WO1998021586A1 - Chemiluminescent detection methods using dual enzyme-labeled binding partners - Google Patents
Chemiluminescent detection methods using dual enzyme-labeled binding partners Download PDFInfo
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- WO1998021586A1 WO1998021586A1 PCT/US1997/019612 US9719612W WO9821586A1 WO 1998021586 A1 WO1998021586 A1 WO 1998021586A1 US 9719612 W US9719612 W US 9719612W WO 9821586 A1 WO9821586 A1 WO 9821586A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
- C07D219/02—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with only hydrogen, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the ring system
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
- C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
- C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
- C07D219/06—Oxygen atoms
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B15/00—Acridine dyes
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- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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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
- G01N33/58—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
- This invention relates to methods of detecting analytes or target species using two enzyme-labeled specific binding partners where the two enzymes function in concert to produce a detectable chemiluminescent signal.
- the invention further relates to a method for detecting analytes or target species using a peroxidase-labeled specific binding partner and a second specific binding partner labeled with a hydrolytic enzyme.
- the invention relates to the use of the method to detect and quantitate with improved specificity various biological molecules including antigens and antibodies by the technique of immunoassay, proteins by Western blotting, DNA by Southern blotting, RNA by Northern blotting. The method may also be used to detect DNA mutations and chromosomal translocations .
- compositions comprising an acridancarboxylate derivative, a peroxide compound, and a protected enhancer compound which produce chemiluminescence by the action of a hydrolytic enzyme and a peroxidase enzyme.
- the hydrolytic enzyme removes a protecting group from the protected enhancer to produce a phenol compound which promotes the chemiluminescent peroxidase-catalyzed oxidation of the acridancarboxylate derivative .
- compositions comprising a dihydrophthalazinedione, a peroxide compound, and a pro-enhancer compound which produce chemiluminescence by the action of a hydrolytic enzyme and a peroxidase enzyme.
- the hydrolytic enzyme removes a protecting group from the pro-enhancer to produce an enhancer compound which promotes the chemiluminescent peroxidase-catalyzed oxidation of the dihydrophthalazinedione .
- the anti-hapten antibodies conjugated with either AP or horseradish peroxidase (HRP) offer versatility of detection because of the availability of different haptens for labeling the probes, the anti-hapten antibodies conjugated with either AP or horseradish peroxidase (HRP) , and their respective chemiluminescent substrates.
- U.S. Patent 5,516,641 discloses DNA probe capture assays employing two probes complementary to different non-contiguous sequence regions of a nucleic acid analyte. In the methods disclosed, one probe is capable of immobilization and is not detectably labeled and the other probe capable of having a detectable label . The two probes are hybridized and then subsequently covalently joined by chemical, photochemical or enzymatic means .
- EP 0 185 494 A2 discloses DNA probe assays employing two probes complementary to different contiguous sequence regions of a nucleic acid analyte. Neither publication discloses or suggests the use of two different enzyme labels on the two probes acting in concert to produce a chemiluminescent signal. IN THE DRAWINGS
- Figure 1 is an image of an x-ray film from an experiment detecting a mixture of AP-labeled and HRP- labeled Hindlll-digested DNA fragments on a nylon membrane with a chemiluminescent reagent composition. Lanes containing only one of the two sets of DNA fragments or unlabeled fragments are not detectable.
- Figure 2 is a an image of an x-ray film from an experiment detecting EcoRI-linearized pBR322 DNA hybridized to AP-labeled and HRP-labeled 17-mer probes on a nylon membrane with a chemiluminescent reagent composition containing acridan ester as the chemiluminescent peroxidase substrate .
- Figure 3 is a schematic drawing of a ligation assay according a to the present invention to detect the ⁇ F508 mutation in samples which are homozygous or heterozygous for the mutation and not in a sample which is homozygous normal .
- Figure 4A and 4B are digital images of x-ray films from an experiment detecting the ⁇ F mutation in samples which are homozygous or heterozygous for the mutation and not in a sample which is homozygous normal by the ligation of 21mer and 24mer probes.
- the electrophoresis gel was run a short distance which did not separate the 21mer and 24mer oligonucleotides.
- the electrophoresis gel was run a longer distance which separated the 21mer and 24mer oligonucleotides.
- Figure 5 is a schematic drawing of a DNA hybridization assay according to the present invention to detect the ⁇ F mutation only in a sample which is heterozygous for the mutation and not in samples which are homozygous normal or homozygous for the mutation.
- Figure 6 is a a digital image of an x-ray film from an experiment detecting the ⁇ F mutation only in a sample which is heterozygous for the mutation and not in samples which are homozygous normal or homozygous for the mutation.
- Figure 7 is a schematic drawing of the immunological reaction used in a Western blot employing dual enzyme- labeled antibodies according to the present invention.
- Figure 8 is a dgital image of an x-ray film from a Western blot of human HIV ⁇ -gpl20 with an AP-labeled 2° antibody and an HRP-labeled 3° antibody chemiluminescent detection conducted according to the present invention.
- the detection medium in the above methods may take the form of e.g. an immunoassay on a test strip or on the surface of a bead, well or test tube, the detection of a band of protein on a Western blotted membrane the detection of a band of DNA on a Southern blotted membrane or in a DNA hybridization assay on a filter.
- Binding pair - two molecules or portions thereof which have a specific binding affinity for one another by virtue of multiple noncovalent attractions.
- Specific binding pairs are well known in the art and include by way of illustration antigen-antibody, hapten-antibody, antibody- antibody, complementary strands of DNA, DNA-RNA duplexes, DNA-complementary oligonucleotide, RNA-complementary oligonucleotide, DNA-anti-DNA antibody, DNA-DNA binding protein, biotin-avidin or streptavidin, receptor-ligand, protein A-IgG and lectin-carbohydrate .
- Chemiluminescent peroxidase substrate - compounds which undergo an oxidation reaction in the presence of a peroxidase and a peroxide which results in the production of visible light.
- chemiluminescent peroxidase substrates are known in the art as described in (Kricka Ref) .
- the most commonly used include the amino-substituted dihydrophthalazinediones such as luminol, isoluminol, N- alkyl and N,N-dialkylamino derivatives of luminol and isoluminol, 5-amino-6, 7 , 8-trimethoxydihydrophthalazinedione and the benzo-fused homologs such as 7- dimethylaminonaphthalazinedione .
