U.S. patent application number 10/287341 was filed with the patent office on 2003-06-19 for antibodies to crosslinkers and methods for using the same.
Invention is credited to DeVico, Anthony L., Pal, Ranajit, Sarngadharan, Mangalasseril G..
Application Number | 20030113780 10/287341 |
Document ID | / |
Family ID | 23742465 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113780 |
Kind Code |
A1 |
Sarngadharan, Mangalasseril G. ;
et al. |
June 19, 2003 |
Antibodies to crosslinkers and methods for using the same
Abstract
The present invention is directed to the discovery of antibodies
that will react to proteins or nucleic acids bound to particular
crosslinkers, but not to the free crosslinkers or free proteins or
nucleic acids. Monoclonal antibodies with such binding specificity
have widespread applications in receptor-ligand binding,
immunodiagnostic and nucleic acid diagnostics.
Inventors: |
Sarngadharan, Mangalasseril G.;
(McLean, VA) ; Pal, Ranajit; (Gaithersburg,
MD) ; DeVico, Anthony L.; (Alexandria, VA) |
Correspondence
Address: |
Samir R. Patel, Esq.
bioMerieux, Inc.
Patent Department
100 Rodolphe Street
Durham
NC
27712
US
|
Family ID: |
23742465 |
Appl. No.: |
10/287341 |
Filed: |
November 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10287341 |
Nov 4, 2002 |
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09438895 |
Nov 12, 1999 |
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6503736 |
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Current U.S.
Class: |
435/6.12 ;
435/7.1; 435/70.21; 530/388.1 |
Current CPC
Class: |
C07K 16/44 20130101;
Y10S 530/809 20130101 |
Class at
Publication: |
435/6 ;
435/70.21; 530/388.1; 435/7.1 |
International
Class: |
C12Q 001/68; G01N
033/53; C12P 021/04; C07K 016/44 |
Claims
We claim:
1. A monoclonal antibody that will specifically bind to a BS3 or
DSS modified protein or nucleic acid, but not to free BS3 or DSS or
unmodified protein or nucleic acid.
2. The monoclonal antibody of claim 1, which is selected from the
group consisting of: 7E3-2E7; 2C3-2E10; 12G9-2C5; and 11F2-2F7.
3. A nucleic acid molecule which has been modified by covalent
bonding to BS3 or DSS.
4. The nucleic acid molecule of claim 3, which is a hybridization
probe in a diagnostic assay.
5. A method of assaying receptor-ligand binding, comprising: (a)
linking BS3 or DSS to the ligand of interest; (b) reacting the BS3
or DSS modified ligand with cells expressing the ligand's receptor;
(c) reacting bound BS3 or DSS modified ligand/receptor with labeled
anti-BS3/DSS monoclonal antibody; and (d) detecting the label on a
complex formed between the labeled antibody and the
receptor/ligand.
6. A method of assaying receptor-ligand binding, comprising: (a)
linking BS3 or DSS to the ligand of interest; (b) reacting the BS3
or DSS modified ligand with cells expressing the ligand's receptor;
(c) reacting bound BS3 or DSS modified ligand/receptor with
anti-BS3/DSS monoclonal antibody, followed by anti-mouse IgG
conjugated to FITC; and (d) detecting the bound FITC-conjugated
antibody by flow cytometry or immunofluorescence.
7. The method of claim 6, wherein the ligand is gp120 and the
receptor is CD4.
8. A method for detecting products of a nucleic acid amplification,
comprising: (a) labelling NTPs that contain a free NH.sub.2 group
with BS3 or DSS; (b) using the labelled NTPs in an amplification
reaction mixture; (c) at the conclusion of the amplification,
reacting the amplification products with anti-BS3/DSS antibody,
which is itself labelled with a detectable moiety or reacted with a
labelled antibody that will bind to the anti-BS3/DSS antibody,
thereby detecting the amplification products.
9. The method of claim 8, wherein the amplification is an
isothermal transcription based amplification reaction and the NTPs
are rNTPs.
10. The method of claim 9, wherein the amplification reaction is
NASBA.
11. A method for detecting a nucleic acid sequence, comprising
hybridizing a probe with complementary regions of said sequence,
wherein said probe comprises a nucleic acid sequence labeled with
BS3 or DSS.
