pH of a solution and thereby stabilize or increase the decay half time of luminescent compounds.
  • Organic compounds such as dimethyl sulfoxide (DMSO), acetonitrile, and various adamantanones can also increase the decay half time of luminescent compounds and stabilize them as can TBQ and BDMQ.
  • Other additives such as various alcohols, including ethanol, methanol, glycerol, and ethyleneglycol, may also be present for various reasons and have little effect on the properties ofthe luminescent compounds.
  • Inorganic or organic base compounds including sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide and tertiary butyl hydroxide may also be present. These basic compounds can be used to reduce the half time of decay ofthe luminescent compound.
  • the type of luminescent molecule such as the mode of use ofthe invention, the type of luminescent molecule, the type of detergent, the type of substrate, the type of enzyme used, the type of buffer used, the desired sensitivity of detection, the type of analyte being tested for, and the assay format selected.
  • the proper types and amounts of these other substances to use can readily be empirically determined by one skilled in the art.
  • the method ofthe present invention is practiced by bringing together a detergent compound or compounds, an enhancer compound or compounds, and a luminescent compound or compounds.
  • the detergent stabilization ofthe luminescent molecules can be accomplished by using a variety of detergents and combinations of detergents can also be effective in this regard. It is probable that a single molecular entity or compound can be constructed which combines any two, or even all three of these types of chemical compounds into one chemical compound.
  • One possibility is to synthesize a single molecule containing a detergent, an enhancer, and an inert luminescent substrate. This molecule could then be activated by an enzyme converting the substrate to a luminescent molecule.
  • Other schemes are also possible and would be apparent to one of skill in the art.
  • the present invention can be practiced in multiple variants ofthe method, which include, but are not limited to, the following: I. Adding the detergent compound or compounds (plus or minus other additives) to a solution, or to a solid surface already containing enhancer molecules and luminescent molecules (plus or minus other additives).
  • a luminescence-enhancing amount of both the enhancer and the detergent in combination can vary, depending on the number, characteristics and concentration ofthe enhancer used, the number and characteristics ofthe detergent used, the number, characteristics and concentration ofthe luminescent molecules, the overall conditions used, and the mode of the invention being practiced.
  • the conditions to be considered by one of skill in the art in determining what constitutes a luminescence-enhancing amount of any given combination of detergent and enhancer compound include pH, buffer type, solvent type, temperature, volume, the presence of other additives, or type of solid surface utilized, if any.
  • the effective and luminescence-enhancing amounts of both the enhancer and the detergent are those which produce the desired increase in luminescent intensity from the chemiluminescent system employed, which increase is greater than the additive effects expected from use ofthe detergent and enhancer alone, and can be determined empirically by one of ordinary skill in the art using routine experimentation.
  • Factors which can effect the magnitude ofthe enhancement obtained when practicing the invention include: the type and concentration of detergent; the type of luminescent molecules utilized; the type and concentration of enhancer; the temperature; the type and concentration ofthe solvent used; the type and concentration of non- detergent, non-enhancer additives present; the molar ratio ofthe enhancer and detergent present; and the relative molar ratios of other non-detergent, non-enhancer additives.
  • the method ofthe present invention can be practiced in a number of variations, including modes I, II, III and IV described above. In addition, as will be evident to one of skill in the art, a large number of permutations can be practiced within each of these modes.
  • the method ofthe invention is very versatile and adaptable, and can be effectively utilized in most areas where a signal generation system is desired to determine the presence, absence, and/or concentration of an analyte, a substance whose presence, absence and or concentration is being determined. Such applications include medical, veterinary and industrial diagnostic tests, as well as pharmaceutical and industrial quality control. This includes enzyme, nucleic acid probe, immuno and receptor assays.
  • One or another mode of practice ofthe invention can be readily adapted to test formats which are commonly used in these assays, which are discussed below.
  • the 1,2-dioxetane is used as a direct label for the analyte of interest. Therefore, in this embodiment an inert form ofthe 1,2-dioxetane is specifically linked to the analyte molecule of interest.