- Other chemiluminescent peroxidase substrates include the pyridazinoquinoxalinones as disclosed in U.S. Patent No. 5,324,835.
- Still other chemiluminescent peroxidase substrates include the hydroxy- substituted dihydrophthalazinediones such as 5-hydroxy- and 6-hydroxyphthalazinediones and the hydroxynaphthalazinedione as disclosed in U.S. 5,552,298 assigned to the assignee of the present application, and a class of N-alkylacridan-9-carboxylate derivatives including esters, thioesters and sulfonimides as disclosed in commonly assigned U.S. Patents 5,491,072 and 5,523,212 and U.S. Application No. 08/205,093.
- This can be accomplished by physically binding the two enzyme labels to the same molecular species by direct or indirect means .
- the closely spaced relationship can also result when a physical mixture of two distinct target molecules each bound to an enzyme-labeled specific binding partner are physically immobilized in the same region of a solid support.
- Enhancer - a substance which promotes or prolongs the oxidative or peroxidative function of a peroxidase enzyme.
- the most effective enhancers are certain aromatic amines and phenols. Phenolic compounds known to enhance peroxidase reactions are described in G. Thorpe, L. Kricka, in Bioluminescence and Chemiluminescence, New Perspectives, J. Scholmerich, et al, Eds., pp. 199-208 (1987), M. Ii, H. Yoshida, Y. Aramaki, H. Masuya, T. Hada, M. Terada, M. Hatanaka, Y. Ichimori, Biochem . Biophys . Res . Comm .
- Preferred enhancers are selected from the group consisting of substituted phenols, unsubstituted and substituted naphthols, including but not limited to: p-phenylphenol, p-iodophenol, p-bromophenol, p-chlorophenol, 2 , 4-dichloro- phenol, p-imidazolylphenol, p-thiazolylphenol, p-hydroxy- acetanilide, p-hydroxycinnamic acid, (p-cyanomethylthio) - phenol and ring halogenated derivatives thereof, phenolindophenol, 2-naphthol, 6-bromo-2-naphthol 6- hydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, firefly luciferin and dehydroluciferin.
- Enzyme label - a functional enzyme associated with a member of a specific binding pair.
- the enzyme may be covalently linked to the specific binding partner, e.g. an enzyme-antibody conjugate or an enzyme-oligonucleotide conjugate.
- the enzyme may be indirectly linked or associated with the specific binding partner of the target by the use of an auxiliary specific binding partner to which the enzyme is covalently linked.
- An example of the latter relationship would be the use of a biotin-labeled oligonucleotide probe for a certain DNA sequence associated with an enzyme-avidin conjugate.
- Genetic disease - pathologic condition caused by a genetic defect such as a mutation or a series of mutations.
- the mutation may be a point mutation, a single base substitution, a deletion, an insertion, a duplication or a transposition of bases or a combination of the above.
- the mutant gene may or may not be expressed, if expressed, it may lead to the production of truncated or non-functional protein products or proteins with an altered amino acid sequence.
- Certain genetic mutations are recessive whereby both mutant alleles or copies of the gene on the homologous chromosomes must be present for disease symptoms to occur. Other genetic diseases are dominant whereby only one copy of the gene needs to bear the mutation for disease symptoms to occur. Individuals with one copy of the recessive mutant gene are carriers without any disease but can still transmit a copy of the mutant gene to offspring.
- Hydrolytic enzyme - are enzymes which catalyze the hydrolytic cleavage of various groups.
- Representative members include; esterases such as carboxyl esterase, acetylcholinesterase, butyrylcholinesterase and cholinesterase, glycosidases, such as galactosidase, glucosidase, glucuronidase, lactase, and N-acetylglucosaminidase, lipase, phospholipase, plant or animal phosphatases, including acid and alkaline phosphatases, protease enzymes such as chymotrypsin, trypsin, papain and pepsin and sulfatase enzymes.
- Peroxidase enzyme - enzymes belonging to class EC 1.11.1.7 including horseradish peroxidase, cytochrome C peroxidase, glutathione peroxidase, microperoxidase, myeloperoxidase, lactoperoxidase, Arthromyces ramosus peroxidase (ARP) and soybean peroxidase.
- Peroxide - compounds which act as a source of hydrogen peroxide which function as the primary substrate of the peroxidase include hydrogen peroxide, urea peroxide and perborate salts, especially sodium perborate.
- Sample - materials upon which the methods of the present invention are performed to detect an analyte includes human and animal bodily fluids, such as blood, serum, urine, saliva, sputum, CSF, seminal fluid and cell lysate, as well as food samples, water samples, plant samples, microbiological specimens and forensic specimens. Other types of samples as would occur to one of ordinary skill in the art are considered to be within the scope of the invention.
- Solid support - test medium on which assay methods of the present invention can be carried out Such supports include test strips, blotting membranes, filters, microwells, test tubes, beads and the like as are known in the art of assays. The supports must be capable of capturing or immobilizing the target species-specific binding agent pair by physical adsorption or covalent linkage or both.
- Target species - molecule or portion thereof whose presence is being probed.
- Target species must be capable of binding with a substance with which there is a specific binding affinity.
- the target species will be bound to two different specific binding partners, each of which has a specific binding affinity.
- Exemplary target species include, nucleic acids such as ssDNA, dsDNA, RNA, oligonucleotides, proteins, antibodies, antigens, haptens, cell surface receptors, ligands, hormones, viruses, bacteria and the like.
- the present invention relates to a method for simultaneously detecting a first and second target species in a sample suspected of containing the two target species by a chemiluminescent reaction comprising:
- the combined action of both a hydrolytic enzyme and a peroxidase enzyme which are maintained in a closely spaced relationship to each other operates to produce a detectable chemiluminescent signal for the purpose of detecting the presence, location or amount of an analyte or target species in a sample. It is believed that the enforced proximity of the two enzymes leads to improved assay sensitivity and specificity. Previous chemiluminescent two enzyme assays using a hydrolytic enzyme as a label and the peroxidase as a reagent in a solution required much higher concentrations of the peroxidase for optimum performance. In the present methods, the ratio of peroxidase/hydrolytic enzyme ranges from about 1:1 to about 1:5.