12. An immunoassay using a labeled antigen or a labeled antibody,
wherein BS3 or DSS is the label.
13. A method for detecting an antigen of interest in a mixture of
antigens, comprising: (a) modifying the antigens in the sample with
BS3 or an analogue thereof; (b) reacting the modified antigens with
a capture antibody, which is an IgA anti-BS3 antibody; (c) reacting
the bound antigen with an IgG antibody that is specific to the
antigen of interest; and (d) detecting bound IgG by reacting with a
labeled anti-IgG antibody, thereby detecting the antigen of
interest.
14. The method of claim 13, wherein the IgA antibody is 7E3-2E7.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to the discovery of
antibodies that will react to proteins or nucleic acids bound to
particular crosslinkers, but not to the free crosslinkers or free
proteins or nucleic acids. Monoclonal antibodies with such binding
specificity have widespread applications in receptor-ligand
binding, immunodiagnostic, molecular amplification assays and other
nucleic acid diagnostics.
BACKGROUND OF THE INVENTION
[0002] DSS (Dissuccinimidyl suberate) is a non-cleavable, membrane
permeable, amine-reactive, homobifunctional crosslinker. BS3
(Bis[Sulfosuccinimidyl]suberate) is a non-cleavable, membrane
impregnable, water soluble analog of DSS. By means of two
bifunctional sulfo-NHS ester reactive groups, BS3 (and DSS) can
serve as a crosslinking reagent between molecules with amino
groups. The BS3 and DSS compounds are sold by Pierce (Rockford,
Ill.) as protein crosslinking reagents. These compounds will
crosslink molecules that are within a certain distance (i.e. spacer
arm length), but otherwise will modify such molecules without
necessarily crosslinking them. Thus, BS3 and DSS can be used as
labels of analytes.
[0003] Such modifications of proteins and nucleic acids by
digoxigenin, for instance, are known in the art. In general,
digoxigenin has been used as a label in bioanalytical assays where
it may be itself radiolabelled or may act as a hapten, for
instance, which reacts with an anti-hapten antibody for detection
of the digoxigenin-labeled analyte. See U.S. Pat. Nos. 3,855,208;
5,198,537; and 5,804,371.
[0004] Digoxigenin and derivatives thereof have also been used in
the field of nucleic acid diagnostics, where in general it is
incorporated as a label into amplificates or probes, and whereby
the labelled moieties are detected by hapten anti-hapten reaction
principle. See, for example, U.S. Pat. Nos. 5,354,657; 5,843,670;
5,929,108; and 5,344,757.
[0005] However, there are drawbacks of the digoxigenin system.
First, the procedure for derivatizing with digoxigenin is
relatively complicated. Second, because digoxigenin is a large
molecule and contains a hydrophobic steroid, modification of a
molecule will perturb the molecule's conformation. Third,
digoxigenin is relatively expensive, as compared to for instance
BS3.
[0006] Therefore, a need exists for a simpler bioanalytical
detection system, which does not require multiple derivatization
steps, is less expensive, and is less likely to disrupt the
conformation of the molecule being modified therewith. A system
which is parallel in many respects to the digoxigenin system has
now been, unexpectedly, discovered. This system can be used in the
same manner as digoxigenin.
SUMMARY OF THE INVENTION
[0007] The present invention is the result of the discovery that
certain monoclonal antibodies produced by hybridomas raised against
BS3-modified gp120-CD4 complexes were actually directed to the BS3
linker itself. These antibodies do not react with the free BS3
molecule alone, and show different binding specificities. For
instance, some of the antibodies appear to react with the "hinge"
formed between amino acid residues and the BS3 molecule in a
modified protein. These antibodies would be expected to crossreact
with proteins treated with other crosslinkers, such as DSG (Pierce)
and DTSSP (a molecule analogous to DSS but with an S-S bridge in
the middle of the methylene chain). Other monoclonals react with
the linear carbon chain that lies between the two end
sulfosuccinimidyl groups of the BS3 molecule. Thus, such
monoclonals would also be expected to react with DSS modified (or
crosslinked) proteins, because it contains the same long methylene
chain, and presumably other crosslinkers such as DMP, DMA, DSG and
MSA (all sold by Pierce).