  • an inert form ofthe 1,2-dioxetane is specifically linked to the analyte molecule of interest.
  • One means of accomplishing this is by covalently linking an inert luminescent molecule to a DNA probe or antibody molecule.
  • any other known method of linking a chemical compound to an antibody or DNA probe can also be used, for instance by means of a bifunctional linking moiety.
  • the DNA probe or antibody molecule can then be allowed to specifically associate with the analyte molecule to form a complex, for instance by spontaneous hybridization ofthe probe to the target DNA or immunoreaction ofthe antibody with its target antigen. Any labeled DNA probe or antibody molecules not specifically and directly attached to an analyte molecule as the result ofthe reaction is then removed, for instance by washing, or by other known methods of separation. The remaining inert luminescent molecules can then be triggered to emit light, and the light is then measured.
  • the 1,2-dioxetanes that are particularly suitable inert luminescent molecules for use in the practice of this embodiment ofthe invention have a high QY and a very short decay tl/2 in aqueous solutions in which the pH is greater than the pK ofthe luminescent form ofthe molecule (i.e., the anion created by dephosphorylation). When triggered, such molecules will, because ofthe short decay tl/2, give a complete light emission in a short time. A flash of light is obtained.
  • Examples of commercial diagnostic assays using this general approach with acridinium ester luminescent compounds are GenProbe, Inc., Pace II DNA probe products, and Ciba-Corning's Magic Light immunoassay products.
  • Luminescent molecules having a long decay tl/2 of light emission, while not as useful as direct labels, may be very useful in an enzyme-mediated luminescent signal generation system.
  • 1,2-dioxetane substrates it will be useful to utilize 1,2-dioxetane substrates to illustrate other methods for utilizing luminescent molecules in diagnostic assays.
  • Most 1 ,2-dioxetane luminescent molecules have a relatively long decay tl/2 of light emission and are therefore not generally used as direct labels.
  • These 1,2-dioxetane compounds have been most useful as substrates in enzyme-mediated luminescent signal amplification assays.
  • an enzyme molecule is linked directly to the analyte molecule.
  • One method of accomplishing direct linking is to covalently link an enzyme molecule to a DNA probe or antibody molecule.
  • the enzyme labeled DNA probe or antibody molecule can then be specifically associated with the analyte molecule to form a complex. Any enzyme labeled DNA probe or antibody molecules not specifically complexed with analyte molecules are then removed for example, by washing.
  • the remaining enzyme-analyte complexes are then detected and quantitated by adding a 1,2-dioxetane substrate and allowing the remaining enzyme to generate product 1,2-dioxetane luminescent product molecules, which then decompose to emit light.
  • the light emission decay tl/2 ofthe luminescent molecule will vary with the type of 1,2-dixoetane used. In most enzyme-mediated assays the substrate molecules are in very great excess over the enzyme molecules. Therefore, luminescent product molecules will continually be produced as long as the enzyme remains active.
  • the enzyme is continuously generating product luminescent molecules from the non-luminescent substrate molecules, and the luminescent molecules produced are continuously decaying and emitting light.
  • the rate of production of luminescent molecules by the enzyme will equal the rate of loss of luminescent molecules.
  • light output from the reaction becomes constant and will remain constant as long as there is sufficient substrate and all ofthe enzyme molecules remain active.
  • This constant light signal is generally termed a "glow” or “plateau” signal, and when the light measuring point ofthe assay is at the plateau, it is said that the assay is a glow or plateau mode assay.
  • An advantage of this mode is flexibility about when the light signal can be measured.
  • the light measurement is done directly on the enzyme substrate solution. Ifthe plateau state is steady for hours, the measurement can be made at any time before the plateau becomes unstable. With this mode of measurement the maximum signal obtainable is at the plateau and the light intensity observed at the plateau is directly proportional to: (a) the QY ofthe luminescent molecule in the solution;
  • An alternate strategy can be used to measure the light emitted from the luminescent product molecules generated by the enzyme.
  • the light is measured at some time after the enzyme substrate reaction is started, and before the plateau is reached.