- a DNA sample which can be crude or purified genomic DNA, a restriction digest, DNA amplified by PCR, LCR or other known amplification methods, or RNA is subjected to the method of the present invention to detect the presence of a mutation selected from a point mutation, a single base substitution, a deletion, an insertion, a duplication, a transposition of bases or a combination of the above .
- Two probes complementary to the target region are hybridized, wherein at least one of the probes is complementary to a region containing either the normal or mutant sequence .
- the probes can be directly labeled with the two requisite enzymes or can be labeled with a member of a specific binding pair and subsequently bound with an enzyme conjugate of the complementary specific binding partner.
- two differently labeled probes of m and n bases, respectively are hybridized such that, after hybridization to the target mutant sequence, the 3 ' -end of the first probe lies immediately adjacent to the 5 '-end of the second probe.
- the two probes are then ligated using a ligase to form a new probe of length m+n bases bearing both labels.
- the presence of a base or several bases in the normal target sequence between the hybridized first and second probes will result in a gap preventing the ligation of the hybridized oligonucleotides.
- the detection of the longer ligated probe indicates the presence of the mutant target sequence (m+n bases) in the sample .
- Another area of application is in a method for the detection of juxtaposed genes in chromosomal translocations.
- Associated with some pathologic conditions, including certain malignancies is the occurrence of altered DNA sequences in which genes that are normally present on two different chromosomes recombine such that the two gene sequences are juxtaposed on the same chromosome.
- a probe for the first gene directly or indirectly labeled with a hydrolytic enzyme and a probe for the second gene directly or indirectly labeled with a peroxidase are hybridized to the DNA suspected of containing the translocation. Reaction with the chemiluminescent reagent produces a signal only when the two sequences are present in the same translocated portion of DNA.
- the methods of the present invention can be used to provide a chemiluminescent method for the differentiation of heterozygotes from homozygotes for a genetic condition. Since two copies of a chromosome containing a DNA sequence of interest are present in a sample, the two enzyme label technology embodied in the present methods provides a means for distinguishing heterozygotes from either homozygote. In many genetic diseases with a recessive gene mutation, the two homozygous genotypes are readily disinguished from each other clinically asbeing normal or having a disease phenotypes. The heterozygous genotype found in phenotypically normal carriers is determined by genetic analysis or analysis of familial inheritance patterns.
- Sandwich immunoassays are well known in the art. Heterogeneous sandwich immunoassays involve the use of a first antibody (capture antibody) immobilized on a solid phase which binds to a first epitope on the analyte. A second antibody binds to a second epitope of the analyte to form the so-called sandwich.
- To adapt the chemiluminescent detection methods of the present invention to this type of assay requires only that one antibody be labeled with a peroxidase enzyme and the other with a hydrolytic enzyme. Reaction of the immune complex with the chemiluminescent reagent produces a signal emanating from the solid phase.
- Various formats for the immunological binding of two antibodies with different enzyme labels are possible. The first of these formats is shown in Figure 7. The two requisite enzyme-labeled antibodies are supplied as a 2° antibody / 3° antibody stack as shown.
- An alternate format would be to use a mixture of hydrolytic enzyme-labeled and peroxidase-labeled 2° antibodies which compete for binding to the 1° antibody.
- a third format would use a 1° antibody with one enzyme label and a 2° antibody -with the other enzyme label.
- the two different enzymes are localized at the site where the antigen is fixed, so that the chemiluminescent reaction can proceed.
- the detection medium in the above methods may take the form of a test strip, the surface of a bead, well or test tube, the detection of a band of protein on a Western blotted membrane the detection of a band of DNA on a Southern blotted membrane or in a DNA hybridization assay on a filter.
- Detecting a target species can take the form of a quantitative assay for determining the amount of the target species in the sample.
- the detection can also be qualitative in nature in which case a simple yes/no type of answer can be obtained.
- the methods of the present invention can also be employed in a test to determine the location of an analyte or target species since the enzyme labels are localized or immobilized in a region of space.
- Preferred enhancers to be generated by the action of the hydrolytic enzyme on the protected enhancer include phenolic compounds. Phenolic compounds known to enhance peroxidase reactions are well known in the art and are listed, in part, in co-pending U.S. Application No. 08/300,367.
- chemiluminescent substrate is typically supplied in an aqueous solution containing other components such as buffer salts, metal ions and preservatives which are necessary or useful for enzyme function and for storage and manufacturing considerations.
- the chemiluminescent substrate is supplied in a reagent composition comprising a buffer, the peroxide and the protected enhancer.
- Buffer pH will be in the range of about 7 to 10 and more preferable about 8 to 9.5.
- the composition can further comprise metal ions beneficial for enzyme function and preservatives to prevent microbial growth.
- the composition can further comprise suppressing agents which reduce background chemiluminescence from the reagent composition.
- Exemplary background suppressing agents include nonionic surfactants, nonionic water-soluble polymers such as polyether polyols and proteins such as serum albumin and non-fat milk.
- the composition can be prepared in advance and stored for later use or be assembled as needed from two or more containers containing the components . For example it may be desirable to store the chemiluminescent substrate separately from the peroxide .
- chemiluminescent peroxidase substrate compounds can undergo the necessary oxidation reaction in the presence of a peroxidase and a peroxide which results in the production of visible light.
- Preferred reagents include luminol 5-hydroxyphthalazinedione and N- alkylacridan-9-carboxylate derivatives including esters, thioesters and sulfonimides as disclosed in commonly assigned U.S. Patents 5,491,072 and 5,523,212 and U.S. Application No. 08/205,093 having the general formula:
- Preferred acridancarboxylate derivatives have the formula:
- Hydrolytic enzymes useful in the practice of the present invention are those which can be linked or conjugated to a specific binding partner and function to convert a protected enhancer compound to the free enhancer.
- Preferred hydrolytic enzymes include alkaline phosphatase, ⁇ -galactosidase, ⁇ -glucosidase, ⁇ -glucuronidase and carboxyl esterase. Alkaline phosphatase is most preferred.
- At least one hydrolytic enzyme must be supplied as the label on one of the two enzyme-labeled specific binding partners .