[0008] The monoclonals of the present invention are useful in
diagnostic immunoassays, such as ELISAs. They are also useful in
ligand-receptor studies. Finally, it is also contemplated that the
BS3/anti-BS3 system can be used as a detection system for nucleic
acid amplification assays. In fact, these anti-hapten antibodies
can be used in the same manner as other hapten/anti-hapten systems
known in the art, such as digoxigenin/anti-digoxigenin.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The term "anti-BS3" in reference to the antibodies of the
present invention is not meant to imply that these antibodies are
only reactive with BS3-modified molecules, but as explained above,
are likely to be reactive with other known crosslinkers used to
modify proteins or nucleic acids. In addition, in the present
specification the term "BS3-modified" includes molecules modified
by the other crosslinkers mentioned above as being crossreactive
with the antibodies. Because of their close similarity, BS3 and DSS
are particularly preferred in the present invention.
[0010] BS3 (Bis[Sulfosuccinimidyl]suberate) is a crosslinker which
contains two bifunctional sulfo-NHS ester reactive groups. See
formula below. 1
[0011] DSS (Disuccinimidyl suberate) is a water insoluble analogue
of BS3, and has the formula below. 2
[0012] When these crosslinkers are reacted with molecules
containing amino groups, the succinimidyl groups on each end of the
molecule are cleaved, leaving essentially a 6 carbon fatty acid
chain, either between two molecules that are being crosslinked or
extending from the modified molecule. This is diagrammatically
depicted below. 3
[0013] In studies involving HIV, BS3 was used to crosslink a
complex of HIV-1 gp120 envelope protein and soluble human CD4. This
covalently crosslinked complex was then used to immunize mice for
the purposes of generating complex specific monoclonal antibodies
(mABs). Although several complex specific mABs were generated from
the immunized mice, several IgG and IgA hydridomas were produced
which are specific for the BS3 component of the complex.
Interestingly, none of the monoclonals were reactive with gp120,
sCD4 (soluble CD4), or free BS3; the reactivity was specific for
BS3 linked to an amino group.
[0014] In its preferred embodiment, this invention is directed to
four murine hydridomas that secrete monoclonal antibodies with
unique specificity. Chemical modification of proteins with BS3
resulted in the exposure of an epitope in the protein or in the
BS3, which showed strong immunoreactivity with these antibodies.
One of the monoclonals is an IgA (designated 7E3-2E7), and three
others are IgGs (2C3-2E10, 12G9-2C5, and 11F2-2F7). These
monoclonals react with BS3 linked to proteins other than gp120-CD4,
and thus have broad utility is the assay arts.
[0015] The hybridomas producing the antibodies have the same
designations as the monoclonals above, and all four were deposited
with the ATCC, Manassass, Va., USA, under the terms of the Budapest
Treaty, on ______, 1999, and given the designations: ______ for
7E3-2E7, ______ for 2C3-2E10, ______ for 12G9-2C5, and ______ for
11F2-2F7.
[0016] The IgA (7E3-2E7) monoclonal is particularly unique and
preferred because of its five binding sites. It can thus amplify
the signal in an immunoassay. It is also particularly useful in a
two antibody system, as a capture antibody. That is, anti-BS3 IgA
can be used as a capture antibody for all BS3-modified antigens in
a sample. An IgG probe antibody can be added which is specific for
a particular antigen that has been captured. Then, a labeled
anti-IgG detector antibody can be added, which will not bind to the
IgA capture antibody.
[0017] Other monoclonal antibodies may be made by methods well
known in the art. See, for instance, Harlow and Lane, ANTIBODIES: A
LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988), ISBN
0-87969-314-2, which is incorporated herein by reference. The
immunogen in such methods would be the crosslinker-modified
molecule. Additionally, the term "antibody" is intended to also
encompass fragments, such as the Fab, Fab', F(ab).sub.2 and
F(ab').sub.2 fragments, or other antibody fragments modified, for
example, by genetic engineering.