  • the measurement is made directly on the enzyme substrate reaction solution.
  • the advantage to this approach is a shorter time to measurement.
  • a disadvantage is that the light signal is smaller than would be seen at the plateau.
  • the light intensity obtained is directly proportional to:
  • Still another strategy can be used to measure the light emitted from the luminescent product molecules generated by the enzyme. This involves obtaining a light measurement soon after causing an enhancement of luminescence by changing the enzyme-substrate solution in some manner, so that an enhancement of luminescence from the solution is effected.
  • This enhancement may arise from an increase in the QY ofthe luminescent molecules present, and/or a decrease in the light emission decay tl/2 which has been induced by the change in solution environment.
  • an increase in the quantum yield ofthe luminescent molecules present can be effected by adding an enhancer to an enzyme-substrate solution not containing enhancer.
  • a decrease in the decay 1 1/2 ofthe chemiluminescent molecules present can be effected by increasing the pH ofthe enzyme-substrate solution over an appropriate range, or by raising the temperature ofthe enzyme-substrate solution or by adding certain additives to the enzyme-substrate solution.
  • This latter method can be utilized to measure light from the sample before, or after, the plateau ofthe enzyme-substrate reaction is reached.
  • the overall advantage of using this method, whether light is measured at or before the plateau, is that it results in a greater luminescent enhancement, and therefore an increased potential for detecting lower numbers of analyte molecules in a diagnostic assay.
  • the present invention provides an improved method for obtaining significant increases in enhancement of luminescence over the additive effects of luminescence enhancement obtainable by use of either surfactants or polymeric enhancers in the prior art enhancement methods.
  • Table 11 illustrates the effect of various detergents on the light intensity emitted from the 1,2- dioxetane anion AMP ' D when various concentrations of prior art detergents are used for enhancement of luminescence in aqueous solutions containing the luminescent anion.
  • Example 8 illustrates one mode of practicing the invention, in which an effective concentration of detergent is inco ⁇ orated into an art known solution containing an effective concentration of enhancer compound and inert luminescent substrate molecules.
  • the first step ofthe method ofthe invention is practiced by forming a solution comprising an art known buffer, a luminescent enzyme substrate compound, an effective concentration of art known detergent compound, and an effective concentration of art known enhancer compound.
  • enzyme is then added to the solution, and in the presence the combination of detergent and enhancer compounds, the enzyme generates luminescent product molecules from the substrate.
  • the QY ofthe resultant product molecules is then enhanced by the presence ofthe detergent and enhancer molecules.
  • an effective concentration of enhancer compound is added to an already formed solution containing luminescent molecules and a detergent compound or compounds (See Examples 4 and 5).
  • the first step of this embodiment ofthe invention the following are brought together into a solution: an art known buffer, one or more are known substrate compound(s), and an effective concentration of one or more detergent compounds.
  • enzyme is added to the solution, and luminescent product molecules are generated from the substrate over time.
  • an effective concentration of one or more enhancer compounds, along with any desired additives is added to the solution, and an enhancement of luminescence occurs relative to an otherwise identical sample not containing detergent.
  • An increase in the QY ofthe luminescent product molecules is directly due to the presence ofthe detergent, and results in enhanced luminescence.
  • the method ofthe invention is independent ofthe mechanism by which the enhancement occurs, and Applicant does not wish to be bound by a theory of mechanism.
  • the presence ofthe detergent in the enzyme substrate reaction solution generates additional, non-QY related luminescent enhancement arising from the ability of detergent compounds and mixtures of different detergent compounds to stabilize the unstable luminescent product molecules, thereby slowing their decomposition.
  • This stabilization is due to the ability ofthe detergents to substantially increase the half time of decay ofthe luminescent compound. In general, the higher the detergent concentration, the greater the stabilization.
  • the increase in luminescent product molecules in the presence of detergent at the time of light measurement results in enhanced luminescence.
  • AMPPD alkaline phosphatase
  • Alkaline phosphatase processes AMPPD to form the product luminescent molecule AMP " D.
