WO2002050537A1 - Absorbance multiplex technology - Google Patents
Absorbance multiplex technology Download PDFInfo
- Publication number
- WO2002050537A1 WO2002050537A1 PCT/US2001/049745 US0149745W WO0250537A1 WO 2002050537 A1 WO2002050537 A1 WO 2002050537A1 US 0149745 W US0149745 W US 0149745W WO 0250537 A1 WO0250537 A1 WO 0250537A1
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- WO
- WIPO (PCT)
- Prior art keywords
- enzyme
- antibody
- conjugated
- substrate
- marker
- Prior art date
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
Definitions
- the present invention relates to immunodiagnostics. More specifically, the invention relates to an immunoassay method for simultaneously detecting and quantifying multiple target molecules from the same sample, in the same reaction well, and at the same time.
- ELISA solid-phase heterogeneous enzyme-linked immunosorbent assay
- the enzyme-labeled ligand is an enzyme-labeled antibody.
- Commonly used enzymes include alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, ⁇ - glucuronidase, luciferase, and urease.
- the enzyme-labeled ligand binds the target molecule/bound antibody complex.
- a substrate specific for the enzyme, is added to the solid phase support.
- Commonly used substrates include 3,3', 5,5'-tetramethyl benzidine (“TMB”), para- nitrophenyl phosphate (“PnPP”), o-phenylene diamine (“OPD”), 2, 2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) (“ABTS”) o-nitrophenyl-D-galatopyranoside (“ONGP”) and 5-bromo-4-chloro-3 indolyl phosphate (“BCIP”).
- TMB 3,3', 5,5'-tetramethyl benzidine
- PnPP para- nitrophenyl phosphate
- OPD o-phenylene diamine
- ABTS 2, 2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid)
- ABTS 2, 2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid)
- ABTS 2, 2
- ELISA has been used in a wide variety of immunodiagnostic applications such as serodiagnositics to detect antigens from a wide range of specific viruses, bacteria, fungi, and parasites. ELISA is also used to monitor factors involved in non-infectious disease such as hormone levels, hematological factors, serum tumor markers, drug levels and antibody levels. ELISA can be used to monitor treatment for an allergy or measure antibodies that develop in response to an infectious disease. Further, ELISA can be used to screen for chemicals in a biological sample or to screen for pesticides or microorganisms in soil or water samples.
- the current invention detects and quantitates multiple target molecules from the same sample, in the same reaction well and at the same time.
- antibodies to different target molecules may be bound to a reaction surface.
- a sample solution believed to contain the target molecules for example, a biological specimen, may then be added to the coated wells and incubated. During incubation, the target molecules form complexes with the bound antibodies.
- an enzyme-bound antibody mixture may be added to the wells.
- the enzyme-bound antibodies have an affinity for the different target molecule/bound antibody complexes.
- the invention may also be performed by coating the antigens directly to a reaction surface such as a coated or noncoated polystyrene well, or to a solid or semisolid phase support. After a washing step, an enzyme-bound antibody mixture may then be added to a single reaction surface.
- the enzyme-bound antibodies have an affinity to existing antigens.
- the mixture may be developed sequentially by the addition of specific enzyme substrates where changes in colors are measured and analyzed using one wavelength, without poisoning any of the enzymes.
- a goal of the present invention is to provide a method for determining the level of several target molecules in the same sample, in the same reaction well and at the same time by a single absorbance immunoassay technique.
- Another aim of the present invention is to provide a new, enhanced and modified enzyme-linked immunosorbent assay (ELISA) or EIA as a kit or individual reagents for the rapid diagnosis and/or prognosis of diseases or the evaluation of responses that involve the measurement of more than one target molecule which can be used on a routine basis in clinical or non-clinical laboratories.
- the same reaction may be used to identify unrelated target molecules from one individual to diagnose multiple diseases.
- the invention thus includes methods of making the kit according to the invention.
