Note: Descriptions are shown in the official language in which they were submitted.
- WO91/0~92 2 0 ~ ~ 7 3 ~ PCT/AU90/00~53
AGGLUTINATION ASSAY
TECHNICAL FIELD
The present invention relates to a reagent and a method
for detecting an antigen, antibody or other analyte in a
sample, such as human or animal blood, by an agglutination
assay. The invention is also directed to a kit containing the
reagents and to processes for preparing the reagents.
BACKGROUND OF THE INVENTION
Description of the Background Art
Immunoassays (and analogous specific binding assays) have
revolutionized human diagnostic and veterinary medicine since
the introduction of techniques such as the radioimmunoassay
(RIA), first reported by Yalow and Berson (Nature 184:1648
(1959)), and the enzyme immunoassay (EIA) which was first
reported by Engvall and Perlman (Immunochem. 8: 871 (1971))
and Van Weeman and Schuurs (FEBS Lett. 15:232 (1971)).
Immunoassays have enabled exquisitely sensitive
measurement of analytes circulating in the blood of a subject,
and have allowed the determination of levels of hormones,
drugs and other compounds present at very low concentrations
(such as picomoles/liter). Such assays, based on antibody-
antigen interactions, usually involve complex detection
systems. The reagents used are generally antigens labeled with
an enzyme or a radioisotope, antibodies or complexes thereof
which require either incubation with specific substrates and
measurement of a color end-point either visually or by means
of a colorimeter, or measurement of radioactive decay with
radiation counters to detect the presence of the analyte being
tested. These assays also involve several washing steps. Most
immunoassays for the detection of analytes in blood are
currently of this nature. Although these assays are sensitive,
they re~uire lengthy and involved procedures and e~pensiva
instrumentation.
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An alternative ~o the RIA and EIA is provided byagglutination immunoassays of the type described by Gupta et
al., J. Immunol. Meth. 80:177-187 (1985)), wherein
erythrocytes and anti~erythrocyte antibodies are used as the
indicator system. Typically, foreign erythrocytes, such as
sheep erythrocytes, are used in such immunoassays. Both direct
and indirect agglutination assays are known in the art. In the
conventional direct assay for an antigen, r~ed cells are coated
with antibody, and reacted with the sample. Multifunctional
antigens act as bridges between the coated red blood cells,
creating an agglutinate. In the conventional indirect assay,
red cells are coated with antigen, and contacted with both a
soluble antibody and with sample. Sample antigen competitively
inhibits the binding of sensitized red cells by the antibody,
and hence the agglutination.
Agglutination assays may use other agglutinable
particles, For example, latex agglutination assays are
described in Castelan et al. (J. Clin. Pathol. 21:638 (1968))
and Singer et al. ~Amer. J. Med. [1956 (Dec)]: 888).
Molinaro, U.S. 4,130,634 describes an agglutination
assay employing exogenous red blood cells precoated with
antibody. The problem of nonspecific agglutination of
erythrocytes by anti-erythrocyte antibodies was noted by
C7-ismas, U.S. 3,639,558, who proposed eliminating all
naturally occurring antigenic sites on the red blood cell by
coating it with protein. Chang, U.S. 4,433,059 precoated
exogenous red blood cells with a covalent, "tail-to-tail"
conjugate of an anti-erythrocy~e antibody (usually univalent)
linked by a heterobifunctional coupling agent to an anti-
analyte antibody. See also Smith, W088/05931, Gibbons, U.S.
4,329,011 and Smith, III, U.S. 4,900,685. In the method of
the latter patent, the agglutinated erythrocytes are
endogenous to the sample.
Hillyard~ Ryla~t, Kemp and Bundesen, U.S. Patent No. 4,~94,347
teach an agglutination immunoassay for whole blood samples
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WO91/04492 PCT/AU90/00453
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featuring the use of sndogenous erythrocytes as indicator
particles, and of an agglutination reagent in which an
erythrocyte binding molecule is conjugated to either an
analyte-
binding molecule (for a direct assay) or to an analyte
analogue (for an indirect assay). They discovered that
nonspecific agglutination could be avoided, even when the
erythrocyte binding molecule was multivalent, i~ it recognized
an abundant, well distributed membrane constituent such as
glycophorin.
In U.S. Patent No, 4,894,347, we pointed out that in an
agglutination assay for an~igenic analytes large enough to
allow simultaneous binding o~ two antibody molecules, but
which lack repeating epitopes, for agglutination to occur, the
antigen must interact with the immunoreagent so that at least
some molecules of antigen act as a bridge between proximate
erythrocytes. We taught that one solution was to employ a
; reagent comprising two or more distinct conjugates, i.e.,
ABMl/ EBM + ABM2/EBM, where EBM denotes an erythrocyte binding
molecule and ABMl and ABM2 are analyte binding molecules
specific for different, non-overlapping, non-repeating
epitopes of the analyte. An agglutination assay of this type
is depicted in Figure l(b).
; This solution, ho~ever, has a defect which is
particularly apparent when analyte concentrations are low,
e.g., for HCG less than lOnM. That is that a molecule o~
ABMl/EBM and a molecule of ABM2/EBM can bind simultaneously to
a single erythrocyte (Figure l(c)). The bound analyte molecule
then does not act as a bridge between proximate erythrocytes
and therefore does not promote agglutination.
Smith, U.S. 4,578,360 and Smith, U.S. 4,401,764 describe
conjugates of an erythrocyte binding molecule and a label
binding molecule. Chu, U.S. 4,493,793 constructed cov~lently
~; coupled lertin-antibody or lectin-antigen conjugates. Segal,
U.S. 4,676,980 prepared an antibody-antibody immuno
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WO91/0~92 2 ~ ~ ~ 7 3 ~ .4. PCT/AU90/00453 _
therapeutic conjugate for associating a target (e.g., tumor)
cell with a cytotoxic effector cell. Freytag, U.S. 4,517,303
describes an immunolytic assay employing a conjugate between
an analyte analogue and a cytolysin, e.g., whole mellitin. Li,
U.S. 4,661,441 referred to a conjugate of an analyte-binding
antibody and an antibody specific for the idiotype of the
analyte-binding moiety.
Wardlaw, U.S. 4,695,553 and Guesdon, U.S. 4,668,637
relate to use of "universal" anti-erythrocyte antibodies (and
cf. McLaughlin, U.S. 4,683,196), while type-specific
antibodies are taught by Lloyd, ~.S. 4,678,747, Graham Jr.,
U.S. 4,358,436, Lu, U.S. 4,550,017, Steplewski, U.S. 4,607,009
and Lennox, W083~03q77. Bigtee, Molec. Immunol., 20:1353-1362
(1983) describes the production and testing of anti-
glycophorin monoclonal antibodies; Wardlaw suggested use of an
anti-glycophorin antibody in clarifying the interface between
erythrocytes and leukocytes in centrifuged whole blood. This
group of references does not disclose conjugating the
erythrocyte binding molecule "tail-to-tail" to another binding
molecule.
It is known in the art that a blood sample may be divided
into a plurality of aliquots which are then assayed separately
for the presence of different analytes. We are not aware of any
reference which teaches or suggests dividing a blood sample,
reacting the aliquots with different agglutination reagents, and
then recombining the sample to obtain in creased sensitivity to a
single analyte.
Theofilopoulos (US 4,342,566), Duermeyer (US 4,292,403) and
Goldenberg (US 4,331,647) demonstrate the use of antigen binding
fragments of antibodies as substitutes for intact antibodies in
immunological assays. The construction of heterobifunctional
antibodies is taught by Auditore-Hargreaves
(US 4,446,233), Paulus (US 4,444,878) and Reading (US 4,474,~93).
Mochida (US 4,200,436) discloses the use of monovalent
antibodies, or antigen-binding fragments thereof, in certain
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W09t/04492 PCT/AU90/004~3
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immunoassays. Forrest (US 4,659,678) mentions that monovalent
antibodies cannot ~orm dimers or more extensive com pleY~es with
the antigen; such monovalent antibody-antigen aggregates were
said to be capable of interfering with the binding of the
antigen-antibody complex to a solid phase support.
Traditional hemagglutination assays are generally faster,
but less sensitive than radioimmunoassays (RIA) or enzyme
immunoassays (EIA). The speed and sensitivity of hemagglutination
assays can be increased by the use of the
au~ologous red cell agglutination technique described in, e.g.,
U.S. 4,894,347. The advantage of such a technique is that blood
does not have to be separated; a single finger prick is
sufficient to provide an assayable sample. For analytes which are
large molecules with repeating epitopes, the sensitivity of such
an assay is sufficient.