- more than one of the same enzyme molecule may be supplied and it is frequently advantageous to do so.
- the specific binding partner to be labeled is a high molecular weight or polymeric species, such as an antibody or nucleic acid
- some labeling techniques do not permit the selective attachment of just one label.
- the enzyme may be indirectly linked or associated with the specific binding partner of the target by the use of an auxiliary specific binding partner to which the enzyme is covalently linked. An example of the latter relationship would be the use of a biotin-labeled oligonucleotide probe for a certain DNA sequence bound with an enzyme-avidin conjugate.
- the probe length may be any suitable length which provides specific binding and is capable of bearing at least one label.
- Synthetic oligonucleotide probes can be from about 10-200 bases, more commonly 15-50 bases . Probes produced by cloning can be up to a few thousand bases in length. In general, shorter probes of 20- 25 bases provide better specificity and longer probes of several hundred bases long increase the sensitivity because of the ability to attach more labels. Methods of probe production and labeling are generally known in the art and do not form a part of the inventive method per se. Suitable target species for which the detection methods of the present invention can be applied include molecules or portion thereof which are capable of binding with another substance with which it has a specific binding affinity.
- Preferred target species include, nucleic acids such as ssDNA, dsDNA, RNA and oligonucleotides, proteins, antibodies, antigens, haptens, cell surface receptors, ligands, hormones, viruses, bacteria and the like. Genetic sequences can represent the target species .
- chromosomal translocation For the identification of a chromosomal translocation, probes to two nucleic acid sequences which will reside on a common fragment as a result of the rearrangement are employed. Exemplary gene rearrangements resulting from chromosomal translocations and the associated disease syndrome are listed in Table 1. A more thorough compilation can be found in T.H. Rabbitts, Nature, 372, 143-9 (1994) .
- the two target species will be the normal sequence region without the mutation and the region with the mutant sequence .
- Table 2 Examples of Single Gene Disorders with Autosomal
- proteins and high molecular weight analytes which present at least two epitopes for immunological recognition include for example antigens, immunoglobulins , hormones, including thyrotropin (TSH) , alpha-fetoprotein (AFP) , carcinoembryonic antigen (CEA) , ferritin, serum albumin, C- reactive protein and insulin.
- TSH thyrotropin
- AFP alpha-fetoprotein
- CEA carcinoembryonic antigen
- ferritin ferritin
- serum albumin C- reactive protein and insulin.
- Methods of the present invention can be applied in a Western blot format or a sandwich immunoassay format using two enzyme-labeled antibodies for the detection or quantitation of such analytes in a sample. Exemplary techniques will be presented below.
- the method may also be used to simultaneously detect the presence of an infectious agent in a clinical specimen along with the expression of host factors such as cytokines in response to the presence of the infectious agent.
- the method may also be used to simultaneously detect the presence of two different infectious agents in a clinical specimen by detecting their DNA, RNA, antigens or the antibodies produced in response to the infection.
- the present invention has as one objective to develop methods for specific and rapid detection of genetic mutations such as the CF ⁇ F mutation and their genotypes, particularly to identify heterozygous patients (carriers) in a single test.
- genetic mutations such as the CF ⁇ F mutation and their genotypes
- One method depends on the specific detection of ligated product of two uniquely labeled oligonucleotide primers that are complementary to the flanking regions of the ⁇ F mutation.
- a pair of oligonucleotides designed to anneal to the flanking regions of ⁇ F mutation are differentially labeled with the haptens biotin or fluorescein and ligated using the template DNA of wildtype (N/N) , heterozygous
- N/ ⁇ F and homozygous ( ⁇ F / ⁇ F ) genotypes.
- the ligated product containing both the labels is detected by first binding with anti-biotin-HRP (or avidin-HRP) and anti- fluorescein-AP followed by reacting with the dual substrate.
- anti-biotin-HRP or avidin-HRP
- anti- fluorescein-AP or anti- fluorescein-AP
- the ligation products were detected only in N/ ⁇ F and ⁇ F / ⁇ F genotypes but not in N/N where the ligation is precluded by the presence of three intervening nucleotides .
- the three genotypes are hybridized on a membrane simultaneously with uniquely labeled (biotin or digoxigenin) oligonucleotides designed to bind the normal and mutant alleles.
- uniquely labeled biotin or digoxigenin
- oligonucleotides designed to bind the normal and mutant alleles.
- HRP and AP- conjugated specific binding partners followed by reacting with a composition comprising a chemiluinescent peroxidase substrate, a peroxide and a protected enhancer, only the band from N/ ⁇ F genotype emitted a strong signal because of the binding of both oligonucleotides.
- the utility of a novel dual probe/dual substrate method for differentiating the genotypes of ⁇ F mutation of cystic fibrosis is demonstrated in the examples presented hereinafter.
- the ligation and hybridization formats are exemplary. Other formats may be devised for the detection of mutations as well.
- An ELISA-type detection format can be used where the sample DNA is immobilized and a pair of enzyme-labeled oligonucleotide probes are hybridized and optionally ligated. Alternatively, one of the enzyme-labeled oligonucleotides can be immobilized as a capture probe. After or concurrently with capture of target DNA, the other enzyme-labeled oligonucleotide is hybridized.
- Example 1 Detection of Two Co-immobilized DNA Sequences .
- the detection scheme is based on our discovery that an enzymatically liberated phenol enhancer markedly increases the intensity and duration of light emission from horseradish peroxidase (HRP) catalyzed oxidation of N-alkylacridan-9-carboxylic acid derivatives.
- HRP horseradish peroxidase
- Binding of a pair of differentially labeled oligonucleotide probes by a target on a solid phase places both enzymes in close proximity.
- An AP-labeled probe generates the phenol enhancer in situ from a phosphate derivative in the vicinity of a peroxidase-labeled probe.
- the phenol enhancer catalyzes the light producing reaction. Nonspecific signal only arises if both probes bind unspecifically at the same site .
- the digoxigenin-labeled and fluorescein-labeled were bound to HRP-anti-fluorescein and AP-anti-digoxigenin and then detected with a reagent containing 2-naphthyl phosphate, peroxide, and 2 , 3 , 6-trifluorophenyl 10-methylacridan-9- carboxylate. Unlabeled DNA, dig-labeled DNA and fluorescein-labeled DNA alone were not detectable.
