US20020031781A1 - Signal enhancement of bispecific antibody-polymer probe for immunoassay use - Google Patents
Signal enhancement of bispecific antibody-polymer probe for immunoassay use Download PDFInfo
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- US20020031781A1 US20020031781A1 US09/380,168 US38016899A US2002031781A1 US 20020031781 A1 US20020031781 A1 US 20020031781A1 US 38016899 A US38016899 A US 38016899A US 2002031781 A1 US2002031781 A1 US 2002031781A1
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- 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
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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Definitions
- An immunoassay utilizes antibodies to detect a compound of choice.
- the sensitivity of this detection is generally limited by the amount of signal that can be carried either on the antibody, for a direct binding assay, or on the probe compound, in a competitive inhibition assay.
- existing immunoassays such as radioimmunoassay, ELISA, immunofluorescent assays or immunochemiluminescent assays
- too many signal entities such as radioisotopes, horse radish peroxidase or alkaline phosphatase, attached to the detection moieties invariably inactivate the antibody or denature the antigen and change the property of the detection probe. Therefore, in order to obtain more signal, additional antibody or probe must be added. This, in turn, reduces the sensitivity of the assay, the capability of the assay to detect minute quantities of the compound in question.
- the invention is directed to a method to increase the sensitivity of an immunoassay, by at least 10,000 fold, without losing specificity. This improvement is achieved by the use of a bispecific antibody complex and a unique detection signal probe capable of recognizing the bispecific antibody complex.
- the invention features an immunoassay method including reacting a sample from a patient with a bispecific antibody, wherein the bispecific antibody includes one antibody specific for a compound to be detected and a second antibody specific for a compound foreign to said patient sample, and subsequently reacting the patient sample with a polymer probe, wherein the polymer probe includes a compound recognized by the second antibody in the bispecific antibody complex and further includes at least two detectable signals.
- the invention also features the bispecific antibody and the polymer probe of the method of the invention.
- the sample from the patient is a blood or serum sample;
- the bispecific antibody includes an antimyosin antibody and an antibody against DTPA;
- the polymer probe is a polylysine polymer and includes DTPA and at least six HRP as the detectable signal compounds.
- FIG. 1 a shows a standard ELISA according to the prior art
- FIG. 1 b shows an immunoassay according to the invention
- FIG. 2 is a graph showing competitive inhibition curves using standard ELISA (R11D10), bispecific antibody complex with standard secondary antibody for signal production (BiMAb (Ab-HRP)), and the method according to the invention (BiMAb(PL-DTPA-HRP)).
- the invention is directed to the development of a new approach to the use of bispecific antibodies in immunoassays.
- the new specific antibody comprises one antibody specific for the compound associated with the pathological state to be detected and another antibody to a chemical or reporter compound that is not found naturally in man. These two are chemically or genetically linked.
- the bispecific antibody complex constitutes the first line of interaction with the compound one is attempting to detect. Normally many antibodies must react with the compound to enable development of sufficient signal intensity for detection.
- a novel detection probe is used, made up of any type polymer, such as polylysine or other polyamino acid, that is amenable to attachment of signal reagents and reporter compounds.
- the amount of signal reagent that can be used in a given assay is limited only by the size of the polymer. Only a few molecules of the detection probe are therefore needed to provide this signal.
- the signal probe is extremely versatile as any type of signal producing compound such as radioactivity, chemical color producing enzymes or fluorescent probes can be attached to the polymer backbone. Signal amplification is not limited by the nature of the bispecific antibody complex itself.
- the immunoassay sensitivity can be amplified by at least 10,000-fold compared to conventional immunoassays or immunosandwich assays. Since early detection of many pathological states, such as acute myocardial infarction and cancer, is limited by the sensitivity of immunoassays to detect minute elevations of the pathologically associated compounds, an method and compounds of the invention will enable diagnosis of disease states at a much earlier time than previous assays, which may allow for better therapeutic intervention.
- Another advantage of the method of the invention is the versatility for adaptation to any antibody.
- the method could be adapted to detect troponin-I or T by using the antibody specific for troponin-I or T attached to a second antibody, such as the antibodies shown herein, that recognizes the detector probe. If higher sensitivity is necessary, the polymer probe could be generated to carry higher numbers of signal compounds.
- the polymer probe can include any kind of signal compound, such as radioisotope, fluorescent, or paramagnetic linked signal compounds.
- Serum immunoassays for intracardiac contractile proteins constitute the mainstay for detection of myocyte necrosis associated with various cardiovascular disorders.
- myosin heavy chain (MHC) fragments can be detected by immunoassay only after 48 h from the onset of chest pain.
- MAb monoclonal antibody
- MAb 4G4-1D5 specific for DTPA.