The present inventors recognized a need in the art for
improved sensitivity in autologous agglutination assays, in
particular for detection of hormones such as human chorionic
gonadotrophin (HCG) in the early stages of pregnancy. Such assays
with increased sensitivity could also serve as alterna tives to
inhibition assays in the immunodetection of small molecules.
Inhibition assays are inherently less sensitive than direct
assays, and the end point is more difficult to define.
No admission is made that any referenca cited in this
specification i9 prior art. All references are hereby
incorporated by reference.
SUMMARY OF THE INVENTION
It is the object of the present .invention to overcome the
deficiencies of the art set forth above. The invention is useful
in assaying low concentrations of antigens lacking repeating
determinants. We provide a method by which these analytes may be
assayed directly. In particular, we descrihe herein a direct
- agglutination assay wherein an analyte which has two nonrepeating
epitopes may be detected without loss of sensitivity due to the
formation of multiple "bridges" between an analyte molecule and a
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WO91/044~2 PCT/AU90/00453 -~
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single agglutinable particle.
~ ccording to a first embodiment of this invention there is
provided a direct agglutination assay for t:he detection of an
analyte in a sample, wherein the analyte includes a first
nonrepeating epitope and a second nonrepeating epitope, the assay
comprising:
(a) providing first and second primary agglutination reagents,
each of which is capable of binding simultaneo~lsly to an
agglutinable particle, and to a nonrepeating epitope of said
analyte, said first and second reagents binding to different,
non-overlapping epitopes of said analyte;
(b) forming a first mixture of a first sample portion, said first .
reagent, and agglutinable particles bindable by said first
reagent;
(c) forming a second mixture of a second sample portion, said :-
second reagent, and agglutinable particles bindable by said
second reagent; and
(d) forming a third mixture by mixing said first and second
mixtures,
wherein agglutination in the third mixture indicates the presence :~
of the analyte in the sample.
According to a second embodiment of this invention there
is provided a direct agglutination assay for the detection of an
analyte in a sample, wherein the analyte includes a flrst binding
site, the assay comprising:
(a) providing a first primary agglutination reagent which is
capable of binding to an agglutinable particle and to said
analyte epitope, and which when bound to said analyte epitope
forms a ~econd epitope;
(b) forming a first mixture of a first sample portion, said first
reagent, and agglutinable particles bindable by said first
reagent;
(c) providing a second primary agglutination reagent which is
capable o~ binding to an agglutinable particle and to said second
epitope;
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(d~ forming a second mixture of a second sample portion, said
second reagent, and agglutinable particles bindable by the second
reagent, said second epitope being ~indable by said second
reagent; and
(e) forming a third mixture by mixing said first and second
mixtures,
wherein agglutination in the third mixture indicates the presence
of the analyte in the sampleO
According to a third embodiment of this invention there is
provided a two site direct agglutination assay for the detection
of the simultaneous presence of two different analytes in a
sample wherein the first and second analytes each comprise at
least one analyte-binding site, the assay comprising:
(a) providing a first primary agglutination reagent which is
capable of binding simultaneously to an agglutinable particle and
to said first analyte;
(b) providing a second primary agglutination reagent which is
capable of binding simultaneously to the same or a different type
of epitope of an agglutinable particle and to said second
analyte;
(c) providing agglutinable particles to which said first and
second primary agglutination reagents can bind, if such particles
are not already present in the sample;
~d) forming a first mi~ture of a first sample portion,
said first agglutination reagent, and bindable
agglutinable particles to ob~ain
first analyte:first agglutination reagent:par~icle
complex;
(e) forming a second mixture of a second sample portion,
second primary agglutination reagent, and bindable
agglutinable particles to obtain
second analyte:second reagent:particle complex;
(f) providing a secondary agglutination reagent which can
bind simultaneously to both said first complex and to said
second complex, in each case being specific for either a
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WO91/04492 PCT/AU90/00453 ^-
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native epitope of the analyte or an epitope formed by the
binding of the primary reagent to said analyte, but not
able to bind to two complexes of the same analyte;
~g) forming a third mixture of said first and second
mixtures and said secondary agglutination reagents.
wherein agglutination of said third mixture indicates the
simultaneous presence of said first and second analytes in
the sample.
According to a fourth embodiment of this invention
there is provided a direct agglutination assay for the
detection of an analyte in a sample, wherein the analyte
includes a first nonrepeating epitope and a second
nonrepeating epitope, the assay comprising:
(a) providing a first primary agglutination reagent which
is capable of binding simultaneously to an agglutinable
particle, and to a nonrepeating epitope of said anàlyte;
(b) forming a first mixture of a first sample portion, ~-
said first reagent, and agglutinable particles bindable by
said first reagent;
(c) providing a primary agglutination agent comprising a
second primary agglutination reagent:agglutinable particle
complex wherein said second primary agglutination reagent
is capable of binding to a nonrepeating epitope of said
analyte and said first and second reagents binding to
different, non-overlapping epitopes of said analytP;
(d) forming a second mixture of said first mixture and
; said agent, and agglutinable particles bindable by said
first reagent;
wherein agglutination in the second mixture indicates the
presence of the analyte in the sample.
According to a fifth embodiment of this invention
there is provided a direct agglutination assay for the
detection of an analyte in a sample, wherein the analyte
includes a first binding si~e, the assay comprising:
(a) providing a first primary agglutination reagent which
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WO 91/0~92 2 ~ PCT/AU90/00453
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is capable of binding to an agglutinable particle and to
said analyte epitope, and which when bound to said analyte
epitope forms a second epitope;
(b) forming a first mixture of a first sample portion,
said first reagent, and agglutinable particles bindable by
said first reagent;
(c) providing a primary agglutination agent comprising a
second primary agglutination reagent:agglutinable particle
complex wherein said second primary agglutination reagent
is capable of binding to said second epitope; and
(d) forming a second mixture by mixing said first mixture
and said agent
wherein agglutination in the second mixture indicates the
presence of the analyte in the sample.
According to a sixth embodiment of this invention
there is provided a two site direct agglutination assay
for the detection of the simultaneous presence of two
different analytes in a sample wherein the first and
second analytes each comprise at least one analyte-binding
site, the assay comprising:
(a) providing a first primary agglutination reagent which
is capable of binding simultaneously to an agglutinable
particle and to said first analyte;
(b) providing agglutinable particles to which said first
primary agglutination reagent can bind, if such particles
are not already present in the sample;
(c) forming a first mixture of a ~irst sample portion,
said first agglutination reagent, and bindable
agglutinable particles to obtain
first analyte:first agglutination reagent:particle
complex;
(d) providing a primary agglutination agent comprising a
second primary agglutination reagent:particle complex
whexein said second primary agglutination reagent is
capable of binding to said second analyte thereby forming
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W091/0~492 ~ 3 6 ~CT/AU90/00453 -
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a second complex;
(e) providing a secondary agglutination reagent which can
bind simultaneously to both said ~irst complex and to said
second complex, in each case being specific for a native
epitope of the analyte or an epitope formed by the binding
of the primary reagent to said analyte, but :not able to
bind to two complexes of the same analyte;
(f) forming a second mixture of said first mixture, said
primary agglutination agent and said secondary
agglutination reagents,
wherein agglutination of said second mixture indicates the
simultaneous presence of said first and second analytes in
the sample.
According to a seventh embodiment of this invention
there is provided a two site direct agglutination assay
for the detection of the simultaneous presence of two
different analytes in a sample wherein the first and
second analytes each comprise at least one analyte-binding
site, the assay comprising:
(a) providing a first primary agglutination agent
comprising a first primary agglutination reagent:particle
complex wherein said first primary agglutination reagent
is capable of binding to said first analyte thereby
forming a first complex;
(b) providing a second primary agglutination agent
comprising a second primary agglutination reagent:particle
complex wherein said second primary agglutination reagent
is capable of binding to said second analyte thereby
forming a second complex;
(e) providing a secondary agglutination reagent which can
bind simultaneously to both said first complex and to said
second complex, in each case being specific for a native
epitope of the analyte or an epitope formed by the binding
of the primary reagent to said analyte, but not able to
bind to two complexes of the same analyte;
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~f) forming a first mixture of said sample, said first and
second primary agglutination agents and said secondary
agglutination reagents,
wherein agglutination of said first mixture indicates the
simultaneous presence of said first and second analytes in
the sample.