  • the anion is unstable and will eventually decompose (see Figure 1). It is believed that the presence of detergent in the enzyme-substrate reaction solution stabilizes the AMP ' D. anion, as well as other 1,2-dioxetane anions, and prevents the anionic complex from decomposing as rapidly as it would in the absence of detergent, resulting in a substantial increase in the light intensity.
  • the luminescent compound Under destabilizing conditions (i.e., conditions that decrease the half-time of light emission ofthe luminescent compound relative to that in the stabilized state).
  • the half-time of light emission can be decreased either by raising the pH, or by effectively lowering the concentration ofthe detergent, or both.
  • Another approach is to contact the stabilized luminescent compound with an additive that partially or wholly neutralizes the stabilizing effect ofthe detergent, thereby decreasing the half-time of light emission ofthe luminescent compound.
  • Example 9 illustrates another embodiment of this invention wherein manipulation ofthe pH ofthe reaction is combined with the use of detergent stabilization to increase the light emission.
  • This procedure is particularly useful for enzyme-substrate reactions usually conducted at high pH.
  • a pH above 9 is preferred for the reaction of the enzyme alkaline phosphatase with the substrate 1,2-dioxetane.
  • a significant fraction of many 1 ,2-dioxetane enzyme products are not protonated and are, therefore, unstable, leading to loss by decomposition of a significant fraction of the 1,2-dioxetane enzyme product molecules.
  • the proportion of product molecules lost increases with the length ofthe incubation time.
  • the addition of an effective concentration of detergent to the enzyme-substrate reaction mixture can greatly reduce the loss ofthe luminescent compound during the incubation.
  • the half time of light emission ofthe detergent stabilized luminescent compound was decreased in two ways before measurement ofthe light intensity: (1) by increasing the pH and (2) by lowering the detergent concentration in the incubation solution.
  • Example 5 illustrates the preferred embodiment ofthe invention in which three factors operate to maximize the output of light from a luminescent system. First, the after the plateau has been established, the degree of enhancement due to the stabilization effect will be at a maximum.
  • An advantage of the method ofthe invention is that it reduces the diversity of signal generation and detection technologies necessary to produce diagnostic tests of all kinds. This can be illustrated by discussing the current state of clinical diagnostic tests used to evaluate the various aspects ofthe human medical condition. Such clinical diagnostic tests require the ability to detect a wide variety of different chemical and biological compounds and organisms, including bacteria and viruses, present in a wide range of different amounts and concentrations. In response to these requirements, a wide diversity of different signal generation and detection technologies and formats are currently used to produce clinical tests of various types.
  • the present invention when combined with other technical advances, is capable, as mentioned in the summary, of detecting very small numbers of enzyme molecules (10-30) in less than one hour and can be easily adapted to detect larger numbers of molecules.
  • the method ofthe present invention will become part of an advanced signal generation and detection technology which can replace many of the existing signal generation and detection technologies, and thereby significantly reduce the technological diversity needed to meet the requirements ofthe clinical laboratory.
  • Such an alternate technology would have applications in any medical or industrial diagnostic area.
  • the present invention when combined with other technical advances, is capable, as mentioned in the summary, of detecting very small numbers of enzyme molecules (10-30) in less than one hour and can be easily adapted to detect larger numbers of molecules.
  • the method ofthe present invention will become part of an advanced signal generation and detection technology which can replace many ofthe existing signal generation and detection technologies, and thereby significantly reduce the technological diversity needed to meet the requirements ofthe clinical laboratory.
  • Such an alternate technology would have applications in any medical or industrial diagnostic area.
  • the light intensity is relative to an equivalent unenhanced system.
  • AMP 2-amino-2-methyl-l -propanol
  • CTAB cetyltrimethylammonium bromide
  • DEA Diethanol amine
  • a buffer LDS Lithium dodecyl sulfate
  • a detergent SLSC Sodium lauryl sarcocinate
  • a detergent Example 1 illustrates one mode of practicing the invention, the incorporation of an effective concentration of detergent into an art known solution containing a fixed concentration of enhancer compound and luminescent molecules.