- the gel was rinsed with water and then treated with 1 M Tris-HCl (pH 7.5) /I .5 M NaCl three times for 15 min each.
- the DNA in the gel was transferred onto the membrane by capillary blotting overnight using 10X SSC.
- the blots were rinsed with 5X SSC twice for 5 min each and then air-dried for 30 min followed by baking at 80°C for 2 hours.
- the blots were rinsed with TBS and then blocked with 0.5% blocking agent (Amersham) in TBS. The fragments were reacted with a mixture of HRP-anti-fluorescein antibody and AP-anti-digoxigenin antibody (Boehringer- Mannheim) in 0.5% bovine serum albumin in TBS for 40 min. The blots were washed with 0.05% Tween-TBS four times for 10 min each and then rinsed with water.
- the membrane was wetted with a detection reagent consisting 1 mM of the protected HRP enhancer 2-naphthyl phosphate, 0.05 mM 2,3,6- trifluorophenyl 10-methylacridan-9-carboxylate, 2.5 mM urea peroxide, 0.5 % Tween 20 in 0.01 M tris buffer, pH 8.8 and incubated for 25 min. Exposure of the membrane to X-ray film for 20 min produced an image of the set of bands in lane 4 of Figure 1. As controls, lane 1 contained 94 ng of unlabeled DNA, lane 2 contained 30 ng of the digoxigenin- labeled DNA, lane 3 contained 66 ng of the fluorescein- labeled DNA.
- Example 2 Specific Detection of Target DNA with Dual Probes .
- This example demonstrates the detection of a target ssDNA using two complementary probes .
- the probes were each labeled with a distinct hapten which was bound to an enzyme conjugated binding partner after the probe hybridization.
- the latter 17-mer was then labeled with digoxigenin-dUTP using a DIG Oligonucleotide Tailing Kit (Boehringer- Mannheim) .
- the yield of dig-labeled P-2100 was estimated by dot blotting with the standard dig-labeled oligonucleotide from the kit and using AP-anti-digoxigenin and NTP/X-P for colorimetry.
- MagnaGraph nylon was soaked sequentially in water and 10X SSC for 2 and 10 min, respectively.
- the gel was rinsed with water, soaked in 0.25 N HCl for 10 min and then treated with 0.5 M NaOH/1.5 M NaCl twice for 15 and 30 minutes, respectively.
- the gel was rinsed with water and then treated with 1 M Tris-HCl (pH 7.5) /l.5 M NaCl three times for 15 min each.
- the DNA in the gel was transferred onto the membrane by capillary blotting overnight using 10X SSC.
- the blots were rinsed with 5X SSC twice for 5 min each and then air-dried for 30 min followed by baking at 80°C for 2 hours .
- the blots were soaked in 6X SSPE for 10 min and then prehybridized in prehybridization buffer (0.5% blocking agent, 0.1% SDS, 6X SSPE, 100 ⁇ g/mL freshly denatured herring sperm DNA) for 1 h at room temperature.
- the hybridization was done in the prehybridization buffer with 0.48 pmol/mL fluorescein-labeled P-2051 and 158 pmol/mL digoxigenin-labeled P-2100 for 15 h at 37 °C .
- the blots were washed with 2.5X SSC twice for 5 min each at room temperature and then with 2.5X SSC, 0.1% SDS for 30 min at room temperature.
- the blots were washed with IX SSC, 0.1% SDS twice for 15 min each at 37 °C and with 2.5X SSC for 5 min at room temperature .
- the blots were soaked in TBS and then blocked with 0.5% blocking agent in TBS for 1 h at room temperature.
- the blots were rinsed with TBS and then reacted with AP-anti- digoxigenin and HRP-anti-fluorescein (Amersham) in 0.5% BSA in TBS for 45 min at room temperature.
- the blots were washed with 0.05% Tween-TBS four times for 10 min each and then rinsed with water.
- the membrane was wetted with a detection reagent consisting of 1 mM of the protected HRP enhancer 2-naphthyl phosphate, 0.05 mM 2 , 3 , 6-trifluorophenyl 10-methylacridan-9-carboxylate, 2.5 mM urea peroxide, 0.5 % Tween 20 in 0.01 M tris buffer, pH 8.8 and incubated for 13 min.
- a detection reagent consisting of 1 mM of the protected HRP enhancer 2-naphthyl phosphate, 0.05 mM 2 , 3 , 6-trifluorophenyl 10-methylacridan-9-carboxylate, 2.5 mM urea peroxide, 0.5 % Tween 20 in 0.01 M tris buffer, pH 8.8 and incubated for 13 min.
- the 40, 20, 10 and 5 ng samples of linearized pBR322 DNA were clearly detected after a 13 min incubation and 10 min exposure . Controls in which one
- Example 3 Detection of the Cystic Fibrosis ⁇ F5Q8 Mutation by Ligation of Enzyme-Labeled Probes.
- This experiment demonstrates the detection and determination of carrier status, of the CF ⁇ F508 mutation.
- the method relies on the hybridization of two differently labeled probes of n and m bases, respectively, flanking the deletion. Ligation of the adjacent bound probes in the mutant allele creates a new probe of m+n bases carrying both labels. Nonligated and ligated probes are resolved by denaturing gel electrophoresis, blotted onto a nylon membrane, reacted with enzyme-antibody conjugates and detected by the dual enzyme-catalyzed luminescence method. The method of detecting and differentiating the ⁇ F508 genotypes is shown schematically in Figure 3.
- the chemiluminescent detection reagent comprised 0.01 M tris, pH 8.8, 1 mM 2- naphthyl phosphate, 2.5 mM urea peroxide, 0.5 % Tween-20, 0.3 mM 2 , 3 , 6-trifluorophenyl 10-methylacridan-9- carboxylate .
- DNA samples of the wildtype (N/N) , heterozygous (N/ ⁇ F 508 ) and homozygous ( ⁇ F 508 / ⁇ F 508 ) genotypes were obtained from the Coriell Cell Repositories, Camden, New Jersey. Samples negative for the ⁇ F 508 mutation were used as negative controls .