According to an eighth embodiment of this invention
there is provided a two site direct agglutination assay
for the detection of the simultaneous presence of three
different analytes in a sample wherein each of said
analytes comprise at least one analyte binding site, the
assay comprising:
(a) providing a first primary agglutination reagent which
is capable of binding simultaneously to a first
agglutinable particle and to a first analyte;
(b) pro~iding a second primary agglutination reagent which
is capable of binding simultaneously to a second
agglutinable particle and to a second analyte;
(c) providing first and second agglutinable particles to
which said said first and secondary primary agglutination
reagents can bind respectively, if such particlas are not
already present in said sample;
(d) forming a first mixture of a first portion of the
sample, said first primary agglutination reagent and said
first agglutinable particles to obtain a first
analyte:first primary agglutination reagent:first particle
complex;
(e) forming a second mixture of a second portion of the
sample, said second primary agglutination reagent and sald
second agglutinable particles to obtain a second
analyte:second primary agglutination reagent:second
particle complex;
~f) providing first and second secondary agglutination
: reagents whereln said first secondary agglutination
rea~ents can bind simultaneous1y to both said first
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WO91/0~92 2 ~ 3 ~ ~- PCT/AU90/00453 _
complex and to a third analyte, and said second secondary
agglutination reagent can bind simultaneously to both said
second complex and to said third analyte;
(g) forming a third mixture of the first mixture, the
second mixture and the first and second secondary
agglutination reagents, : ;
wherein agglutination of said third mixture indicates the
simultaneous presence of said first, second and third
analytes in the sample.
According to a ninth embodiment of this invention
there is provided a two site direct agglutination assay
for the detection of the simultaneous presence of a
plurality of different analytes in a sample wherein each
of said analytes comprise at least one analyte binding
site, the assay comprising:
(a) providing a first primary agglutination reagent which
is capable of binding simultaneously to a irst
agglutinable particle and to a first analyte;
(b) providing a second primary agglutination reagent which
is capable of binding simultaneously to a second
agglutinable particle and to a second analyte;
(c) pro~iding first and second agglutinable particles to
which said said first and secondary primary agglutination
reagents can bind respectively, if such particles are not
already present in said sample;
(d) forming a first mixture of a first portion of the
sample, said first primary agglutination reagent and said
firs~ agglutinable particles to obtain a first
analyte:first primary agglutination reagent:first particle
complex;
(e) forming a second mixture of a second portion of the
sample, said second primary agglutination reagent and said
second agglutinable particles to obtain a second
analyte:second primary agglutination reagent:second particle
complex;
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WO91~0~92 -13- PCT/AU90/00453
(f) providing first and second secondary agglutination
reagents wherein said first secondary agglutination
reagent can bind simultaneously to both said first comple~
and to a third analyte, and said second secondary
agglutination reagent can bind simultaneously to both said
second complex and to a fourth analyte;
(g) providing (n+l)th tertiary agglutination reagents
which can bind simultaneously with two diffe:rent of (n)th
additional analytes wherein a first tertiary agglutination
reagent binds to said third analyte and a first additional
analyte and a second tertiary agglutination reagent binds
to said fourth analyte and a second additional analyte and
wherein n is an integer of 2 or more;
(h) forming a third mixture of said first and second
mixtures, said first and second agglutinable particles,
and said (n+l)th tertiary agglutination reagents,
wherein agglutination of said third mixture indicates the
simultaneous presence of said plurality of different
analytes in said sample. 'n' is typically from 2 to 100.
According to a tenth embodiment of this inventlon there
is pro~ided an agglutination assay for the detection of an
analyte in a particulate-containing sample wherein the analyte
comprises at least three binding sites, the assay comprising:
mixing a first conjugate comprising a particulate-
binding molecule and an analyte binding molecule capable of
binding to first and second binding sites on the analyte with
a first particulate-containing sample to form a first mixed
sample,
mixing a second conjugate comprising a particulate-
binding molecule and an analyte binding molecule capable of
binding to a third binding site on the analyte with a second
particulate-containing sample to form a second mixed sample,
mixing the first mixed sample with the second mixed
sample wherein agglutination of the combined mixed samples
indicates presence of analyte in the first particulate-
WO9l/0~92 2 ~ PC~/AU90/00453
-14-
containing sample.
According to an eleventh embodiment of this
invention there is provided an assay for an allergen-
specific IgE antibody in whole blood comprising:
(a) providing a heterobispecific anti-erythrocyte and
anti-IgE antibody specific for the IgE of the species from
which said whole blood is obtained, said heterobispecific
antibody being capable of binding to only one site on the
IgE molecule;
(b) providing a known quantity of a multivalent allergen
bound by said IgE antibody;
(c) reacting the whole blood sample with said
heterobispecific antibody and said allergen, whereby
erythrocytes endogenous to said whole blood sample are
agglutinated if and only if an IgE antibody which binds
said allergen multivalently is present.
According to a twelfth embodiment of this invention
there is provided an assay for an allergen-specific IgM
antibody in whole blood comprising:
~a) providing a heterobispecific anti-erythrocyte and
anti-lgM antibody specific for the IgM of the species from
which said whole blood is obtained, said heterobispecific
antibody being capable of binding to only one site on the
IgM molecule;
(b) providing a known quantity of a multivalent allergen
bound by said IgM antibody;
(c) reacting the whole blood sample with said
heterobispecific antibody and said allergen whereby
erythrocytes endogenous to said whole blood sample are
agglutinated if and only if an IgM anti~ody which binds
said allergen multivalently is present.
Generally, mixing of the first conjugate with the first sample
and the second conjugate with the second sample are typically
done over 1 - 2 minutes~ The first mixed sample and the
second mixed sample are typically mixed together for 1 - 2
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minutes. A strong positive will agglutinate in lO - 20
seconds.
In one embodiment, the sample is divided into two portions.
A first primary agglutination reagent ~Rl) which can
simultaneously bind both an agglutinable indicator particle and a
first, nonrepeating epitope analyta of interest is combined with
the analyte in the first portion and with indicator particle
(which may be endogenous or exogenous to the sample). A second
primary agglutination reagent (R2), similar in function except
that it binds a ~econd, nonrepeating epitope of the analyte, is
combined with the second portion and with indicator particle. The
stoichiometry of the reaction is such that there is essentially
no uncomplexed analyte in either portion. Also, es~entially all
the excess reagent is particle bound (P-Rl or P-R2).
The two sample portions are now recombined. The Particle-
Reagentl-Analyte complex which has a free second epitope,-reacts
with the ReagentZ~Particle (P-R2) comple~. The Particle-Reagent2-
Analyte complex which has a free first epitope, reacts with the
Reagentl-Particle (P-Rl) complex. Each reagent molecule is
already bound to one particle (from one portion), but is free to
bind only to an analyte molecule complexed to another particle
(from the other portion). Multiple bridges between a single
particle and a single analyte molecule are avoided.
In a second embodiment, the assay is adapted to the
situation where the analyte initially has only a single,
nonrepeating epitope. It relies on the fact that the immune
complex of the first reagent and the analyte has epitopes which
are not found on either the reagent or the analyte alone. The
second reagent binds to such a formed epitope, rather than to an
epitope native to the analyte.
In a third embodiment, we also employ a third, bispecific
secondary agglutination reagent which conjugates a first compleY
comprising analyte (from the first sample portion) bound to a
particle through the first reagent, and a second complex
comprising analyte (from the second sample portion) bound to a
wo 9l/044g2 2 ~ ~ ~ 7 3 ~ PCT/AU90/00453
16~
particle through the second reagent. Each valency of the third
reagent may be for either a native epitope of the analyte or one
formed by the analyte-Rl or analyte-R2 compleY~.
If the specificities of the third reagent are chosen so
that it is incapable of conjugating first complex-to-first
complex or second complex-to-second complex, it will not be able
to conjugate two analyte molecules conjugated already to the same
particle, and therefore, will more efficiently contribute to the
formation of the desired agglutinate.
According to a thirteenth embodiment of this invention
there is provided a test kit for use in a direct agglutination
assay, for the detection of an analyte in a particulate-
containing sample, which comprises:
(a) a first conjugate comprising a particulate-binding
molecule and a first analyte binding molecule
capable of binding to a first binding site on the
analyte, and
~b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule
capable of binding to a second binding site of the
analyte.
~ ccording to a fourteenth embodiment of this invention
there is provided a test kit for use in a direct agglutination
aqsay for the detection of an analyte in a particulate-
containing sample, comprising a single binding site, ~hich
comprises:
(a) a first conjugate comprising a particulate-binding
molecule and a first analyte binding molecule
capable of binding to the single binding site of
the analyte, and
(b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule
capable of binding to the first analyte binding
molecule bound to the analyte.
~ccording to a fifteenth embodiment of this invention
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-- WO91/0~92 17- PCT/AU90/OOqS3
there is provided a test kit for use in a two site direct
agglutination assay for the detection of an analyte in a
particulate-containing sample, wherein the analyte comprises
at least two binding sites, which comprises:
(a) a first conjugate comprising a particulate-binding
molecule and a first analyte binding molecule capable of
binding to a first binding site on the analyte,
(b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule capable of
binding to a second binding site of the analyte, and
(c) a crosslinking reagent which comprises a dimeric
analyte binding molecule capable of binding to a third binding
site on the analyte.