  • a solution is first formed comprising an art known buffer solution containing a luminescent enzyme substrate compound, an effective concentration of art known detergent compound, and an effective concentration of art known enhancer compound. Enzyme was then added to this solution, and in the presence of both detergent and enhancer compounds the enzyme generated luminescent product molecules from the substrate. The QY ofthe resultant product molecules was enhanced by the presence of the combination of detergent and enhancer molecules.
  • Another mode of practice ofthe invention is to add an effective concentration of enhancer compound to an already formed solution containing a detergent compound or compounds, and luminescent molecules.
  • the practice of this mode, using enzyme generated luminescent molecules includes the following: First, bringing together in one solution, an art known buffer, one or more art known substrate compound(s) and an effective concentration of a detergent compound or compounds. Next, add enzyme to this solution and over time luminescent product molecules will be generated from the substrate. Then, just before signal measurement, an effective concentration of enhancer compound or compounds is added to the solution and luminescent enhancement occurs, relative to an otherwise identical sample not containing detergent.
  • an increase in the QY ofthe luminescent product molecules is directly due to the presence ofthe detergent, and results in enhanced luminescence.
  • the presence ofthe detergent in the enzyme substrate reaction solution results in additional, non-QY related, luminescent enhancement.
  • the additional enhancement arises from the ability of detergent compounds and mixtures of different detergent compounds to stabilize the unstable luminescent product molecules and prevent them from decomposing as rapidly. The result of this is that more luminescent product molecules are present at the time of light measurement when the detergent is present. This source of increased luminescence is also directly due to the presence ofthe detergent and results in enhanced luminescence.
  • Alkaline phosphatase processes the substrate AMPPD to form the product luminescent molecule AMP ' D anion.
  • the anion is unstable and will eventually decompose (see Figure 1). If the anion is protonated it becomes stable and will not decompose.
  • the presence of detergent in the enzyme-substrate reaction solution can also stabilize the AMP ' D anion, as well as other 1,2-dioxetane anions, and prevent the anionic complex from decomposing as rapidly as it would in the absence of detergent.
  • the analyte detection assays utilizing luminescent molecules are performed in aqueous solution.
  • Many electronically excited luminescent molecules, including dioxetanes, will emit light in aqueous solution.
  • the efficiency of light generation in aqueous solution is low, and only a small fraction ofthe potentially luminescent molecules present emit a photon.
  • the quantum yield hereafter designated (QY)
  • QY the quantum yield
  • An increase in the QY will increase the total number of photons that are emitted from a given population of luminescent molecules in solution in a given period of time.
  • This experiment illustrates an alternate method designed to determine the effective concentration ofthe detergent, lithium dodecyl sulfate (LDS), necessary to practice the invention with the enhancer, TBQ used at a concentration of 0.044% (w/v).
  • LDS lithium dodecyl sulfate
  • the AMPPD substrate, the enhancer, and the detergent were mixed together, with different concentrations ofthe detergent in different tubes, and the enzyme was then added last.
  • Each reaction mixture contained 450 microliters of a solution composed of: (a) O.l M DEA pH IO (b) 0.044% TBQ (w/v)
  • the AMPPD substrate was quickly and essentially totally converted to luminescent
  • Each reaction mixture contained 450 microliters of a solution composed of:
  • This example illustrates that compounds which are not normally regarded as detergents, but which have detergent-like or amphipathic properties, in this case a commonly used buffer compound, CHES [2-(N(cyclohexylamino)-ethanesulfonic acid], can act as the detergent in enhancing luminescence in concert with a polymeric enhancer compound in the practice ofthe invention.
  • CHES 2-(N(cyclohexylamino)-ethanesulfonic acid
  • the control solution contained no detergent ⁇ like compound and was comprised of a mixture of 50 ⁇ l of 0.01M DEA buffer, a known amount of CSPD and alkaline phosphatase, and 400 ⁇ l of 3.8 M NaOH 0.05% TBQ. Each reaction mixture contained 450 ⁇ l of a solution composed of:
  • This example illustrates one mode of practice ofthe invention wherein an enhancer compound is added to a solution containing a luminescent compound and a detergent compound.