[0018] For the assay of protein analytes, a free amino group can be
reacted with the BS3, in the manner set forth in, for instance,
Example 1. Once labelled, these proteins can be detected with
anti-BS3 antibodies, which will specifically bind with the
methylene spacer of the BS3 or the "hinge" between the protein and
the BS3 molecule (i.e., the part containing the C.dbd.O group). The
antibodies themselves can be labelled with a conventional label,
such as an enzyme, fluorescent, chromogenic, metal particle or
radioactive label, or can be detected using a labeled anti-IgA or
anti-IgG antibody.
[0019] Essentially, any method in which the
digoxigenin/anti-digoxigenin system can be employed in the field of
antigen assay is also a method in which the BS3/anti-BS3 can be
used. In this regard, U.S. Pat. Nos. 3,855,208, 5,804,371, and
5,843,670, which disclose various assays with
digoxigenin/anti-digoxigenin, are incorporated by reference.
[0020] In one embodiment, the BS3/anti-BS3 system is useful in
diagnostic immunoassays that employ two antibodies (the capture
antibody and the detector antibody). In these assays, the detector
antibody is treated with BS3 and any unreacted BS3 is blocked. The
BS3-conjugated antibody bound to the antigen that has been captured
by the capture antibody can be recognized by an anti-BS3 antibody
that contains a label, such as HRP. It is contemplated that this
assay system would have an increased senstivity over an ELISA using
two murine monoclonal antibodies and a secondary anti-mouse
antibody-enzyme conjugate, because the secondary antibody will also
react with the capture antibody on the solid phase. See further,
above, regarding the IgA antibody of the present invention.
[0021] The relevance for nucleic acid diagnostics of the anti-BS3
mABs is as a detection reagent. Given the reactivity of BS3 to
dNTPs or rNTPs with amino groups, the same chemical process that is
used for protein labeling/crosslinking can be used. Once the
nucleotide is modified with the BS3, it can still serve as a
monomer in a nucleic acid polymerization reaction, and the
resulting product would be reactive with the anti-BS3 mABs. The
bound Ab can then be detected either with an appropriately labeled
second Ab, or by incorporating a label into the anti-BS3 Ab
directly. Essentially, any DNA detecting method in which the
digoxigenin/anti-digoxigenin system can be employed is also a
method in which the BS3/anti-BS3 can be used. In this regard, the
nucleic acid detection assays disclosed in U.S. Pat. Nos.
5,843,670, 5,198,537, 5,354,657, 5,843,670, 5,929,108 and 5,344,757
are incorporated by reference.
[0022] Experiments have been conducted on this aspect of the
invention by making dGTP monomer complexes with BS3, which were
then used in an in vitro transcriptase reaction on a polyc
template. The product was transferred to nitrocellulose and
analyzed in Western Blot-type analysis with anti-BS3. The product
was successfully detected by this method, indicating that (1) dGTP
can be labeled with BS3, and (2) the modified dGTP can serve as a
reactive monomer for reverse transcription.
[0023] Several formats are envisaged in a nucleic acid diagnostic
system using these monoclonals. First, BS3 modified NTP's (those
containing an amino group, or else modified to contain an amino
group) could be used as monomers in an amplification reaction (such
as PCR, NASBA, etc.). The monomers would be incorporated into the
amplicons (amplificates), which can then be detected with the
anti-BS3 antibodies. Second, one could modify the 5' end with the
P2 primer in a transcription based amplification reaction such as
NASBA, which in combination with the monoclonals can be a generic
capture method. Third, modification of the 5' end of a known
capture probe and using the monoclonal to a linker between a
particle or solid substrate as a means to bind the capture probe to
the surface. Fourth, nucleic acid can be labelled with the BS3 (or
other crossreactive crosslinker) and used as a probe for
hybridization. The nucleic acid to be assayed can be detected by
allowing it to hybridize with the probe to form a nucleic acid
hybrid, removing the free probe from the system, and detecting the
label contained in the hybrid. In the present invention, the BS3
label can be detected using an enzyme-bound anti-BS3 antibody. The
nucleic acid to be assayed is usually immobilized on a membrane or
nitrocellulose prior to use.
[0024] For hybridization in the nucleic acid detection method of
the present invention, any common hybridization method can be used,
including colony hybridization, plaque hybridization, dot blot
hybridization, southern or northern hybridization, and the like.
The nucleic acid to be assayed may be either DNA or RNA. The
nucleic acid probe may also be DNA or RNA.