- the PCR was performed in 100 ⁇ l reaction consisting of IX buffer (10 mM Tris-HCl, pH 8.3, 50 mM KC1) , 200 UM of each deoxynucleotide, 1 ⁇ M of each primer, 2.5 mM MgCl 2 , 2.5 units of Amplitaq DNA polymerase (Roche Molecular Systems, Branchburg, NJ) , and 0.5 ⁇ g of genomic DNA template.
- the DNA was amplified in Perkin-Elmer 480 (Norwalk, CT) thermal cycler for 35 cycles with an overlay of mineral oil. Each cycle consisted of 1 min each of denaturation, primer annealing, and extension at 94 °C , 60 °C and 72 °C, respectively, with a 10 min extension at the end of cycling. Prior to the cycling, the template DNA was denatured at 95 °C for 3 min, quickly cooled on ice and added to the PCR reaction tube. Specific amplification of the correct size product was confirmed on an 1% agarose gel containing ethidium bromide.
- the amplified DNA was purified using QIAquick purification kit (QIAGEN, Chatsworth, CA) to remove unincorporated deoxyribonucleotides and oligonucleotide primers. Purified DNA was quantitated spectrophoto- metrically before use in the ligation and hybridization experiments described below.
- oligonucleotides Two oligonucleotides, one labeled with biotin and the other with fluorescein, and differing in length by three nucleotides (21 and 24mers) were designed to anneal to the sense strand of the DNA template and are designated antisense upstream:
- the ligation reaction consisted of 200 ng of purified PCR amplified template of each genotype, 10 ng each of the oligonucleotides, IX ligation buffer (30 mM Tris-HCl, pH 7.8, 10 mM MgCl 2 , 10 mM DTT and 0.5 mM ATP) and 5 units of T4 DNA ligase. These concentrations of the template and the oligonucleotides provided for optimum discrimination of genotypes.
- the template-oligonucleotide mix was first heated at 95 °C for 3 min and quickly cooled on ice before adding to it the ligation buffer and ligase. The ligations were performed in 20 ⁇ l reaction at 15°C overnight.
- a negative control ligation reaction of the labeled oligonucleotides in the absence of template was also performed.
- the ligation reactions were fractionated on 16% or 20% denaturing polyacrylamide gels (7 M Urea) along with a dual labeled (biotin and fluorescein) oligonucleotide as size marker (45mer) .
- the electrophoresed ligation reactions were capillary transferred onto a 0.2 ⁇ m MagnaGraph nylon membrane in 10X SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.2) .
- the blotted membrane was baked at 80 °C for 2 h.
- the working concentrations of all the enzyme conjugates were 1:5000 dilutions in 2% blocking buffer. Following treatment with the enzyme conjugate, the membranes were washed twice for 20 min each in IX wash buffer and then reacted with the chemiluminescent substrate for 5 min in the dark.
- Example 4 Detection of the Cvstic Fibrosis ⁇ F508 Mutation by Hybridization of Enzvme-Labeled Probes.
- the hybridization method of detecting and differentiating the ⁇ F508 genotypes is shown schematically in Figure 5.
- a pair of differentially labeled (biotin and digoxigenin) oligonucleotide probes one complementary to the normal and the other to the mutant allele, were hybridized simultaneously to the membrane bound DNA templates and reacted sequentially with the antibody-enzyme conjugates followed by the chemiluminescent substrate.
- the labeled oligonucleotides are 5' biotin - ATA TCA TCT TTG GTG TTT CCT 3' (Seq. ID#7) (normal) and 5' digoxigenin - GAA AAT ATC ATT GGT GTT TCC 3' (Seq. ID#8) (mutant) .
- the oligonucleotides were custom synthesized by Oligos etc., ( ilsonville, OR) .
- the amplified PCR products of each genotype (100 ng) were electrophoresed on a 1% agarose gel for a short distance (1 cm) so that the PCR products of normal and ⁇ F 508 alleles differing by three nucleotides migrate as a single band.
- the gel was depurinated (0.25 M HCl), denatured (0.5 N NaOH, 1.5 M NaCl), neutralized (0.5 M Tris-HCl, pH 7.5, 3 M NaCl) , and vacuum blotted onto a neutral nylon membrane (Hybond N, Amersham, Arlington Heights, IL) .
- the blotted membrane was baked at 80 °C for 2 hours and hybridized with the biotin and digoxigenin labeled oligonucleotide probes as described below.
- the blot was prehybridized and hybridized at 52 °C for 1 h and overnight, respectively, using a buffer containing 6X SSC (0.9 M sodium chloride, 0.09 M sodium citrate, pH 7.0), 0.01 M EDTA, pH 8.0, 5X Denhardt's solution (0.1% Ficoll Type 400, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 0.5% SDS and 100 ug/ml denatured salmon sperm DNA (GIBCO BRL, Life Technologies, Gaithersburg, MD) .
- 6X SSC 0.9 M sodium chloride, 0.09 M sodium citrate, pH 7.0
- 0.01 M EDTA 0.01 M EDTA
- pH 8.0 pH 8.0
- 5X Denhardt's solution 0.1% Ficoll Type 400, 0.
- the working concentrations of all the antibody-enzyme conjugates were 1:5000 dilutions in 2% blocking buffer. Following the antibody-enzyme treatment, the membranes were washed twice for 20 min each in IX wash buffer and then reacted with the chemiluminescent substrate for 5 min in the dark. Excess substrate from the membrane was removed by placing and gently pressing the membrane between transparency films. The membrane was then exposed to an X- ray film for a time period, generally ranging from a few seconds to minutes, to obtain optimal signal and background.
- Example 5 Random testing of specimens of non-CF individuals . Both the ligation and hybridization methods were used to test DNA specimens of unknown CF genotype for ⁇ F 5_. capita08 mutation using the method of Example 3 and 4. None of the four specimens tested produced positive signals by either the ligation and hybridization methods indicating lack of the ⁇ F mutation. Conversely, known heterozygotes yielded a strong positive signal by both methods.
- the dual substrate and probe systems presented here can also be utilized for the detection of more than one nucleic acid entity such as the presence of two different infectious agents in a clinical specimen.
- Sequence specific oligonucleotide probes are designed to a gene sequence that is unique to each of the infectious agent.
- One probe is labeled with digoxigenin and the other with fluorescein.