  • This example further illustrates a variation of this mode in that an inorganic base additive is added to the enhancer compound for the purpose of reducing the decay half time of light emission ofthe luminescent compound in the final solution.
  • This mode is the presently preferred mode ofthe invention for obtaining the highest QY's for the luminescent molecules and the highest light intensities from the solutions containing the chemiluminescent molecules. Specifically, in this example the following steps are performed at 25 °C:
  • step (b) To the solution of step (a) were added and rapidly mixed, 2 ⁇ l of a solution comprised of 10 "5 M alkaline phosphatase (Biozyme ALPI 12g), 5x1 O ⁇ M TRIS-
  • This example illustrates a mode of practice ofthe invention in which the presence of detergent in the enzyme-substrate solution results in an increase in light intensity obtained from the luminescent system both by causing an increase in the QY ofthe luminescent compound and by causing an increase in the amount of luminescent compound present at the time of light measurement.
  • the presence of a base additive at the time of light measurement results in a further increase in light intensity by decreasing the half time of light emission ofthe luminescent compound relative to the half time in the absence ofthe base additive.
  • This mode is presently the preferred embodiment ofthe invention for obtaining the highest light intensities from luminescent systems where an enzyme is used to convert a luminescently inert compound to a luminescent compound over a period of time.
  • this example ofthe practice of this mode comprises:
  • step (d) Then to 25 ⁇ l ofthe solution of step (a) were added 25 ⁇ l of 0.25% LDS and immediately thereafter were added 400 ⁇ l of 1.9M NaOH, 0.05% TBQ equilibrated to 25 °C. Immediately after mixing the resulting solution, the light intensity ofthe resultant luminescent system was measured.
  • step (e) Then into 25 ⁇ l ofthe solution formed in step (b), the control solution, were mixed 25 ⁇ l of 0.5% LDS and immediately thereafter were added 400 ⁇ l of 1.9M NaOH, and 0.05% TBQ equilibrated to 25 °C. Immediately thereafter the light intensity ofthe resultant luminescent system was determined. The alkaline phosphatase was slightly less active in the presence ofthe 0.25% LDS than it was in the absence ofthe detergent (data not shown). Further, the enhancement of QY ofthe CSP ' D luminescent molecule was the same in the solutions of steps d) and e) at the time of light measurement. The results are presented in Table 17 herein. See Example 4, Table 16 for the QY increase seen in this system.
  • This example illustrates an embodiment ofthe invention wherein a solution containing effective concentrations of enhancer and detergent is added to a solution containing luminescent molecules.
  • a solution containing effective concentrations of enhancer and detergent is added to a solution containing luminescent molecules.
  • the following solutions at 37 °C were rapidly mixed in a 12x75 mm glass or plastic tube:
  • step (a) 10 ⁇ l of a solution comprised of 0. IM TRIS.HC1 buffer pH 7.4 and a known concentration of CSPD substrate was formed.
  • step (b) To the solution formed in step (a) were added and rapidly mixed in, 2 ⁇ l of a solution containing 10 "5 M alkaline phosphatase (Biozyme ALPI 12G), 5x1 O ⁇ M
  • Example 4 As a control for this experiment, the above described process steps were followed to determine the QY for a solution identical to the one resulting from step (c) above except that it did not contain detergent.
  • This example illustrates an embodiment ofthe invention in which the enhancer, detergent and substrate are mixed into a single solution and then the enzyme is inco ⁇ orated into the solution.
  • This embodiment ofthe invention is compatible with a variety of sandwich type DNA probe and immunoassays which utilize a signal generation and detection system based on enzyme generation of a luminescent signal.
  • the glow mode of light signal measurement can be utilized.
  • step (a) 1 ml of a solution was formed containing 0.09M DEA.HCl buffer, pH 9.48, 0.06% sodium hexylsulfate, 0.1% TBQ, 0.1 milligrams/ml sodium fluorescein, and lO ⁇ M CSPD substrate.