[0025] Other, even more sophisticated uses of the monoclonal
antibodies would be apparent to those skilled in the art.
[0026] The monoclonal antibodies of the present invention are also
superior in that they can be used in column purification of
molecules. For instance, an anti-BS3 column can be used to purify
BS3 (or other crossreactive crosslinker) modified proteins/nucleic
acids from a mixture thereof. In addition, in an immunoassay system
that uses two mouse monoclonals, for instance, one could not use a
labelled antimouse antibody to detect complex formation. However,
one of the mouse antibodies could be labelled with BS3, and then
detected with a labeled anti-BS3 antibody.
[0027] The following examples are not intended to limit the scope
of the present invention.
EXAMPLE 1
[0028] Preparation of Monoclonal Antibodies Reacting to BS3
Modified Protein
[0029] Hybridomas secreting BS3 specific antibodies were isolated
from mice immunized with BS3 crosslinked HIV-1 gp120-CD4 complexes.
Equimolar quantities of purified gp120 from an HIV-1.sub.IIIB
isolate and recombinant soluble CD4 were incubated at 37.degree. C.
for 60 min. in PBS. A stock solution of BS3 (5 mM) was prepared in
distilled water. Physically associated complex formed after such
incubation was crosslinked by adding BS3 to a final concentration
of 0.5 mM. The solution was incubated for 30 min at room
temperature and unreacted BS3 was blocked by adding Tris buffer (pH
8.0) to a final concentration of 50 mM.
[0030] Five mice were immunized with BS3 labeled gp120-CD4 complex
(20 ug/mouse) in complete Freund's adjuvant. Subsequent
immunizations were administered in incomplete Freund's adjuvant
until the animals developed high titered antibody response against
gp120 and CD5. Splenic lymphocytes from these immunized animals
were fused with NS1 cells and the hybridomas resulting from such
fusion were screened against uncrosslinked and BS3 cross-linked
gp120-CD4 complex. Single cell cloning of such hybridomas reacting
specifically with BS3 cross-linked complex resulted in the
isolation of four stable hybridomas as shown below:
1 TABLE 1 Hybridomas Clone Isotype 7E3-2E7 IgA 2C3-2E10 IgG1
12G9-2C5 IgG1 11F2-2F7 IgG1
[0031] Supernatant from these hybridoma clones were tested at
different dilutions for immunological reactivity against BS3-linked
and unlinked gp120-CD4 complex by ELISA and the results (expressed
as optical density at 450 nm) are shown below:
2TABLE 2 Hybridoma supernatant Hybridoma supernatant reacted with
BS3- reacted with un- crosslinked gp120-CD4 crosslinked gp120-CD4
Hybridoma complex at dilutions complex at dilutions Clone Undiluted
1:1 1:10 Undiluted 1:1 1:10 7E3-2E7 >3.00 >3.00 >3.00
0.125 0.108 0.105 2C3-2E10 2.741 2.728 2.486 0.125 0.120 0.095
12G9-2C5 2.644 >3.00 2.885 0.095 0.097 0.086 11F2-2F7 2.824
>3.00 2.728 0.252 0.104 0.091
[0032] It is clear from the results presented above that the
antibodies secreted from these hybridoma clones react specifically
with BS3-crosslinked gp120-CD4 complex and had no reactivity with
uncrosslinked complex.
[0033] In order to determine whether these antibodies will react
with other proteins labeled with BS3, a 5% solution of dry milk in
PBS was coated onto an ELISA plate. After binding the proteins for
60 min at 37.degree. C., the wells were treated with 0.5 mM BS3 for
30 min at room temperature. Unreacted BS3 was then blocked with
Tris buffer as described above and the wells were reacted with the
supernatant from the four hybridoma clones listed above and also
with a hybridoma secreting non-BS3 antibody (8F10-2E11). As shown
below, all four supernatants exhibited strong reactivity with
BS3-modified dried milk proteins. However as expected, no
reactivity was observed with the non-BS3 antibody.
