U.S. patent application number 10/071397 was filed with the patent office on 2002-08-29 for signal enhancement of bispecific antibody-polymer probe for immunoassay use.
Invention is credited to Khaw, Ban-an, Narula, Jagat.
Application Number | 20020119582 10/071397 |
Document ID | / |
Family ID | 21903743 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119582 |
Kind Code |
A1 |
Khaw, Ban-an ; et
al. |
August 29, 2002 |
Signal enhancement of bispecific antibody-polymer probe for
immunoassay use
Abstract
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 bispecific antibody;
and the polymer probe of the immunoassay method are disclosed.
Inventors: |
Khaw, Ban-an; (Milton,
MA) ; Narula, Jagat; (Bryn Mawr, PA) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Family ID: |
21903743 |
Appl. No.: |
10/071397 |
Filed: |
February 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10071397 |
Feb 6, 2002 |
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09380168 |
Oct 6, 1999 |
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60039111 |
Feb 26, 1997 |
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Current U.S.
Class: |
436/526 |
Current CPC
Class: |
Y10S 435/912 20130101;
A61K 49/0002 20130101; G01N 33/54306 20130101; Y10S 435/817
20130101; A61K 31/415 20130101; G01N 33/53 20130101; C07K 2317/31
20130101; G01N 33/58 20130101; A61K 31/74 20130101; A61K 47/62
20170801; G01N 33/6887 20130101; C07K 16/44 20130101; C07K 16/18
20130101; G01N 33/532 20130101 |
Class at
Publication: |
436/526 |
International
Class: |
G01N 033/553 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 25, 1998 |
US |
PCT/US98/03638 |
Claims
What is claimed is:
1. An immunoassay method comprising reacting a sample from a
patient with a bispecific antibody, said bispecific antibody
comprising 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 said sample with a polymer probe,
said polymer probe comprising a polymer backbone, attached to said
polymer backbone, a compound recognizable by said second antibody
in said bispecific antibody, and at least two detectable signal
compounds further attached to said polymer backbone.
2. The immunoassay method of claim 1, wherein said polymer probe
comprises at least ten detectable compounds.
3. The immunoassay method of claim 1, wherein said detectable
signal in said polymer probe is selected from the group consisting
of radioisotope, fluorescent probe and paramagnetic probe.
4. The immunoassay method of claim 1, wherein said sample from said
patient is a blood or serum sample; said bispecific antibody
comprises an antimyosin antibody and an antibody against DTPA; and
said polymer probe is a polylysine polymer and comprises DTPA and
at least 6 HRP as said detectable signal compounds.
5. A bispecific antibody for use in an immunoassay comprising one
antibody specific for a compound to be detected in said
immunoassay; and a second antibody specific for a compound foreign
to a sample to be assayed in said immunoassay.
6. A polymer probe comprising a polymer backbone; a compound
recognizable by an antibody in a bispecific antibody attached to
said polymer backbone, and at least two detectable signal compounds
attached to said polymer backbone. a compound recognizable by said
second antibody in said bispecific antibody and at least two
detectable signal compounds.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
BACKGROUND OF THE INVENTION
[0001] 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.
[0002] 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 CK-MB, 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.
SUMMARY OF THE INVENTION
[0003] 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.
[0004] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] 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:
[0006] FIG. 1a shows a standard ELISA according to the prior
art;
[0007] FIG. 1b shows an immunoassay according to the invention;
and
[0008] 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)).
DETAILED DESCRIPTION OF THE INVENTION
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
EXAMPLE I
[0014] Serum immunoassays for intracardiac contractile proteins
constitute the mainstay for detection of myocyte necrosis
associated with various cardio-vascular 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 .mu.g/ml) was used to
coat the microtiter wells. After overnight incubation and washing,
three times, 50 .mu.l each of 5 .mu.g/ml BiMAbor MAb and serial
dilutions of PCM (0.001 to 100 .mu.g/ml) or 50 .mu.l of serial
dilutions (1/1 to 1/10000) of patient sera pre-incubated for 1 h at
37.degree. C. were added and incubated for 2 h at 37.degree. C.
After washing, the wells were incubated with goat anti-mouse
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.times.10.sup.9L/mole. The sensitivity of BiMAb was
0.5 ng, whereas that of R11D10 was 0.5 .mu.g (1 .mu.g/ml). BiMAb
developed with the conventional goat anti-mouse IgG-HRP had a
sensitivity of 0.05 .mu.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 .mu.l serum at 1/10.sup.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.
EXAMPLE II
[0015] 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.sup.-5 to 100 .mu.g/ml) was at least 10,000 fold
better than the conventional immunoassay (0.1 .mu.g/ml).
[0016] 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.
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