According to a sixteenth embodiment of this in~ention
there is provided a test kit for use in a two site direct
agglutination assay for the detection of an analyte in a
particulate-containing sample, wherein the analyte comprises
at least two binding sites, which comprises:
(a) a first conjugate comprising a particulate-binding
molecule and a first analyte binding molecule capable of
binding to a first binding site on the analyte,
(b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule capable of
binding to a second binding site on the analyte, and
(c) a crosslinking reagent which comprises a divalent
hybrid analyte binding molecule capable of binding to third
and fourth binding sites on the analyte.
. According to a seventeenth embodiment of this invention
: there is provided a test kit for use in a two site direct
agglutination assay for the detection of an analyte in a
particulate-con~aining sample, wherein the analyte comprises
at least two binding sites, which comprises:
(a) a first conjugate comprising a particulate-binding
molecule and a first analyte binding molecule capable of
binding to a first binding site on the analyte,
WO91/0~92 PCT/AU90/004~3 ~
2 ~
(b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule capable of
binding to a second binding site on the analyte, and
(c) a crosslinking reagent which comprises a divalent
hybrid analyte binding molecule capable of binding to a third
binding site on the analyte and to a site generated by the
binding of the first analyte binding molecule or the second
analyte binding molecule to the analyte.
According to an eighteenth embodiment of this invention
there is provided a test kit for use in a two site direct
agglutination assay for the detection of an analyte in a
particulate-containing sample, wherein the analyte comprises
at least two binding sites, which comprises:
(a) a first conjugate comprising a particulate~binding
molecule and a first analyte binding molecule capable of
binding to a first binding site on the analyte,
(b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule capable of
binding to a second binding site on the analyte, and 6
(c) a crosslinking reagent which comprises a divalent
hybrid analyte binding molecule which is capable of binding to
a site generated by the binding of the first analyte binding
molecule and to a site generated by the binding of the second
analyte binding molecule to the analyte.
According to a ninteenth embodiment of this in~ention
there is provided a test kit for use in a two site direct
agglutination assay for the detection of an analyte in a
particulate-containing sample wherein the analyte comprises at
least two binding sites, which comprises:
(a) a first conjugate comprising a particulate-binding
molecule and a first analyte binding molecule capable of
binding to a first binding site on the analyte,
(b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule capable of
binding to a second binding site on the analyte, and
23~73~
WO91/04492 PCT/AU90/0045
-19 -
(c) a crosslinking reagent which comprisas a divalent
hybrid analyte binding molecule capable of binding to the
first binding site on the analyte and to the second binding
site on the analyte.
According to a twentieth embodiment of this invention
there is provided a test kit for use in a two site dlrect
agglutination assay for the detection of an analyte in a
particulate-containing sample wherein the analyte comprises at
least two binding sites, which comprises:
(a) a first conjugate comprising a particulate-blnding
molecule and a first analyte binding molecule which binds to a
first binding site on the analyte,
(b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule capable of
binding to a second binding site on the analyte, and
(c) a crosslinking reagent comprising a divalent hybrid
analyte binding molecule capable of binding to the second
binding site on the analyte and to a site generated by the
binding of the second analyte binding molecule to the second
binding site.
According to a twenty first embodiment of this inventlon
thexe is provided a test kit for use in a two site direct
agglutination assay for the simultaneous detection of two
different analytes in a particulate-containing sample, wherein
the first and second analytes each comprise at least one
analyte binding site, which comprises:
~ a) a first conjugate comprising a particulate-binding
molecule and a first analyte binding molecule capable of
binding to a first binding site on the first analyta,
~ b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule capable of
binding to a second binding site on the second analyte, and
(c) a crosslinking reagent which comprises a dimeric
analyte binding molecule capable of binding to a third binding
site on the fi~st analyte and to a fourth binding site on the
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WO91/04492 2 ~ 3 ~ PCT/AUgO/00453
20-
second analyte.
Accordlng to a twenty second embodiment of this
invention there is provided a test kit for use in a two site
direct agglutination assay for the simultaneous detection of
two different analytes in a particulate-cont:aining sample,
wherein the first and second analytes each comprise at least
one analyte binding site, which comprises:
(a) a first conjugate comprising an erthrocyte binding
molecule and a first analyte binding molecule capable of
binding to a first binding site on the first analyte,
(b) a second conjugate comprising an erthrocyte binding
molecule and a second analyte binding molecule capable of
binding to a second binding site on the second analyte, and
(c) a crosslinking reagent which comprises a divalent
hybrid analyte binding molecule capable of binding to a third
binding site on the first analyte and to a fourth binding site
on the second analyte generated by the binding of the second
analyte binding molecule to the second analyte.
~ ccording to a twenty third embodiment of this invention
there is provided a test kit for use in a two site direct
agglutination assay for the simultaneous detection of two
different analytes in a particulate-containing sample, wherein
the first and second analytes each comprise at least one
analyte binding site, which comprises:
(a) a first conjugate comprislng a particulate-binding
molecule and a first analyte binding molecule capable of
binding to a first binding site on the first analyte molecule,
(b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule capable of
binding to a second binding site on the second analyte, and
(c) a crosslinking reagent which comprises a divalent
hybrid analyte binding molecule capable of binding to a third
binding site on the first analyte generated by the binding of
the first analyte bindiny molecule to the first analyte and to
a fourth binding site on the second analyte generated by the
3 ~
W091/~4492 21- PCT/AU90/00~53
binding of the second analyte binding molecule to the second
analyte.
According to a twenty fourth embodiment of this
invention there is provided a test kit for use in a two site
direct agglutination assay for the simultaneous detection of
two different analytes in a particulate-containing sample,
wherein the first and second analytes ea~h comprise at least
one analyte binding site, which comprises-
~ a) a first conjugate comprising a particula~e-bindlng
molecule and a first analyte binding molecule capable of
binding to a first binding site on the first analyte,
(b) a second conjugate comprising a particulate-binding
molecule and a second analyte binding molecule capable of
binding to a second binding site on the second analyte, and
(c) a crosslinking reagent which comprises a divalent
analyte binding molecule capable of binding to a third bindiny
site present on each of the first and second analytes.
According to a twenty fifth embodiment of this invention
there is provided a test kit for an agglutination assay for
~he detection of an analyte in a particulate-containing sample
wherein the analyte comprises at least three binding sites,
comprising:
(a) a ~irst conjugate comprising a particulate-binding
molecule and an analyte binding molecule capable of binding to
first and second binding sites on the analyte; and
(b) a second conjugate comprising a particulate-binding
molecule and an analyte binding molecule ~apable of binding to
a third binding site on the analyte.
According to a twenty sixth embodiment of this invention
there is provided a test kit for use in a direct agglutination
assay for the detection of an analyte in a first particulate-
containing sample, wherein the analyte comprises a first
binding site and a second binding site, which comprises:
(a) a first conjugate comprising a first particulate
binding molecule and an analyte binding molecule
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WO9J/04492 2 ~ 3 ~ -22- PCT/AU90/00453
capable of binding to the first binding site; and
(b) a reagent prepared by miYing/reactlng a second
particulate-containing sample with a second conjugate
comprising an analyte binding molecule capable of binding to
the second binding site and a second particulate binding
molecule.
According to a twenty seventh embodiment of this
invention there is provided a test kit for a direct
agglutination assay for the detection of an analyte in a first
particulate-containing sample, wherein the analyte comprises a
binding site, which comprises:
(a) a first conjugate comprising a first particulate-
binding molecule and an analyte binding molecule
capable of binding to the binding site, and thereby
forms a second binding site; and
(b) a reagent prepared by mixing/reacting a second
particulate-containing sample with a second
conjugate comprising an analyte binding molecule
capable of binding to the second binding site and a
second particulate binding molecule.
According to a twenty eigh~h embodiment of this
in~ention there is provided a test kit for a two site direct
agglutination assay for the detection of an analyte in a first
particulate-containing sample, wherein the analyte comprises
at least two binding sites, which comprises:
(a) a first conjugate comprising a first particulate-
binding molecule and an analyte binding molecule
capable o~ binding to a first binding site on the
analyte;
(b) a reagent prepared by mi~ing/reacting a second
particulate-containing sample with a second
conjugate comprising an analyte .binding molecule
ca~able of binding to the second binding site and a
second particulate binding molecule; and
(c) a crosslinking reagent;
;
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2~73~
W~91/04492 PCT/AU90/0~453
23-
In one form the cross linking reagent comprises adimeric analyte binding molecule capable of binding to a third
analyte binding site on the analyte.
In another form the crosslinking reagent comprises a
divalent hybrid analyte binding molecule capable of binding to
a third analyte binding site on the analyte and on a fourth
analyte binding site on the analyte.