  • step (b) To the solution of step (a) was added 10 ⁇ l of a solution containing O.lM CHES buffer and 6x10 5 alkaline phosphate molecules (Biozyme ALPI 12G). The resulting solution was incubated at 37 °C. At specified times after adding the enzyme, the light intensity ofthe solution was measured in order to determine when the light output reached a plateau stage.
  • step (c) The control for this example was performed using a solution identical to the solution of step (a) above, except that it did not contain detergent.
  • This example illustrates the embodiment ofthe invention wherein an effective concentration of enhancer compound is added to a solution containing both an effective concentration of detergent and luminescent molecules.
  • this example illustrates using a detergent to stabilize a luminescent compound as it is produced, and to cause its accumulation over time. Specifically, in the practice of this embodiment ofthe invention, the following steps were taken:
  • step (b) A solution identical to the solution in step (a) was prepared, except that it contained no TX-100.
  • step (g) Immediately after step (f), the light intensity ofthe solutions was measured.
  • step (b) To 10 ⁇ l ofthe solutions formulated in step (a) were added and rapidly mixed in, 400 ⁇ l of 0.09M DEA.acetate pH 9.9, and 0.1% TBQ equilibrated to about 25 °C.
  • step (c) After mixing, the light intensity ofthe solutions of step (b) were immediately measured over a period of one second.
  • step (d) A control for this experiment was made by forming a solution identical to the those described in step (a) except that no detergent was present. After the control solution reached its plateau light output, steps (b) and (c) were performed using 10 ⁇ l ofthe control solution. The results of this experiment are shown below in Table 22:
  • This example illustrates an embodiment ofthe invention wherein a solution containing an effective concentration of detergent compound is added to a solution containing luminescent molecules and an effective concentration of enhancer compound.
  • step (a) A lO ⁇ l solution containing O.lM TRIS.HC1 buffer pH 7.5 and a known concentration of CSPD substrate was formed in an appropriate container,
  • step (b) To the solution of step (a) were added and rapidly mixed in, 2 ⁇ l of 10 '5 M alkaline phosphatase (Biozyme ALPI 12G), 5x10 ⁇ 1 MgCl 2 , 10 '5 M ZnCl 2 , and 5% glycerol. The solution was incubated at room temperature for 20 seconds. Essentially all of the substrate CSPD was converted to luminescent product molecules within seconds.
  • step (c) To the solution of step (b) 0.4 ml of 0.09M DEA.HCl buffer pH 9.9, 0.1 % (w/v) of TBQ were immediately added and rapidly mixed in.
  • step (d) Immediately thereafter, 0.4 ml of 0. IM DEA.HCl buffer pH 9.9, 0.05% lithium dodecyl sulfate were mixed into the solution of step (c). The initial light intensity ofthe resultant mixture was measured, and the decay tl/2 and QY ofthe luminescent product molecules were determined as described above.
  • Example 4 This example is similar to Example 4 above, except that a neutral detergent, DDS (dodecanoyl sucrose) was used. Specifically, in this embodiment ofthe invention the following steps were taken at a temperature of 25 °C:
  • step (c) To the solutions formed in steps (a) and (b) were added and rapidly mixed in, 2 ⁇ l of a solution of 10 '5 M alkaline phosphatase (Biozyme ALPI 12G), pH 7.0, TRIS buffer, 5x1 O ⁇ M MgCl 2 , 10 '5 M ZnCl 2 , and 5% glycerol. Essentially all ofthe dioxetane substrate was converted to product molecules within seconds.
  • Example 10 illustrates the same embodiment ofthe invention as does Example 10 except that a cationic detergent, cetyltrimethylammonium bromide (CTAB), was used. Specifically the following steps were taken:
  • step (c) Then 20 ⁇ l ofthe solution of step (a) was removed and mixed with 20 ⁇ l of 0.1 M DEA-acetate buffer pH 9.46, 0.006% (w/v) CTAB. To the resulting mixture was added and mixed in 1 ml of 1.9 M NaOH, 0.05% TBQ.