- the target DNA of the specimen can be amplified by PCR.
- the probes are hybridized to their respective target DNA, extracted from a clinical specimen, which is either bound to a nylon membrane or is in solution.
- the amplified DNA target is biotinylated (by using the biotinylated primers for the amplification) so that the hybridized probe-target DNA complexes are captured onto streptavidin coated microtiter wells.
- the hybridized DNA is treated with anti-hapten AP and HRP antibodies followed by reacting with the dual substrate for chemiluminescent detection.
- Example 7 Detection of Gene Rearrangements .
- the dual substrate and probe methods of the present invention are utilized for the detection of gene rearrangements where two DNA segments are fused as a consequence of a chromosomal translocation.
- the genes are on separate chromosomes. Following the chromosomal translocation the genes are fused to form a contiguous piece of DNA.
- SUBSTiTUIE SHEET (RULE 26)
- the target gene regions involved in the gene rearrangement are amplified by PCR using a forward sense primer of gene 1 and a reverse antisense primer of gene 2.
- This primer pair amplifies only the contiguous DNA resulting from the gene rearrangement and no amplification results from the untranslocated DNA.
- the size of this amplified DNA will depend on the site of interchange between the two genes involved in the translocation.
- the detection of the amplified target can be acheived in two ways.
- the target is immobilized in wells of a streptavidin coated microtiter well in which case the primers used for the amplification are labeled with biotin.
- the amplified product is immobilized on a nylon membrane without the need to use biotinylated primers for amplification.
- the target is hybridized to a digoxigenin-labeled probe to gene 1 and a fluorescein-labeled probe to gene 2.
- the membrane or well is blocked to prevent non-specific binding of antibodies and then anti-digoxigenin-AP and anti-fluorescein-HRP antibodies are bound to the immobilized probes as described above.
- the reagent of Example 3 is added and chemiluminescence detected with x-ray film or a CCD camera when the method is performed on a blotting membrane or with a microtiter plate luminometer when the method is performed in a microtiter plate.
- the specimen with gene rearrangement is positively identified with the chemiluminescent signal.
- hybridization and ligation formats used can be adapted for automation to reduce ' turnaround time which is desirable for clinical laboratory setting.
- Example 8 Western Blot Assay Using Dual Enzyme Labels .
- the dual substrate and probe systems presented here was utilized in a Western blot assay of the HIV ⁇ -gpl20 protein.
- the assay format is shown in Figure 7. Materials and Methods. Affinity purified rabbit IgG anti-mouse IgG2a-AP conjugate and sheep IgG anti-rabbit IgG-peroxidase conjugate were obtained from Cappel Products (Durham, NC) .
- HIV-l- ⁇ gpl20 50 ⁇ g/500 ⁇ L
- mouse IgG2a anti-human HIV-l- ⁇ gpl20 MAblCL
- the HIV-l- ⁇ gpl20 and IgG samples were centrifuged at 10,000g for 2 min and the supernatant was used in the immunological reaction.
- IMMOBILON-P PVDF membrane was from Millipore (Bedford, Mass . ) SDS-PAGE was performed utilizing the buffer system described by Laemmli (U.K. Laemmli, Nature (London) , 227, 680-5 (1970)).
- the stacking gel was 4.38% acrylamide : 0.12% bisacrylamide.
- the separating gel was 6.81% acrylamide : 0.19% bisacrylamide.
- the transfer buffer which contained 20 mmol/L Tris, 153 mmol/L glycine and 20% (v/v) methanol.
- the gel sandwiched between a sheet of membrane and a sheet of Whatman 3MM chromatography paper, was placed in the transfer unit (Bio-Rad Laboratories, Richmond, CA) .
- the proteins in the gel were electroeluted for 25 min at 4° C at 100 V constant voltage.
- the membrane was then placed in 50 mmol/L Tris-HCl buffered saline at pH 7.4 (TBS) at 4° C overnight.
- Blocking was performed with 0.05% Tween-20 in TBS, pH 7.4 (T-TBS) containing 1% non-fat powdered milk (NFM) for 1 h at room temperature .
- T-TBS TBS, pH 7.4
- NFM non-fat powdered milk
- the blocked membrane was incubated for 90 min at room temperature with 1° antibody (1:250 dilution of mouse IgG2a anti-human HIV-l- ⁇ gpl20) using T-TBS containing 1% NFM.
- the membrane was then rinsed and washed three times for 5 min each with T-TBS at room temperature.
- the washed membrane was incubated for 75 min at room temperature with 2° antibody (1:250 dilution of rabbit IgG anti-mouse IgG2a-AP) using T-TBS containing 1% NFM.
- the membrane was rinsed and washed four times for 5 min each with T-TBS.
- the washed membrane was next incubated for 60 min at room temperature with 3° antibody (1:20,000 dilution of sheep IgG anti-rabbit IgG-peroxidase) using T-TBS containing 1% NFM.
- the membrane was rinsed and washed four times for 5 min each with T-TBS followed by a 5 min wash with TBS.
- the washed membrane was incubated in the detection reagent of Example 1 for 10 min, drained and placed between sheets of transparency film.
- the amount of antigen loaded in the five lanes of the gel was 1) 8 ng, 2) 4 ng, 3) 2 ng, 4) 1 ng, 5) 0.5 ng.
- all five bands were clearly detectable over the background in a 3 min exposure less than one hour after the membrane was contacted with detection reagent. Images could be easily obtained over the course of a day. Additional experiments under identical conditions have demonstrated detection of 30 pg in under 3 hours .
- the specificity of the assay was evaluated by performing it without the HRP-antibody conjugate in one experiment and without the AP-antibody conjugate in another experiment. No signal was detected for any of the five bands in each experiment. In addition, an experiment using both enzyme-labeled antibodies but omitting the 2-naphthyl phosphate from the detection reagent also produced no signal.
- Example 9 Immunoassay Using Dual Enzyme Labels.
- a procedure for a sandwich immunoassay for the detection of hCG according to the methods of the present invention is as follows. Mouse anti-hCG antibody (Sigma) is labeled with
- mouse anti-hCG antibody conjugated to AP by the periodate method or using a bifunctional coupling agent containing succinimide and maleimide moieties.