- the probe consisted of DTPA-modified polylysine (28:1 molar ratio) covalently linked to horse-radish peroxidase (6 moles/mole polylysine)(PL-DTPA-HRP).
- Porcine cardiac myosin (PCM, 1 ⁇ g/ml) was used to coat the microtiter wells. After overnight incubation and washing, three times, 50 ⁇ l each of 5 ⁇ g/ml BiMAbor MAb and serial dilutions of PCM (0.001 to 100 ⁇ g/ml) or 50 ⁇ l of serial dilutions (1/1 to 1/10000) of patient sera pre-incubated for 1 h at 37° C. were added and incubated for 2 h at 37° C. After washing, the wells were incubated with goat-antimouse IgG-HRP or PL-DTPA-HRP for 2 h. A chromogen, dinitrobenzidine was used to develop the assay.
- PCM Porcine cardiac myosin
- BiMAb and R11D10 were the same at 1.5 ⁇ 10 9 L/mole.
- the sensitivity of BiMAb was 0.5 ng, whereas that of R11D10 was 0.5 ⁇ g (1 ⁇ g/ml)
- BiMAb developed with the conventional goat anti-mouse IgG-HRP had a sensitivity of 0.05 ⁇ g. Therefore, BiMAb assay has a 1000 fold increase in sensitivity compared to the conventional immunoassay in the sera of 3 heart transplant patients. Using the BiMAb assay, 2.5, 1.25 and 1.3 ng MHC/50 ⁇ l serum at 1/10 3 dilution, were detected.
- This BiMAb technology can be used in RIA or ELISA by interchanging the HRP probe for radiolabeled probe and should provide more specific in vitro diagnosis of acute myocardial infarction since detection of MHC is not feasible at the present time of day 1 of myocardial infarction by conventional immunoassays.
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Abstract
Description
- An immunoassay utilizes antibodies to detect a compound of choice. However, the sensitivity of this detection is generally limited by the amount of signal that can be carried either on the antibody, for a direct binding assay, or on the probe compound, in a competitive inhibition assay. For example, in existing immunoassays, such as radioimmunoassay, ELISA, immunofluorescent assays or immunochemiluminescent assays, too many signal entities, such as radioisotopes, horse radish peroxidase or alkaline phosphatase, attached to the detection moieties invariably inactivate the antibody or denature the antigen and change the property of the detection probe. Therefore, in order to obtain more signal, additional antibody or probe must be added. This, in turn, reduces the sensitivity of the assay, the capability of the assay to detect minute quantities of the compound in question.
- For all existing immunoassays, there is lag time for the compound of interest to reach a high enough concentration in the serum to become detectable for diagnostic purposes. In the case of heart attacks, there is a delay of 4-6 hours from the onset of chest pain until the diagnostic detection of CKMB, Troponin-T or I is possible. Myoglobin is detectable earlier, but its specificity is low. If there were an assay that could detect very minute increases of these indicator compounds in the blood at an earlier point in time, then therapeutic intervention could be started earlier and thereby bring about greater myocardial salvage. In the case of cancer detection, where, e.g., tumor associated antigens related to breast cancer or colon cancer, etc., are detected, treatment might be more effective if minute elevations of these antigens could be detected at an early stage. Therefore, there is a need to increase the sensitivity of the assay without adversely affecting the specificity of the assay system.
- The invention is directed to a method to increase the sensitivity of an immunoassay, by at least 10,000 fold, without losing specificity. This improvement is achieved by the use of a bispecific antibody complex and a unique detection signal probe capable of recognizing the bispecific antibody complex.
- In one aspect, the invention features an immunoassay method including reacting a sample from a patient with a bispecific antibody, wherein the bispecific antibody includes one antibody specific for a compound to be detected and a second antibody specific for a compound foreign to said patient sample, and subsequently reacting the patient sample with a polymer probe, wherein the polymer probe includes a compound recognized by the second antibody in the bispecific antibody complex and further includes at least two detectable signals. The invention also features the bispecific antibody and the polymer probe of the method of the invention. Preferably, the sample from the patient is a blood or serum sample; the bispecific antibody includes an antimyosin antibody and an antibody against DTPA; and the polymer probe is a polylysine polymer and includes DTPA and at least six HRP as the detectable signal compounds.
- Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims, taken in conjunction with the accompanying drawings, in which:
- FIG. 1a shows a standard ELISA according to the prior art;
- FIG. 1b shows an immunoassay according to the invention; and
- FIG. 2 is a graph showing competitive inhibition curves using standard ELISA (R11D10), bispecific antibody complex with standard secondary antibody for signal production (BiMAb (Ab-HRP)), and the method according to the invention (BiMAb(PL-DTPA-HRP)).