In a further form the crosslinking reagent comprises a
divalent hybrid divalent analyte binding molecule capable of
binding to a third analyte binding site on the analyte and to
a site generated by the binding of the first analyte binding
molecule to the first binding site or the second analyte
binding molecule to the second binding site.
In yet another form the crosslinking reagent comprises a
divalent hybrid analyte binding molecule capable of binding to
a site generated by the binding of the analyte binding
molecule to the first binding site, and to a site generated by
the binding of the second analyte binding molecule to the
second binding site.
In yet a further form the crosslinking reagent comprises
a divalent hybrid analyte binding molecule capable of binding
to the first binding site in the analyte and to the second
binding site on the analyte.
In another form the crosslinking reagent comprises a
divalent hybrid analyte binding molecule capable of binding to
the second binding site on the analyte and to a site generated
by the binding of the second analyte binding molecule to the
second binding site.
According to a twenty ninth embodiment of this invention
there is provided a test kit for a two site direct
agglutination assay for the simultaneous detection of two
different analytes in a first particulate-containing sample
wherein the first and second analytes each comprise at least
one analyte bindiny site, which comprises:
~a) a first conjugate comprising a first particulate-
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WO~1/044~2 2 ~ $ ~ 7 ~ 24- PCT/AU90/00453
binding molecule and a first analyte binding molecule capable
of binding to a flrst analyte binding site on the first
analyte;
~ b~ a reagent prepared by mixing/reacting a second
pa~ticulate-containing sample with a second conjugate
comprising an analyte binding molecule capable of binding to
the second binding site and a second particulate binding
molecule; and
(c) a crosslinking reagent.
In one form the crosslinking reagent comprises a
divalent hybrid analyte binding molecule capable of binding to
a third analyte binding site on the first analyte and a fourth
analyte binding site on the second analyte.
In another form, the crosslinking reagent comprises a
divalent hybrid analyte binding molecule capable of binding to
a third analyte binding site on the first analyte and to a
fourth analyte binding site generated by the binding of the
second analyte binding molecule to the ~econd analyte.
In yet another form, the crosslinking reagent comprises
a divalent hybrid analyte binding molecule capable o~ binding
to a third analyte binding site generated by the blnding of
the first analyte binding molecule to the first analyte, and
to a fourth analyte binding site generated by the binding of
the second analyte binding molecule to the second analyte.
In a further form, the crosslinking reagent comprises a
divalent analyte binding molecule capable of binding to a
third analyte binding site present on each of the first and
second analytes.
According to a thirtieth embodiment of this invention
there is provided test kit for an agglutination assay for the
detection of an analyte in a first particulate-containing
sample wherein ~he analyte comprises at least thre~ binding
sites, which comprises:
(a) a first conjugate comprising a first particulate-
binding molecule and an analyte binding molecule capable of
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W091/0~9~ 2 ~ 3 ~ PCT/AUgo/00453
25~
binding to first and second binding sites on the analyte; and
tb) a reagent prepared by miY~ing/reacting a second
particulate-containing sample with a second conjugate
comprising an analyte binding molecule capable of binding to a
third binding site on the analyte and a particulate-binding
molecule.
Generally,in the twenty sixth to thirtieth embodiments
the particles in the second particulate-containing sample are
different from the particles in the first particulate-
containing sample. Generally, the particulate-containing
samples can be whole blood, semen, culture of hybridoma cells,
samples obtained from microbial fermentation and tissue
culture, for example. Alternatively, the particulate-
containing samples can contain synthetic or artificial
particles such as carbohydrates, latex (natural or synthetic
rubber or plastic), glass beads, carbohydrates (cellulose)
liposomes and metal oxide particles, for eY.ample.
BRIEF DESCRIPTION OF T~E DRAWIN~S
Figure l(a~ shows the three components of an agglutination
assay described in the Hillyard patent: a particle with multiple
epitopes, an analyte with two different nonrepeating epitopes,
and two primary agglutination reagents, both of which bind the
particle, but which also bind the analyte at the di~ferent
epitopes.
Figure l(b) shows an agglutinate formed by the sequential
or simultaneous combination of the aforementioned reaction
components.
Figure l(c) reveals how sensitivity can be reduced through
the formation of multiple bridges between one molecule of analyte
and one erythrocyte.
Figure 2 illustrates the present invention. The sample is
divided into two portions, each of which is reacted with
particles and with one or the other of the two primary
agglutination reagents. When the two portions axe recombined,
only "interparticle" bridges can form.
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WO91/0~92 ~ 3 ~ ~26- PCT/AU90/00453 _
Figure 3 depicts a variant on the above in which the
analyte need only have a single, nonrepeating epitope. In the
reaction shown on the left side, the binding of the first reagent
to the analyte creates a new epitope which :is recognized by the
second reagent shown on the right side when the sample portions
are recombined
Figure 4 illustrates uses of a secondary agglutination
reagent. While drawn so as to suggest that the epitope recognized
by the reagent is one generated by formation of the analyte-
primary reagent comple~., this is not in fact required, as may be
seen in, e.g., the neYt Figure.
Figure 5 depicts various ways in which the secondary
agglutination reagent can bridge two analytes, including (a)
binding the same epitopes recognized by the primary reagents, (b)
binding a third epitope on each analyte, (c) binding a third and
fourth epitope, on each analyte, (d) bridging a third epitope and
a complex-formed epitope, (e) binding two complex-formed
epitopes, and (f) binding an epitope recognized by a primary
reagent and an epitope formed by the compleYing of the analyte
with the other primary reagent.
Figure 6 shows an adaptation of the assay for simultaneous
measurement of two different analytes.
Figure 7 illustrates use of a first primary agglutination
reagent as previously described, in conjunction with a particle-
bound analyte binding molecule, the latter replacing the second
primary agglutination reagent.
Figure 8 depicts an assay for antibody employing a first
PBM-ABM reagent wherein the analyte binding molecule is an
an~igen specifically bound by the analyte antibody and a second
reagent which is a carrier particle bound anti-immunoglobulin.
Note that this anti immunoglobulin may be covalently bound to the
carrier particle (as shown) or conjugated through a particle
binding moiety (such as an anti-glycophorin antibody moiety, in
the case of an erythrocyte).
Figures 9 and 10 are schematic representations of a
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W~91/04492 2 ~ 6 ~ 7 3 ~ PCT/AU90/00~53
further type of assay which detects specific immunoglobulin
subtypes, such as IgM or allergen-specific IgE. This assay also
allows the detection of IgG antibodies to large antigens, which
would be difficult to couple directly to the anti erythrocyte
antibody. The EBMs can be the same or bind to different sites on
the erythrocyte.
Figure 9 represents the assay for allergen specific IgE.
The conjugate consists of a bi-specific anti-erythrocyte-anti IgE
antibody which binds to only one site on the IgE. The allergen is
added as a multivalent moiety, either alone or bound to a carrier
such as latex or a protein. Agglutination occurs when IgE
antibodies, specific to the allergen are present in the sample.
Figure l0 represents the assay for specific IgM. The
conjugate consists of a bi-specific anti erythrocyte-anti IgM
antibody which binds to only one site on the IgM. The antigen is
added as a multivalent moiety, either alone or bound to a carrier
such as latex or a protein. Agglutination occurs when IgM
antibodies, specific to the antigen are present in the sample.
BEST MODE AND OTHER MODES FOR CARRYING OUT THE INVENTION
The term "epitope" is used herein to refer to a binding
site on a target molecule which is specifically recognized by a
binding molecule, and is not limited to binding sites recogni2ed
by antibodies.
Agglutinable Indicator Particle
~ ny par~icle which is capable of agglutination to form
detectable agglutinate, and which can be provided in a form for
which a specific particle binding molecule exists, may be used in
the present agglutination assay. The particle may be a biological
or non-biological particle, and it may be natural or artificial
in origin. If natural it may be modified to render i~ more
suitable for assay use. If artificial it may be organic or
inorganic in composition. A variety o~ particles, i~cluding
particles of latex, charcoal, kaolinite, or bentonite, as well as
both microbial cells and red blood cells, have been used as
agglutinable carriers (see Mochida, US 4,308,026). A mixture of
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W O 91/04492 PC~r/AU90/00453 --
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different sizes or types of particles may he used, and different
particles may be used as lndicators in different portions o
sample. The particles may be colored or fluorescent to facilitate
their visualization. The particles may be native to the sample or
added to it. If added, they may be added before, during or after
the aliquoting of the sample and before, during or after the
addition of the agglutination reagent.
The use of erythrocytes as indicator particles was strongly
criticized by Patel (US 3,882,225) on the ground that it is
difficult to standardize the indicator erythrocytes. Nonetheless,
erythrocytes are preferred indicator particles. When the sample
is a blood sample, it is especially preferred that the indicator
particles be the erythrocytes endogenous to the sample.