- the wells of a white microplate are coated with mouse anti-hCG antibody-HRP conjugate, washed and aspirated. Aliquots of hCG standards and samples are added and incubated for 1 h. The wells are washed 3x and aspirated. The wells are incubated a solution containing mouse anti-hCG antibody-AP conjugate and 0.1 % blocking agent for 1 h, washed 3x and aspirated.
- the detection reagent of Example 1 is added and chemiluminescence intensity measured at 10 min.
Abstract
Description
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EP97913856A EP0938677A4 (en) | 1996-11-15 | 1997-11-07 | Chemiluminescent detection methods using dual enzyme-labeled binding partners |
AU50940/98A AU726512B2 (en) | 1996-11-15 | 1997-11-07 | Chemiluminescent detection methods using dual enzyme-labeled binding partners |
JP52259598A JP2001504226A (en) | 1996-11-15 | 1997-11-07 | Chemiluminescence detection using dual enzyme-labeled binding partners |
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US08/749,595 | 1996-11-15 | ||
US08/749,595 US5843666A (en) | 1994-09-02 | 1996-11-15 | Chemiluminescent detection methods using dual enzyer-labeled binding partners |
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EP (1) | EP0938677A4 (en) |
JP (1) | JP2001504226A (en) |
AU (1) | AU726512B2 (en) |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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Families Citing this family (43)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6322980B1 (en) | 1999-04-30 | 2001-11-27 | Aclara Biosciences, Inc. | Single nucleotide detection using degradation of a fluorescent sequence |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5306621A (en) * | 1989-10-17 | 1994-04-26 | British Technology Group Limited | Enhanced chemiluminescent assay |
US5491072A (en) * | 1993-05-17 | 1996-02-13 | Lumigen, Inc. | N-alkylacridan carboxyl derivatives useful for chemiluminescent detection |
US5686258A (en) * | 1993-05-17 | 1997-11-11 | Lumigen, Inc. | Chemiluminescent detection of hydrolytic enzymes using an acridan |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4563417A (en) * | 1984-08-31 | 1986-01-07 | Miles Laboratories, Inc. | Nucleic acid hybridization assay employing antibodies to intercalation complexes |
US4883750A (en) * | 1984-12-13 | 1989-11-28 | Applied Biosystems, Inc. | Detection of specific sequences in nucleic acids |
US4810631A (en) * | 1986-05-12 | 1989-03-07 | Becton, Dickinson And Company | Signal enhancement in immunoassay by modulation of chemical catalysis |
US4835099A (en) * | 1986-11-20 | 1989-05-30 | Becton, Dickinson And Company | Signal enhancement in immunoassay by modulation of enzymatic catalysis |
US4943525A (en) * | 1987-11-02 | 1990-07-24 | Bioventures, Inc. | Simultaneous immunoassay for the determination of antigens and antibodies |
EP0343955A3 (en) * | 1988-05-27 | 1990-04-18 | Bioventures, Inc. | Process for complex binding of targeted molecular species |
US5185243A (en) * | 1988-08-25 | 1993-02-09 | Syntex (U.S.A.) Inc. | Method for detection of specific nucleic acid sequences |
DE68911633T2 (en) * | 1988-09-30 | 1994-04-07 | Fujirebio Kk | Method for determining peroxidase activity using chemiluminescence. |
JPH0786096B1 (en) * | 1989-04-28 | 1995-09-20 | ||
US5324835A (en) * | 1990-03-30 | 1994-06-28 | Biosensor Laboratories Co., Ltd. | Pyridazinoquinoxalinones for use as chemiluminescent agents |
US5552298A (en) * | 1992-10-23 | 1996-09-03 | Lumigen, Inc. | Enzyme-catalyzed chemiluminescence from hydroxyaryl cyclic diacylhydrazide compounds |
US5523212A (en) * | 1993-05-17 | 1996-06-04 | Lumigen, Inc. | Aryl N-alkylacridanthiocarboxylate derivatives useful for chemiluminescent detection |
-
1996
- 1996-11-15 US US08/749,595 patent/US5843666A/en not_active Expired - Fee Related
-
1997
- 1997-11-07 CA CA002259963A patent/CA2259963A1/en not_active Abandoned
- 1997-11-07 WO PCT/US1997/019612 patent/WO1998021586A1/en not_active Application Discontinuation
- 1997-11-07 EP EP97913856A patent/EP0938677A4/en not_active Withdrawn
- 1997-11-07 JP JP52259598A patent/JP2001504226A/en active Pending
- 1997-11-07 AU AU50940/98A patent/AU726512B2/en not_active Ceased
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5306621A (en) * | 1989-10-17 | 1994-04-26 | British Technology Group Limited | Enhanced chemiluminescent assay |
US5491072A (en) * | 1993-05-17 | 1996-02-13 | Lumigen, Inc. | N-alkylacridan carboxyl derivatives useful for chemiluminescent detection |
US5686258A (en) * | 1993-05-17 | 1997-11-11 | Lumigen, Inc. | Chemiluminescent detection of hydrolytic enzymes using an acridan |
Non-Patent Citations (1)
Title |
---|
See also references of EP0938677A4 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1119554A1 (en) * | 1998-09-11 | 2001-08-01 | Tropix, Inc. | Xanthan-ester and acridan substrates for horseradish peroxidase |
EP1119554A4 (en) * | 1998-09-11 | 2002-05-08 | Tropix Inc | Xanthan-ester and acridan substrates for horseradish peroxidase |
GB2355791B (en) * | 1999-10-28 | 2004-10-20 | Molecular Light Tech Res Ltd | Detection of mRNA expression using chemiluminescent labelled probes |
US6682903B2 (en) | 2000-10-14 | 2004-01-27 | Alex M. Saunders | Ligand based solution assay for low concentration analytes |
US6936429B2 (en) | 2000-10-14 | 2005-08-30 | Chronomed, Inc. | Ligand based solution assay for low concentration analytes |
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AU5094098A (en) | 1998-06-03 |
EP0938677A1 (en) | 1999-09-01 |
AU726512B2 (en) | 2000-11-09 |
EP0938677A4 (en) | 2000-09-27 |
JP2001504226A (en) | 2001-03-27 |
US5843666A (en) | 1998-12-01 |
CA2259963A1 (en) | 1998-05-22 |
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