- The invention is directed to the development of a new approach to the use of bispecific antibodies in immunoassays. The new specific antibody comprises one antibody specific for the compound associated with the pathological state to be detected and another antibody to a chemical or reporter compound that is not found naturally in man. These two are chemically or genetically linked. The bispecific antibody complex constitutes the first line of interaction with the compound one is attempting to detect. Normally many antibodies must react with the compound to enable development of sufficient signal intensity for detection. However, in the method of the invention, a novel detection probe is used, made up of any type polymer, such as polylysine or other polyamino acid, that is amenable to attachment of signal reagents and reporter compounds. The amount of signal reagent that can be used in a given assay is limited only by the size of the polymer. Only a few molecules of the detection probe are therefore needed to provide this signal. The signal probe is extremely versatile as any type of signal producing compound such as radioactivity, chemical color producing enzymes or fluorescent probes can be attached to the polymer backbone. Signal amplification is not limited by the nature of the bispecific antibody complex itself.
- Therefore, the immunoassay sensitivity can be amplified by at least 10,000-fold compared to conventional immunoassays or immunosandwich assays. Since early detection of many pathological states, such as acute myocardial infarction and cancer, is limited by the sensitivity of immunoassays to detect minute elevations of the pathologically associated compounds, an method and compounds of the invention will enable diagnosis of disease states at a much earlier time than previous assays, which may allow for better therapeutic intervention.
- Another advantage of the method of the invention is the versatility for adaptation to any antibody. For example, the method could be adapted to detect troponin-I or T by using the antibody specific for troponin-I or T attached to a second antibody, such as the antibodies shown herein, that recognizes the detector probe. If higher sensitivity is necessary, the polymer probe could be generated to carry higher numbers of signal compounds. Furthermore, the polymer probe can include any kind of signal compound, such as radioisotope, fluorescent, or paramagnetic linked signal compounds.
- All previously existing ELISA radioimmunoassays, dip-stick assays for cancer, pregnancy, serum enzymes and probes and any assays utilizing antibodies could be modified according to the method of the invention to provide enhanced sensitivity. In addition, in vivo application to enhance target signal by using the method of the invention is also possible.
- The following examples are presented to illustrate the advantages of the present invention and to assist one of ordinary skill in making and using the same. These examples are not intended in any way otherwise to limit the scope of the disclosure.
- Serum immunoassays for intracardiac contractile proteins constitute the mainstay for detection of myocyte necrosis associated with various cardiovascular disorders. However, myosin heavy chain (MHC) fragments can be detected by immunoassay only after 48 h from the onset of chest pain. To enhance immunodetection of MHC, monoclonal antibody (MAb) R11D10 specific for cardiac MHC was covalently linked to MAb 4G4-1D5 specific for DTPA. The probe consisted of DTPA-modified polylysine (28:1 molar ratio) covalently linked to horse-radish peroxidase (6 moles/mole polylysine)(PL-DTPA-HRP). Porcine cardiac myosin (PCM, 1 μg/ml) was used to coat the microtiter wells. After overnight incubation and washing, three times, 50 μl each of 5 μg/ml BiMAbor MAb and serial dilutions of PCM (0.001 to 100 μg/ml) or 50 μl of serial dilutions (1/1 to 1/10000) of patient sera pre-incubated for 1 h at 37° C. were added and incubated for 2 h at 37° C. After washing, the wells were incubated with goat-antimouse IgG-HRP or PL-DTPA-HRP for 2 h. A chromogen, dinitrobenzidine was used to develop the assay. The affinity of BiMAb and R11D10 were the same at 1.5×109 L/mole. The sensitivity of BiMAb was 0.5 ng, whereas that of R11D10 was 0.5 μg (1 μg/ml) BiMAb developed with the conventional goat anti-mouse IgG-HRP had a sensitivity of 0.05 μg. Therefore, BiMAb assay has a 1000 fold increase in sensitivity compared to the conventional immunoassay in the sera of 3 heart transplant patients. Using the BiMAb assay, 2.5, 1.25 and 1.3 ng MHC/50 μl serum at 1/103 dilution, were detected. This BiMAb technology can be used in RIA or ELISA by interchanging the HRP probe for radiolabeled probe and should provide more specific in vitro diagnosis of acute myocardial infarction since detection of MHC is not feasible at the present time of
day 1 of myocardial infarction by conventional immunoassays. - In a subsequent experiment the DTPA-modified polylysine probe of Example I was covalently linked to 12 moles of horse-radish peroxidase per mole of polylysine. The results of the study show that the sensitivity of the bispecific assay of the invention (10−5 to 100 μg/ml) was at least 10,000 fold better than the conventional immunoassay (0.1 μg/ml).