- Target molecules include, but are not limited to, antigens, or fragments thereof, including antigens derived from microorganisms and other pathogens, antibodies or fragments thereof, including antibodies produced in response to antigens derived from microorganisms and other pathogens, tumor markers, oligonucleotides, nucleic acids, carbohydrates, proteins, chemicals, drugs, receptors, haptens, hormones, gamma globulins, allergens, viruses, virus subunits, bacteria, toxins such as those associated with tetanus and with animal venoms, enzymes, nucleic acid molecules including DNA fragments, RNA fragments, and artificial nucleic acid fragments, and self-antibodies generated in autoimmune disease, or any mix thereof.
- absorbance immunoassays are only capable of detecting one target molecule at a time because the buffers and substrates used to react with one enzyme are often toxic to another enzyme.
- Several antigenic substances or markers are often associated with a pathological or physiological disorder and can be early indicators of disease.
- the accurate diagnosis of infection or disease may require several different specimens and several different screening processes.
- a toxicology screening may search for several different chemicals or drugs in a blood or urine sample.
- researchers are often interested in more than one target molecule.
- detection antibodies or fragments thereof
- any antagonist that irreversibly binds the target molecule including antigens, primers, nucleic acids, or fragments thereof, that recognize specific proteins, markers, receptors or antibodies, or fragments thereof, to be detected.
- an artificial or naturally occurring sequence having affinity for the target nucleic acid sequence may be used.
- the detecting sequence need not necessarily be complementary to the target sequence. The detecting sequence should have sufficient affinity for the target sequence so that the two sequences remain bound during the detection process.
- antigens When using antigens to detect target molecules, several antigens, each specific for a different target molecule of interest, are bound to a reaction surface.
- the antigen may be bound to any reaction surface, solid or semisolid phase support, including, but not limited to, polystyrene wells of microtitre plates, glass or plastic strips, glass or plastic beads, membranes, microplates, magnetic particles, latex particles, nitrocellulose particles, polyacrylamide beads, magnetic beads, polystyrene, polyurethane, agarose, collagen, gelatin, SEPHAROSE, SEPHADEX, SEPHARON, nylon, rayon, and test tubes.
- the antigen is bound to a single reaction well of the reaction surface.
- reaction well is intended to include reaction wells, for example, in a microtitre plate, as well as any localized area on a reaction surface such that the multiple markers are detected and quantitated on the same general section.
- the present invention does not require that the antigen be bound to separated areas of a reaction surface based on the specificity of the antigen.
- the reaction well having bound antigen may be washed and a sample believed to contain the target molecules may be introduced to the reaction well.
- the target molecules bind to the bound antigen in the reaction well.
- a first enzyme-conjugated antibody having an affinity for a first target molecule and a second enzyme-conjugated antibody having an affinity for a second target molecule are added to the reaction well.
- an enzyme substrate specific for the first enzyme may be added and any enzymatic activity, for example, a color change, may be measured.
- a second enzyme substrate specific for the second enzyme may be added and any color change may be measured. The changes in color that are visualized, for example, using dip sticks or light microscopy, indicate the presence of the target molecules.
- An embodiment of the invention includes a method of simultaneously detecting and quantitating multiple markers in a single reaction well of a reaction surface.
- the reaction well may be coated with at least three molecules, each having an affinity for different markers.
- the molecules can be any antagonist having a binding affinity for the target molecules, including, but not limited to, single chain antibody fragments, antibody, antibody fragments, artificial antibodies, peptides, chemical entities, lipids, carbohydrates, artificial nucleic acid sequences, DNA, RNA, antigen or fragments thereof.
- the molecules are antigen or antibody or fragments thereof.
- the reaction well or reaction surface may be coated with the molecules by any method known in the art.
- the reaction surface having molecules bound thereon may be purchased commercially.
- attachment to the reaction surface may be non-covalent wherein the molecule binds to the reaction surface through adsorption.
- the attachment may be covalent wherein the molecule is chemically coupled to the reaction surface with a linker molecule.
- a wash step may be performed after the reaction well is coated.
- a sample suspected of containing at least one marker may be added to the reaction well.
- sample can be any sample of interest, including, but not limited to, a biological sample, including, but not limited to, serum, blood, urine or saliva, a soil sample, or any liquid sample, including water.
- Biological samples may be of either human or non-human origin.
- the markers bind different, specific bound molecules.