There are several advantages of endogenous over eY.ogenous
erythrocytes. First, no pretreatment of endogenous erythrocytes
is required. This is in contrast to methods taught in U.S. Patent
4,433,059, which uses blood group 0, Rh cells, which have been
centrifuged, reacted with an antibody conjugate for 15-30 minutes
and washed 3 times in by centrifugation; or U.S. Patent
4,668,647, which uses sheep erythrocytes which had been washed
and resuspended in PBS, reacted with an antibody on a solid
support, and then fixed.) Secondly, there is no need to
centrifuge sample; whole blood, collected in the presence of a
suitable anticoagulant, is used instead of serum or plasma. The
assay is therefore more suitable for field use. For other
ad~antages of using endogenous erythrocytes as indicator
particles, see Hillyard, U.S. 4,894,347.
Particle ~inding Moiety or Molecule (PBM)
Since, in a preferred embodiment, the particle is an
erythrocyte, the preferred particle binding moiety or molecule is
one which binds erythrocytes. Erythrocyte membranes are lipid
bilayers with a variety of proteins. Some proteins occur only on
outer portion of the membrane. O~her proteins contain a
hydrophobic portion allowing the protein to anchor in or pass
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WO91/0~92 2 ~ ~ ~ 7 3 ~ PCT/AU90/00453
-29-
through the membrane. A membrane-tra~ersing protein, also called
a transmembrane protein, may also have an intracellular or
cytoplasmic portion. Glycophorin A is an example of a
transmembrane protein.
Known proteins of erythrocyte membranes (with their blood
groups shown in parentheses) include Glycophorin A (MN, Ena,
Wrb), Glycophorin B ~Ss, 'N', U), and the minor constituents such
as integral membrane protein 1 (Rh), membrane attached
glycoprotein C~ (Chido ~ Rodgers), integral membrane glycoprotein
(anion channel), ankyrin, spectrin, protein 4.1 and F-actin.
Associated with the membrane proteins are glycolipids (Lewis) and
glycosphingolipids (ABH, Ii, P, Tk).
The following general publications disclosiny basic
information about cell membranes, in particular erythrocyte
membranes, are hereby incorporated by reference: S.B. Shohet et
al., The Red Cell Membrane, In: Hematology, 3rd ed. Eds: Williams
et al., 1983; Marchesi, V.T. The Red Cell Membrane Skeleton,
Blood 61: 1~11, 1983.
~ here the particle is an erythrocyte, there are numerous
erythrocyte binding molecules (EBM), available, including
antibodies, lectins, and other binding proteins. Erythrocyte
membranes contain various antigenic surface constituents
including proteins, glycoproteins, glycolipids and lipoproteins.
Antibodies which recogni~e these constituents may be prepared by
conventional techniques using the membrane, or the purified
constituents thereof, as immunogens. These antibodies may be
monoclonal or polyclonal in nature. Either the intact antibody,
or specific antigen-binding fragments thereof, may be used as an
erythrocyte binding molecule (EBM). The antibody or antibody
fragment may be polyvalent, divalent or univalent.
A preferred EBM is an antibody (or specific binding
fragment thereof) recognizing glycophorin. This molecule
comprises 131 amino acids with 16 oligosaccharide chains. When
erythrocyte sialoglycopeptides are extracted from membranes, the
main fraction (approximately 75% of total) is glycophorln. Thus,
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WO91/0~92 PCT/AU90/00453
-30-
2 ~ r~ 3 ~
this is an abundant moiety, which could allow antibody
attachment without agglutinating the red cells. It is also
readily available in a relatively pure form commercially. (for
example, from Sigma Chemical Company). (See: H. Furthmayr et al.,
Biophys. Biochem. Res. Comm. 65:113-122 (1975)).
The design and use of conjugates of an erythrocyte binding
antibody and an analyte-binding antibody as an agglutination
reagent has been constrained by the need to avoid "auto-
agglutination." By this term, is meant the phenomenon
attributable to the ability of such a reagent, acting alone, to
bind two or more erythrocytes simultaneously, and thereby to
cross-link the erythrocytes into an agglutinate.
The general understanding in the art is that "auto
agglutination" can be avoided only by the use of univalent
erythrocyte-binding molecules. See, e.g. Chang, U.S. 4,533,~59.
Surprisingly, certain intact (e.g. bivalent or pentavalent)
antibodies, notably certain anti-glycophorin mAbs, do no~ cause
significant "auto-agglutination." It is believed that the anti-
glycophorin antibody is non-autoagglutinating for steric reasons;
either the antigen binding sites of the intact antibody are able
only to bind adjacent epitopes on the same erythrocyte ~and not
span the distance between two erythrocytes~ or only one of the
two antigen binding sites can bind to glycophorin at one time.
When the EBM iq multivalent, as is the case of a typical
antibody, it is desirable that the molecule recognize an
erythrocyte membrane constituent which is abundant and well
distributed; the binding site should be in such a position that
crosslinking between cells is inhibited by steric hindrance.
thereby avoiding premature red cell agglutination.
Alternatively, crosslinking may be inhibited by the selection of
an EBM that recognizes a surface constituent present in
sufficient quantity so that the epitopes are sufficiently close
for the binding sites on the EBM to be bound by only the one
erythrocyte.
One aspect of the present invention is thus the use of an
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WO91/0~92 2 ~ ~ ~ 7 3 ~ PCT/AUgo/00453
-31-
intact non-auto-agglutinating anti-erythrocyte antibody (or
multivalent binding fragments thereof) in conjugates in
erythrocyte agglutination immunoassay. The use of such antibodies
is advantageous because the conjugates are easier to prepare in
high yields and there is no requirement to make Fab/Fab'
fragments, which are difficult to purify to t:he necessary degree
purity from a mixture which also contains agglutinating
antibodies.
The use of univalent derivatives of these inherently non-
auto-agglutinating erythrocyte-binding antibodies is also within
the scope of this invention. The advantage of using non-auto-
agglutinating antibody to prepare univalent binding fragments is
that it is difficult to purify Fab' from a mixture of F~ab)2 and
Fab' without some contamination with F(ab)2 fragments.
Furthermore, some F(ab)2 "dimers" reform from the Fab univalent
fragments.
If the original antibody was itself auto agglutinating,
then any contaminating F(ab)2 will cause some agglutination. This
will reduce the sensitivity of the assay. The problem is avoided,
however, by deriving the Fab' fragments only from a non-auto-
agglutinating antibody, as taught herein.
In addition, glycoproteins, glycolipids and other
carbohydrate structures on the surface of erythrocytes are
recognized by proteins known as lectins, which have an affinity
for particular saccharides. Lectins may therefore also be used as
EBMs, as contemplated by the present invention.
Other molecules with specificity and af~inity for the erythrocyte
surface also may be used. These could also include molecules with
an affinity for the lipid bilayer of the membrane. Examples of
such molecules are: protamine, the membrane binding portion of
the bee venom, mellitin, and other highly basic peptides.
Applicants have also discovered a new class of
agglutination reagent in which the erythrocyte-binding moiety is
a non-immunoglobulin peptide derived from the bee venom toxin,
mellitin. Thus, th~ invention provides a conjugate between
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WO91/04492 32- P~T/AU90/00453
mellitin 7-26, or another peptide with similar nonlytic,
univalent erythrocyte-binding property with an analyte-bindiny
molecule to form a direct agglutination assay
reagent. In contrast to an anti-erythrocyte antibody, mellitin 7-
26 can be synthesized much more easily and economically by
chemical or recombinant DNA techniques. However, the present
invention is not limited to any particular method of preparing
the EBM.
The preferred EBMs of the present invention will recognize
erythrocyte membrane constituents found on all, or nearly all
erythrocytes in a sample, so that erythrocytes endogenous to the
blood sample may be used as the agglutinating particles. Such
constituents include the so-called "public antigens".
The known blood group specificities are typically
conferred by carbohydrate or glycolipid moieties, which are
associated with membrane proteins. For its utility in the present
invention, it is thus desirable that an EBM recogni7e either the
protein part of a membrane glycoprotein constituent, which is
common to all erythrocytes of a particular species, or another
common structure. The ability of a divalent EBM to agglutinate
erythrocytes will depend on steric factors such as the mobility
of the molecule and the position of the binding sites relati~e to
the lipid bilayer.
It is preferable but not necessary that a single EBM be
used that recognizes essentially all erythrocytes. Several EBMs
may be used, either in the same or in separate reagents, each of
which recognizes a particular group of erythrocytes, but which in
aggregate recognize essentially all erythrocytes.