3TABLE 3 Immunoreactivity of anti-BS3 antibodies to BS3 modified 5%
Dry Milk Solution in PBS (Blotto) ELISA reactivity of different
dilutions of hybridoma Hybridoma supernatant in duplicate (Optical
Density 450 nm) Clone Undiluted 1:1 1:20 1:200 1:2000 12G9
>3.00, >3.00 >3,00, >3.00 >3,00, >3.00, 0.406,
>3.00 >3.00 0.383 7E3 >3.00, >3.00 >3.00, >3.00
>3.00, >3.00, 1.574, >3.00 >3.00 1.611 2C3 >3.00,
2.803 2.683, 2.774 1.417, 0.284, 0.090, 1.160 0.236 0.079 11F2
>3.00, >3.00 >3.00, >3.00 0.812, 0.138, 0.067, 0.857
0.141 0.068 DMEM 0.052, 0.052 Not Tested Not Not Not Medium Tested
Tested Tested (Negative control) Unrelated 0.170, 0.169 Not Tested
Not Not Not Hybridoma Tested Tested Tested (8F101) (Negative
control)
EXAMPLE 2
[0034] Application of Anti-BS3 Antibodies:
[0035] 1. Receptor-ligand Binding Study
[0036] A BS3/anti-BS3 system will have useful applications in the
area of receptor-ligand binding assays. Such assays can be
performed both on solid phase ELISA format and on the cell surface.
For binding assays on cell surface, BS3-linked ligand will be
reacted with cells expressing its receptor. The binding of
BS3-labeled ligand to the cell surface will then be detected using
anti-BS3 monoclonal antibody followed by anti-mouse IgG conjugated
to FITC. Binding of FITC conjugated antibody to the cell surface
ben be detected either by flow cytometry or by immunofluorescence
assay. An example of such binding assay using BS3-linked ligand is
described below:
[0037] Binding of HIV-1 gp120 to its receptor CD4 on the cell
surface was examined using anti-BS3 antibody. Purified gp120 was
labeled with BS3 by incubating purified protein with 0.5 mM BS3 in
PBS at room temperature for 30 min. Unreacted BS3 was blocked by
adding Tris buffer (pH 8.0) to a final concentration of 50 mM. The
labeled protein was then incubated with Sup T1 cells at 4.degree.
C. for 30 min. Sup T1 cells have been shown to express high level
of CD4 on the cell surface and are highly susceptible to HIV-1
infection. Cells were then washed with PBS and reacted with
anti-BS3 antibody for 30 min at 4.degree. C.
[0038] The binding of anti-BS3 antibody to the cell surface was
detected using FITC conjugated goat anti-mouse antibody. FIG. 1A
shows the FACS profile on the binding of BS3 labeled gp120 to the
sup T1 cell membrane. Binding of unmodified gp120 to Sup T1 cells
was also examined for comparison (FIG. 1B). It is clear from the
figure that the binding of BS3-labeled gp120 to CD4 exposes a
complex-specific epitope recognized by the monoclonal antibody
8F101. This experiment further demonstrates that the modification
of gp120 with BS3 did not affect the binding specificity of the
glycoprotein. In addition, an anti-V3 loop monoclonal antibody (M
77) also reacted with BS3-labeled gp120 bound to the cell
surface.
[0039] Receptor ligand binding can also be performed by solid phase
ELISA using BS3/anti-BS3 system. In this assay the receptor can be
absorbed on a solid phase plastic surface and can be then reacted
with BS3-labeled ligand. The binding of BS3 labeled ligand to the
receptor can be detected using anti-BS3 antibody followed by an
HRP-conjugated goat anti-mouse antibody.
EXAMPLE 3
[0040] Immunodiagnostic Assays
[0041] Another utility of a BS3/anti-BS3 system will be in the area
of immunodiagnostic assays that use a two-antibody detection
system. In this assay the capture antibody coated on a plate will
be used to capture the antigen to be detected. The BS3 labeled
detector antibody will be reacted with the antigen captured on the
plate by the capture antibody. An anti-BS3 that is conjugated with
a label such as HRP can then recognize BS3 labeled detector
antibody bound to the antigen. Using anti-BS3 antibody it will thus
be possible to design an assay using two mouse antibodies at the
same time. It is anticipated that the antigen capture assay using
BS3 labeled detector antibody will have an increased sensitivity
over an ELISA which uses two murine monoclonal antibodies and a
secondary anti-mouse antibody-enzyme conjugate.
* * * * *