- a wash step may be performed after the bound molecule/marker complexes are formed to remove unbound molecules.
- the ligand may be any antagonist having a binding affinity for the reactant, including, but not limited to, single chain antibody fragments, antibody, antibody fragments, artificial antibodies, peptides, chemical entities, lipids, carbohydrates, artificial nucleic acid sequences, DNA, RNA, antigen or fragments thereof.
- the sample and solution are incubated for a sufficient time to allow the first marker to bind with the first conjugated ligand, the second marker to bind with the second conjugated ligand and the third marker to bind with the third conjugated ligand.
- the first reactant is ⁇ -galactosidase and the first substrate is ONGP
- the second reactant is horseradish peroxidase ("HRP") and the second substrate is ABTS
- the third reactant is alkaline phosphatase (“AP”) and the third substrate is PnPP.
- a wash step occurs before the first marker is detected.
- the presence of the first marker may be detected by adding a first substrate, specific for the first reactant, to the reaction well. After incubation for a sufficient time to allow a reactant/substrate reaction, the reaction well can be assayed for reactant activity.
- the reactant is an enzyme
- the reactant activity is preferably a color change detectable with light microscopy.
- a wash step occurs before the second marker is detected.
- the presence of the second marker may be detected by adding a second substrate, specific for the second enzyme, to the reaction well. After sufficient incubation, the reaction well may be assayed for a color change.
- a wash step may occur before the third marker is detected.
- the presence of the third marker may be detected by adding a third substrate, specific for the third enzyme, to the reaction well. After sufficient incubation, the reaction well may be assayed for a color change.
- the present invention can be used to identify two or more markers of interest.
- the invention also includes a kit for the simultaneous identification of multiple markers in a biological sample.
- the kit may include a reaction surface having a coating of at least three molecules immobilized thereon. Each molecule may be specific for a different marker of interest.
- the kit may also include a conjugated antibody solution including a first antibody having affinity for a first marker, a second antibody having affinity for a second marker, and a third antibody having affinity for a third marker.
- the first antibody may be conjugated to a first enzyme
- the second antibody may be conjugated to a second enzyme
- the third antibody may be conjugated to a third enzyme.
- the kit may also include reagents for detecting the labeled antibodies, a first substrate specific for the first enzyme, a second substrate specific for the second enzyme and a third substrate specific for the third enzyme.
- the first reactant is ⁇ -galactosidase and the first substrate is GNGP
- the second reactant is HRP and the second substrate is ABTS
- the third reactant is AP and the third substrate is PriPP.
- the invention further includes a method of making the kit and a method of using the kit to diagnose a disease or identify the presence of multiple target molecules.
- 0.5 mg of dialyzed antibody was added to 1.5 mg of the enzyme in 10 ml of 10 mM PBS.
- 80 ⁇ l 25% glutaraldehyde was added and mixed gently.
- the solution was let stand at room temperature for 2 hrs.
- the reaction was stopped by adding an equivalent volume (10 ml) of PBSLE (10 mMPBS containing 100 mMlysine and 100 mM ethanolamine).
- PBSLE 10 mMPBS containing 100 mMlysine and 100 mM ethanolamine.
- the solution was desalted with a SEPHADEX G25 column in PBSN (10 mMPBS with 0.05MNaN 3 ).
- the antibody-coated plate was rinsed by flooding with washing buffer (20 mM TrisHCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5) a minimum of three times. Each well was filled with blocking buffer (0.2% BSA, 0.01% Gelatin in TBS, pH 7.5) dispensed using multichannel pipettes and incubated 15 min at 37° C. The plate was rinsed three times with washing buffer and any residual liquid was removed by gently flicking it face down onto paper towels.
- washing buffer (20 mM TrisHCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5
- blocking buffer (0.2% BSA, 0.01% Gelatin in TBS, pH 7.5
- Step I 200 ⁇ l aliquots of 1/50 serum dilution in PBS of the test antigen sample solutions (patient serum or plasma enzyme) or the standard antigen dilutions were added to the antibody-coated wells and incubated for 30 minutes at 37° C. The plates were rinsed three times in wash buffer and residual liquid was removed by blotting.