While it is preferable that the EBM recognize a natural surface
constituent of the erythrocyte, it is possible to coat
erythrocytes with a ligand recognl~ed by the EBM, or to treat the
erythrocytes so as to expose a normally cryptic ligand.
- Non-erythrocyte particles may likewise be functionalized so that
they may be bound by an antibody or other binding molecule.
Samples and Analytes
. . .
WO~ ~92 2 ~ 3 ~ PCT/AU90/004S3
The sample may be a biological fluid or a non biological
fluid. Non-limiting examples of biological fluids obtained in
vivo include whole blood, a separated blood fraction, urine,
semen, saliva, cerebrospinal fluid, amniotic fluid, ascites
fluid, pleural effusion, cyst fluid, pus, tissue extracts, etc.
Non-limiting examples of biological fluids obtained in vitro
include tissue culture supernatant, such as that of hybridoma
cells, or microbial fermentation medium.
The sample may also be a non-biological fluid such as
drinking water, wastewater, groundwater, or a nonaqueous fluid.
Preferably, the sample is a particulate-containing sample such
that the particles are suitable for use as agglutinable indicator
particles. However, if the sample does not natively contain
suitable particles, they may be added to the sample to obtain a
"particle-containing sample''. This addition may occur before,
after or simultaneously with the addition of the primary
agglutination reagent as described herein.
This invention is not limited to the detection of any
particular analyte, however, it is especially useful for
detection of analytes lacking repeating epitopes. The analyte may
be a substance normally found in blood, such as a blood protein
or a hormone, or it may be a foreign substance, such as a drug
(including both therapeutic drugs and drugs of abuse~. Typical
analytes include hormones (e.g. HCG, LH, FSIl, insulin), enzymes
~e.g. amylase, trypsin, CX isoforms), other polypeptides such as
myoglobin, steroids (e.y. sex hormones, corticosteroids, anabolic
steroids), drugs (e.g. theophylline, digoxin, paracetamol,
barbiturates, cannabinoids, opioids), venoms, antibodies (e.g. of
the classes IgG, IgE, IgM, etc.).
~ Analyte Binding Moiety or Molecule (ABM)
; The analyte-binding moiety or molecule (ABM) may be any
substance having a preferential affinity for the analyte.
including monoclonal or polyclonal antibodies lectins, enzymes,
; or other binding proteins or substances (or binding fragments
thereof). Where the analyte is an antigen, the ABM is usually an
.
.. . ..
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.
WO9l/0~92 2 ~ 3 ~34- PCT/A~90/004S3 _
antibody, or an antigen~binding fragment of an antibody, such as
a F(ab')2, Fab, Fv or V~ fragment. Where the analyte is an
antibody, the ABM is usually an antigen or hapten recognized by
that antibody, or a second antibody raised against the
immunoglobulin isotype of the analyte antibody.
First and Second Primary Agglutination Reagents (PBM-ABM)
The assay employs conjugates of a particle binding moiety
or molecule (PBM), especially an erythrocyte binding molecule
(EBM), with an analyte binding moiety or molecule (ABM). The
conjugate may be a sinsle molecule with particle and analyte-
binding moieties, or a complex of two or more molecules.
Hereafter, the term "molecule" will be used to cover both
moieties and molecules, and "conjugate" to cover both a single
hybrid molecule with two bindiny moieties and two conjugated
molecules which each have specific binding behavior. In one
embodiment, the conjugate is obtained by coupling an EBM (or
other PBM) to an ABM.
The PBM and the ABM may be coupled together directly or
indirectly, and by covalent or non-covalent means (or a
combination thereof). Below are listed reagents and references
disclosing some of the covalent coupling methods known in the
art.
l.SPDP ~N-Succinimidyl-3,2-(pyridyldithio) propionate)
Neurath et al., 1981, J. Virol. Meth. 3:155-165.
2.MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester)
Kitagawa et al~, 1976, J. Biochem. 79:223 236.
3.SIAB (N-succinimidyl-4-iodoacetylaminobenzoate) Weltman
et al., 1383, Bio. Techniques 1:148-152.
Selective Bifunctional Reagents
P-isothiocyanatobenzoylchloride
~U.S. Patent 4,680,338)
Bifunctional Reagents
1. BSOCOES - Bis[2-
, . . .
.
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WO91/VM92 2 ~ ~ ~ 7 3 ~ PCT/AU90/00453
~35-
(succinimidooxycarbonyloxy)ethyl]sulfone
Zarling et al., 1980, J. Immunol. 124:913-920.
2. BS - Bis~sulfosuccinimidyl)suberate
Staros, 1982, Biochemistry 21:3950-3~55.
Other Reagents
1. Glutaraldehyde - Avrameas, 1969, Immunochem. 6: 43.
2. PeriGdate Oxidation - Nakane et al., 1974, J.
Histochem. Cytochem. 22:1084-1091
3. Carbodiimide
4. Disulfide Exchange
The PBM and the ~BM may also be coupled nonco~alently,
for example, by ~a) attaching biotin to one and avidin ~or
strepta~idin) to the other), (b) attaching an anti antibody to
one, which then binds the other, (c) attaching Protein A to
one. which then binds the Fc portion of the other, or (d)
attaching a sugar to one and a corresponding lectin to the
other.
It should be understood that, in coupling the PBM and
the ABM, the binding characteristics should be changed as
little as possible. It may be advantageous to provide a spacer
moiety between the PBM and the ABM to reduce steric hindrance.
The PBM/ABM conjugate may be a hybrid antibody. One
method of constructing such a conjugate is the following:
(a) preparing F(ab)'2 fragments of a selected antibody by
pep~in digestion;
(b) reducing and treating the fragments with Ellman's reagent
to produce Fab' fragments of the ~elected antibody;
(c) thiolysing a selected analyte-~pecific antibody or a
selected anti-erythrocyte antibody; and
(d) coupling the thioylated Fab' fragment to the Ellman's
reagent-treated Fab' fragment to produce a hybrid anti-
erythrocyte antibody antigen specific antibody conjugate.
Another method for constructing an EBM/ABM conjugate,
in the form of a chimeric antibody, comprises:
: ~ , ,,' , ,
.. , . , . :
:~ : ,, " ,
.: .
W091~0~9~ PCT/AU90/00453
2 ~ 3 ~ 36-
(a) treating an anti-erythrocyte mAb-producing hybridoma
and an analyte-specific mAb-producing hybridoma with a
distinct site-specific irreversible inhibitor of
macromolecular biosynthesis; preferably the inhibitor is
selected from the group consisting of emetine, actinomycin
D, hydro~yurea, ouabain, cycloheximide, edine and
sparsomycin;
(b) fusing the two different mAb-producing hybridomas with
polyethylene glycol to produce a heterohybridoma;
(c) cloning the fused cells by any of a number of cloning
methods such as isolation in soft agarose or by limiting
dilution;
(d) selecting cloned heterohybridomas secreting chimeric
anti-erythrocyte antibody antigen specific antibody with a
screening assay appropriate to the antibodies;
(e) purifying the antibody product by affinity
purification to free it from non-chimeric antibodies. The
hybrid or chimeric antibody of the present invention thus
comprises two "half moleculas," one with specificity for
erythrocytes (the EBM) and the other with specificity for
the analyte (the ABM). In this case the antibody's own
disulfide bonds couple the ABM to the EBM to form an
appropriate conjugate.
Such a hybrid antibody has advantages over a tail-to-
tail conjugate as taught in the prior art, which is formed by
a bifunctional coupling agent, conjugating an anti-analyte
antibody and a univalent fragment of anti-erythrocyte
antibody. The advantages include ease of preparation, the
preservation of the correct stoichiometry and stereochemistry
of both antibodies and the retention of the binding affinity
of each fragment.
When the analyte-binding molecule is also a peptide or
protein, the use of a peptide to bind the erythrocyte has the
further advantage that the entire conjugate may be prepared
without any need for a bifunctional coupling agent. Instead,
~ .
WO91/04492 2 ~ ~ ~ 7 3 ~ PCT/AU90/00453
-37-
:
a DNA sequence encoding the erythrocyte-binding peptide and
the analyte-binding peptide as a single transcriptional unit
is provided, and the desired conjugate is e~pressed as a
fusion protein, with the two moieties joined by a simple
peptide bond, or with a peptide spacer of desired length. One
particularly preferred example is a DNA sequence encoding an
erythrocyte-binding antibody fragment (which could be ~ust
the binding site, not a complete Fab) and analyte antibody
(which could be just the binding site, not a complete Fab) or
antigen as a single transcriptional unit is provided, and the
desired conjugate is expressed as a fusion protein, with the
two moieties joined by a simple peptide bond, or with a
peptide spacer of desired length. Alternatively, the divalent
peptide may be prepared by direct chemical synthe~is.
As intended herein, the particles in or added to the
samples assayed according to the methods of the present
invention have a plurality of binding sites.