  • step (d) 20 ⁇ l ofthe solution of step (b) was removed and mixed with 20 ⁇ l of 0.1 M DEA-acetate buffer pH 9.46, 0.012% (w/v) CTAB. To the resulting mixture was added and mixed in 1 ml of 1.9 M NaOH, 0.05% TBQ.
  • TX-100 causes a significant enhancement of luminescence due to the stabilization of AMP ' D anion at pH 8 was performed using a comparable format to that used in prior art report (Jain and Magrath, supra). As expected, the presence ofthe 0.02% TX-100 produced no significant luminescent enhancement, relative to an identical sample treated in a parallel manner that did not contain detergent.

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Abstract

La présente invention concerne un procédé permettant d'obtenir un meilleur accroissement de la luminescence que celui obtenu par les systèmes de luminescence connus selon l'état actuel de la technique, et ce en incorporant dans ces systèmes de luminescence un ou plusieurs détergents ainsi qu'un ou plusieurs améliorants. Une telle amélioration de la luminescence peut s'obtenir dans une solution ou sur une surface solide. Le procédé peut se mettre en ÷uvre en utilisant des composés tensioactifs ou détergents à l'état anionique, cationique ou zwitterionique ou non ionisés. Le procédé convient aux nombreux domaines d'application où l'on a besoin d'un système de génération de signal: diagnostics médicaux, vétérinaires, agricoles et industriels ainsi que le contrôle de qualité. Cela recouvre tous types de techniques conçues pour détecter et/ou quantifier la présence d'un élément à analyser, et notamment les composés industriels et pharmaceutiques ainsi que les composés et organismes biologiques de tous types tels que les protéines, les glucides, les lipides, les acides nucléiques, la bactéries et les virus. Parmi les exemples de tels tests on peut citer ceux utilisant des sondes d'acide nucléique ainsi que les immunodosages et les dosages de récepteurs.
PCT/US1996/012300 1995-07-28 1996-07-26 Procede d'accroissement de la chimioluminescence WO1997005209A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999002191A1 (fr) * 1997-07-08 1999-01-21 Canji, Inc. Compositions et procedes permettant d'accroitre l'apport d'agents therapeutiques a des cellules
US5994073A (en) * 1990-08-30 1999-11-30 Tropix, Inc. Enhancement of chemiluminescent assays
EP1113077A2 (fr) * 1999-12-28 2001-07-04 Fujirebio Inc. Essai chemioluminescent
US6392069B2 (en) 1996-01-08 2002-05-21 Canji, Inc. Compositions for enhancing delivery of nucleic acids to cells
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Cited By (11)

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US5994073A (en) * 1990-08-30 1999-11-30 Tropix, Inc. Enhancement of chemiluminescent assays
US6392069B2 (en) 1996-01-08 2002-05-21 Canji, Inc. Compositions for enhancing delivery of nucleic acids to cells
US7534769B2 (en) 1996-01-08 2009-05-19 Canji, Inc. Compositions and methods for enhancing delivery of therapeutic agents to cells
US7538093B2 (en) 1996-01-08 2009-05-26 Schering Corporation Compositions and methods for therapeutic use
US8022044B2 (en) 1996-01-08 2011-09-20 Canji, Inc. Compositions and methods for therapeutic use
WO1999002191A1 (fr) * 1997-07-08 1999-01-21 Canji, Inc. Compositions et procedes permettant d'accroitre l'apport d'agents therapeutiques a des cellules
EP1113077A2 (fr) * 1999-12-28 2001-07-04 Fujirebio Inc. Essai chemioluminescent
EP1113077A3 (fr) * 1999-12-28 2004-04-07 Fujirebio Inc. Essai chemioluminescent
US7355056B2 (en) 2003-06-04 2008-04-08 Canji, Inc. Transfection agents
US7691822B2 (en) 2003-12-10 2010-04-06 Canji, Inc. Methods and compositions for treatment of interferon-resistant tumors
US9439977B2 (en) 2003-12-10 2016-09-13 Fkd Therapies Oy Methods and compositions for treatment of interferon-resistant tumors

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