- Step II 200 ⁇ l of specific antibody-enzyme conjugate, IgA-AP aliquots of 1/500 dilution, IgG-HRP and IgM- ⁇ -Gal aliquots of 1/250 dilution in diluent buffer (0.1% BSA, 0.01% Gelatin in TBS, pH 7.5) were added and incubated for 15 min at 37° C. The plate was washed as in Step I. Step III. 200 ⁇ l of substrate solution was added sequentially to each well depending on the enzyme activity used. First, ⁇ -Gal substrate (ONGPTM) solution was added to a final concentration of 2mg/ml and incubated for at least 15 minutes in darkness at room temperature.
- diluent buffer (0.1% BSA, 0.01% Gelatin in TBS, pH 7.5
- the OD obtained from the sequential addition of the substrates on the solid phase was determined as seen in Tables 2a-2f.
- the signal for ⁇ -Gal substrate was read at 15 minutes, wells were washed to remove the enzyme, HRP substrate was then added and read at 5 minutes, then wells were washed to remove the enzyme, and then AP substrate was added and read at 5-10 minutes.
- the assay illustrates the ability to detect and quantitate at least three markers from the same sample, in the same reaction well and at the same time on a solid phase support following the addition of the specific sequence of substrates.
- the sequence of addition as shown in Tables 2a to 2f are as follows:
- ONGP's OD was quenched almost 10 fold from 1.025 to 0.174 due to interference from previous enzymes or their buffers.
- series 2 (Table 2b), ABTS was added first. OD reading at 405nm was followed by a washing step. PnPP was added second and followed by a washing step after reading the OD at 405nm. ONGP was added third and the OD was read at 405nm. This sequential addition of substrates did not work because the OD values obtained at 405nm were 1.039, 1.800 and 0.175 for ABTS, PnPP and ONGP respectively as compared with 0.992, 1.816 and 1.025 obtained from reading the OD of the same marker plated individually in single wells. ONGP's OD was quenched almost 10 fold from 1.025 to 0.175 due to interference from previous enzymes or their buffers.
- ONGP was added first. OD reading at 405nm was followed by a washing step. PnPP was added second and followed by a washing step after reading the OD at 405nm. ABTS was added third and the OD was read at 405nm. This sequential addition of substrates did not work because the OD values obtained at 405nm were 1.089, 1.962 and 0.266 for ONGP, PnPP and ABTS respectively as compared with 1.025, 1.816 and 0.992 obtained from reading the OD of the same marker plated individually in single wells. ABTS's OD was quenched almost 4 fold from 0.992 to 0.266.
- ONGP was added first. OD reading at 405nm was followed by a washing step. ABTS was added second and followed by a washing step after reading the OD at 405nm. PnPP was added third and the OD was read at 405nm. This sequential addition of substrates worked, as the OD values obtained at 405nm were 1.069, 1.282 and 1.727 for ONGP, ABTS and PnPP respectively as compared with 1.025, 0.992 and 1.816. This series showed OD levels of absorbance of all the three markers compatible with the OD readings seen in Table 1. Table 1 : Single marker data from single wells.
- Table 2a Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
- Table 2b Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
- Table 2c Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
- Table 2d Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
- Table 2e Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
- Table 2f Data of sequential addition of substrates of the 3 immunoglobulins added simultaneously in the same well.
- Table 2a ABTS was added first, read at 405 nm and followed by a washing step. ONGP was added next, read at 405 nm and followed by a washing step. PnPP was added last and read at 405 nm. ODs were 1.038, 0.174 and 1.883 for ABTS, ONGP, and PnPP respectively as compared to the 0.992, 1.025 and 1.816 obtained from reading the single marker OD in Table 1.
- Table 2b ABTS was added first, read at 405 nm and followed by a washing step. PnPP was added next, read at 405 nm and followed by a washing step. ONGP was added last and read at 405 nm. ODs were 1.039, 1.800 and 0.175 for ABTS, PnPP and ONGP respectively as compared to the 0.992, 1.816 and 1.025 obtained from reading the single marker OD in Table 1.
- ONGP was added first, read at 405 nm and followed by a washing step.