- While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.
Claims (6)
Priority Applications (3)
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US09/380,168 US6451980B1 (en) | 1997-02-26 | 1998-02-25 | Signal enhancement of bispecific antibody-polymer probe for immunoassay use |
US09/727,421 US20010024795A1 (en) | 1997-02-26 | 2000-12-01 | Immunoassay technique using multispecific molecules |
US10/071,397 US20020119582A1 (en) | 1997-02-26 | 2002-02-06 | Signal enhancement of bispecific antibody-polymer probe for immunoassay use |
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US3911197P | 1997-02-26 | 1997-02-26 | |
PCT/US1998/003638 WO1998038513A1 (en) | 1997-02-26 | 1998-02-25 | Signal enhancement of bispecific antibody-polymer probe for immunoassay use |
US09/380,168 US6451980B1 (en) | 1997-02-26 | 1998-02-25 | Signal enhancement of bispecific antibody-polymer probe for immunoassay use |
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US09/727,421 Continuation-In-Part US20010024795A1 (en) | 1997-02-26 | 2000-12-01 | Immunoassay technique using multispecific molecules |
US10/071,397 Division US20020119582A1 (en) | 1997-02-26 | 2002-02-06 | Signal enhancement of bispecific antibody-polymer probe for immunoassay use |
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US20020031781A1 true US20020031781A1 (en) | 2002-03-14 |
US6451980B1 US6451980B1 (en) | 2002-09-17 |
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US10/071,397 Abandoned US20020119582A1 (en) | 1997-02-26 | 2002-02-06 | Signal enhancement of bispecific antibody-polymer probe for immunoassay use |
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US10/071,397 Abandoned US20020119582A1 (en) | 1997-02-26 | 2002-02-06 | Signal enhancement of bispecific antibody-polymer probe for immunoassay use |
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WO2005019820A1 (en) * | 2003-08-25 | 2005-03-03 | Marc Ramael | A method and kit for the quantitative and/or qualitative detection of components in a sample |
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EP2185725A4 (en) * | 2007-08-02 | 2011-01-12 | Biodesic | Compositions and methods for analyte detection and quantitation |
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Publication number | Priority date | Publication date | Assignee | Title |
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US6753189B1 (en) | 1998-06-04 | 2004-06-22 | Mizuho Medy Co., Ltd. | Detection apparatus and method for the same |
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Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5223242A (en) | 1985-11-05 | 1993-06-29 | The General Hospital Corporation | Negatively charged specific affinity reagents |
US5851527A (en) * | 1988-04-18 | 1998-12-22 | Immunomedics, Inc. | Method for antibody targeting of therapeutic agents |
AU5853590A (en) * | 1989-06-14 | 1991-01-08 | General Hospital Corporation, The | Assay for human ventricular myosin lc1 and monoclonal antibody thereto |
DE3920358A1 (en) * | 1989-06-22 | 1991-01-17 | Behringwerke Ag | BISPECIFIC AND OLIGO-SPECIFIC, MONO- AND OLIGOVALENT ANTI-BODY CONSTRUCTS, THEIR PRODUCTION AND USE |
US5332567A (en) * | 1989-08-24 | 1994-07-26 | Immunomedics | Detection and treatment of infections with immunoconjugates |
AU1929292A (en) * | 1991-05-14 | 1992-12-30 | Hybritech Incorporated | Polymeric compositions having bound antibodies |
AU5729294A (en) * | 1992-11-25 | 1994-06-22 | Tanox Biosystems, Inc. | Conjugates and constructs including anti-cd28 and anti-cd3 binding molecules |
US5482698A (en) * | 1993-04-22 | 1996-01-09 | Immunomedics, Inc. | Detection and therapy of lesions with biotin/avidin polymer conjugates |
US5686578A (en) * | 1994-08-05 | 1997-11-11 | Immunomedics, Inc. | Polyspecific immunoconjugates and antibody composites for targeting the multidrug resistant phenotype |
-
1998
- 1998-02-25 US US09/380,168 patent/US6451980B1/en not_active Expired - Lifetime
- 1998-02-25 WO PCT/US1998/003638 patent/WO1998038513A1/en not_active Application Discontinuation
- 1998-02-25 EP EP98908707A patent/EP0981748A4/en not_active Withdrawn
-
2002
- 2002-02-06 US US10/071,397 patent/US20020119582A1/en not_active Abandoned
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Also Published As
Publication number | Publication date |
---|---|
EP0981748A1 (en) | 2000-03-01 |
EP0981748A4 (en) | 2002-09-18 |
US6451980B1 (en) | 2002-09-17 |
WO1998038513A1 (en) | 1998-09-03 |
US20020119582A1 (en) | 2002-08-29 |
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