- ABTS was added next, read at 405 nm and followed by a washing step.
- PnPP was added last and read at 405 nm.
- ODs were 1.069, 1.282 and 1.727 for ONGP, ABTS and PnPP respectively as compared to the 1.025, 0.992 and 1.816 obtained from reading the single marker OD in Table 1.
- the assay involves the simultaneous measurement of three cytokine markers (IL-1)
- the assay detects and quantitates at least three markers from the same sample, in the same well and at the same time on a solid phase support following the specific sequential addition of substrates as illustrated in Example I.
- Table 4 Triple marker data from single wells.
- the assay involves the simultaneous measurement of two chemokine markers (IL- 8
- the assay illustrates the ability to detect and quantitate at least three markers from the same sample, in the same reaction well and at the same time on a solid phase support following the addition of the specific sequential addition of substrates as illustrated in Example I. Table 5. Single marker data from single wells.
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Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002432398A CA2432398A1 (en) | 2000-12-21 | 2001-12-21 | Absorbance multiplex technology |
AU2002232722A AU2002232722A1 (en) | 2000-12-21 | 2001-12-21 | Absorbance multiplex technology |
EP01992259A EP1352241A1 (en) | 2000-12-21 | 2001-12-21 | Absorbance multiplex technology |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US25828600P | 2000-12-21 | 2000-12-21 | |
US60/258,286 | 2000-12-21 | ||
US94782301A | 2001-09-06 | 2001-09-06 | |
US09/947,823 | 2001-09-06 |
Publications (1)
Publication Number | Publication Date |
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WO2002050537A1 true WO2002050537A1 (en) | 2002-06-27 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US2001/049745 WO2002050537A1 (en) | 2000-12-21 | 2001-12-21 | Absorbance multiplex technology |
Country Status (4)
Country | Link |
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EP (1) | EP1352241A1 (en) |
AU (1) | AU2002232722A1 (en) |
CA (1) | CA2432398A1 (en) |
WO (1) | WO2002050537A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3217176A1 (en) * | 2016-03-11 | 2017-09-13 | Scienion AG | Immunoenzymatic method allowing the sequential detection and identification of analytes |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4948726A (en) * | 1986-06-02 | 1990-08-14 | Longoria Claude C | Enzyme immunoassay based on membrane separation of antigen-antibody complexes |
US5180806A (en) * | 1988-05-16 | 1993-01-19 | The Scripps Research Institute | Polypeptides and compositions of human papillomavirus latent proteins, diagnostic systems and methods |
-
2001
- 2001-12-21 EP EP01992259A patent/EP1352241A1/en not_active Withdrawn
- 2001-12-21 CA CA002432398A patent/CA2432398A1/en not_active Abandoned
- 2001-12-21 AU AU2002232722A patent/AU2002232722A1/en not_active Abandoned
- 2001-12-21 WO PCT/US2001/049745 patent/WO2002050537A1/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4948726A (en) * | 1986-06-02 | 1990-08-14 | Longoria Claude C | Enzyme immunoassay based on membrane separation of antigen-antibody complexes |
US5180806A (en) * | 1988-05-16 | 1993-01-19 | The Scripps Research Institute | Polypeptides and compositions of human papillomavirus latent proteins, diagnostic systems and methods |
Non-Patent Citations (2)
Title |
---|
LAMAN ET AL.: "Synthetic peptide conjugates with horseradish peroxidase and beta-galactosidase for use in epitope-specific immunocytochemistry and ELISA", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 145, no. 1-2, 1991, pages 1 - 10, XP002950342 * |
LIU ET AL.: "Cell-ELISA using beta-galatosidase conjugated antibodies", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 234, February 2000 (2000-02-01), pages P153 - P167, XP002950343 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3217176A1 (en) * | 2016-03-11 | 2017-09-13 | Scienion AG | Immunoenzymatic method allowing the sequential detection and identification of analytes |
Also Published As
Publication number | Publication date |
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CA2432398A1 (en) | 2002-06-27 |
AU2002232722A1 (en) | 2002-07-01 |
EP1352241A1 (en) | 2003-10-15 |
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