The first and second primary agglutination reagents
may recognize the same or different binding sites ~epitopes)
on the surface of the particle, and may be prepared by the
same or different procedures.
Secondary Agglutination Reagent (CBM-CBM)
The secondary agglutination reagent, as previously
stated conjugate~ the first complex (formed by the particles
and analyte of the first sample portion and the first primary
agglutination reagent) and the second complex. It thus
possesses two complex-binding moieties or molecules (CBM).
The CBMs may be the same or differe~t, and they may
bind to an epitope found on the analyte originally, or one
generated by the binding of an ABM-PBM conjugate to its
analyte epitope. This epitope may be the same epitope as is
recognized by the PBM-ABM conjugate, or a different analyte
epitope. The same techniques may be used to prepare the
secondary agglutination reagent as were described for the
.. . . . . . ........................................... . .
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WO91/0~92 PCT/AU90/00~53 -
~S~73~ 38-
primary reagent.
Test Kits
Test kits for use in the present invention comprise
first and second ~BM-ABM conjugates as previously described,
and may optionally include a third reagent as described.
Assay Formats
In a preferred embodiment, the contemplated direct
agglutination assay compris~s:
(a) dividing the sample into a first portion and a second
portion;
(b) forming a first mixture of a first primary agglutination
reagent comprising a particle binding molecule (PBM) and an
analyte binding molecule (A~Ml) capable of binding to a first
binding site of the analyte, a first sample portion and
agglutinable particles;
(c) forming a second mixture of a second primary
agglutination reagent comprising a particle binding molecule
or moiety and an analyte binding molecule or moiety (ABM2)
capable of binding to a second and different binding site of
the analyte, a second sample portion, and agglutinable
particles; and
(d) mixing the first mixture with the second mixture to
obtain a third mixture, wherein agglutination of the third
mi~.ture indicates the presence of the analyte in the sample.
Surprisingly, the separate mixing according to the
above embodiment has been found to increase the sensitivity
by as much as 20- to 30-fold as compared to an assay where
two conjugates are mixed together with a single blood sample
such as in the assay described by the present inventors in
Australian Patent Application No. 24182/88 and U.S. Patent
~,894,347.
It should be understood that this embodiment relies on
the fact that th PPM is present in exce:s compared with the
'
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2~6~3~
WO91/04492 PCT/AU90/00~53
-39-
number of antigenic molecules being detected. The PBM should
thus recognize an particle surface epitope which is abundant.
The affinity of the interactions does not appear to be
a significant factor at analyte concentrations likely to be
present in a blood sample. The basis for the increased
sensitivity using the two spot system is more likely to be
stoichiometric. Thus, in the assay of Example , there were 10
ug/mL of anti-erythrocyte antibody conjugate and
approximately 700,000 glycophorin mol0cules on the surface of
a red cell and the amounts of HCG being measured was in the
order of 25~100,000 IU/L or 2.5-1.0000 mg/L. The molecular
weight of HCG is about 50 kDa and that of the conjugate is
about 100 kDa. Therefore, many more conjugate molecules are
bound to erythrocyte surfaces than there are HCG molecules in
the blood sample. Affinity may have some influence; analysis
of a series of antibody conjugates with varying affinities
for HCG indicated a decrease in sensitivity with decreasing
affinity.
The anti- erythrocyte antibody has an affinity of 1 x
109 and the highest affinity for HCG was 5.9 x 109.
When we speak of forming a mixture of several
components, the component may be brought together
simultaneously, or sequentially in any order.
When one of the components of a formed mixture is a
set of agglutinable particles and another component is a
portion of a sample, it should be understood that the
particles may be endogenous to the sample so that, in effect,
sample analyte and agglutinable particles are mixed
simultaneously with the remaining reaction component(s), or
the particles may be exogenous, in which case they may be
added before or after the division of the samplP, and at any
stage in the formation of the recited mixture.
For the measurement of monoepitopic haptens, such as
drugs or steroids, one may use two conjugates, one consisting
.
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WO91/04492 2 ~ ~ ~ 7 3 6 40- PCT/AU90/00453
of anti-erythrocyte antibody/antihapten antibody and the
other anti-erythrocyte antibody/antibody to the
hapten:antihapten immune complex.
This assay thus comprises:
(a) dividing the sample into a first and a second portion;
(b) forming a first mixture of a first primary agglutination
reagent comprising a particle binding molecule and an
analyte-binding molecule capable of binding to the binding
site, and thereby forming a second binding site, a first
sample portion and agglutinable paxticles;
(c) forming a second mixture of a second primary
agglutination reagent comprising a particle binding molecule
and an analyte-binding molecule capable of binding to the
second binding site a second sample portion and agglutinable
particles; and then
(d) mixing the first mixture with the second mixture to
obtain a third mixture, wherein agglutination of the third
mixture indicates the presence of the analyte in the sample.
The second epitope may comprise at least part of the
first epitope bound by the first analyte-binding molecule or
may result from a conformational change taking place in the
analyte molecule upon binding the first analyte-binding
molecule.
In a further enhancement of these two basic
embodiments, the first and second mixtures may be mixed with
secondary agglutination reagent. Use of a secondary reagent
makes it less important to employ àn agglutinable particle
with a large number of non-overlapping epitopes. Six
different (and non-limiting) types of secondary reagents
which can be used in this version of the assay are described
below.
(l) A dimeric analyte-binding molecule capable of
binding to a third site on each analyte molecule.
12) A divalent hybrid analyte-binding molecule capable of
~ '~' '
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W091/0~92 2 ~ ~ ~ 7 3 ~ PCT/AUgo/0~453
41~
binding to a third site on one analyts molecule and a fourth
si~e on tha other analyte molecule.
(3) ~ divalent hybrid analyte-binding molecule capable of
binding to a third site on one analyte molecule and to a
fourth site which is genarated by the binding of an analyte-
binding molecule to a binding site on another analyte
molecule.
(4) A divalent hybrid analyte-binding molecule capable of
binding to a site generated by the binding the first analyte-
binding molecule (of a primary reagent) to a first analyte
molecule, and to a site generated by the binding of the
second analyte binding molecule to a second molecule of
analyte.
(5) A divalent hybrid analyte-binding molecule capable of
binding to the first binding site of the analyte and to the
second binding site of the analyte.
(6) A divalent hybrid analyte binding molecule capable of
binding to the second binding site of the analyte and to a
site generated by the binding of the second analyte binding
molecule to the second binding site.
In another embodiment, the invention is directed to a
two site direct agglutination assay for the detection of the
simultaneous presence of two different analytes in a particle
containing sample wherein the first and second analytes each
comprise at least one analyte-binding site, the assay
comprising:
(a) dividing the sample into a first portion and a second
portion;
(b) forming a mixture of a first primary agglutination reagent
comprising a particle binding molecule and a first analyte-
binding molecule capable of binding to a first binding site on
the first analyte, the first sample portion, and agglutinable
particles;
(c) forming a second mi~ture of a second primary agglutination
reagent comprising a particle binding molecule and a second
:
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WO91/04492 P~T/AV90/00453 _
2 ~ 3 ~ -42-
analyte-binding molecule capable of binding to a second binding
site on the second analyte, the second sample, and portion and
agglutinable particles;
(d) mi~ing the first mixture with the seconcl miY.ture and with a
secondary agglutination reagent wherein agglutinatlon of the
combined mixtures in the presence of the sec-ondary agglutination
reagent indicates the pres~nce of both the lirst and second
analytes in the sample.
For this embodiment, the secondary agglutination
reagent must be heterobispeci~ic. Generally, mixing of the
first conjugate with the first sample and the second
conjugate with the second sample are performed over a 1-2
minute interval. The first mixed sample and the second mixed
sample are typically mixed together for 1-2 minutes. A strong
positive test will show agglutination in 10-20 seconds.
In a variation of the various embodiments of the
contemplated direct agglutination assay, the blood is mixed
with the first A3M-EBM conjugate. A second reagent is added,
which comprises an added particle, such as exogenous red
blood cells bound to a conjugate of an EBM and a different
ABM which binds to a different site on the analyte. The
latter reagent can also be an added particle, such as latex
or exogenous red blood cells to which an ABM, which binds to
a different site on the analyte, has been coupled chemically.
It will be appreciated that the function of particle/ABM and
particle:EBM ABM is the same, vis-a-vis the binding of
analyte.
In the agglutination assay for an antibody analyte
described by Hillyard, a whole blood sample containing the
analyte ~and endogenous erythrocytes serving as the indicator
particle) is reacted with an EB~-ABM conjugate, wherein the ABM
is typically an antigen specifically bound by the antibody or a
second, anti-idiotypic antibody against the analyte antibody. We
have found that the sensitivlty of such an assay may be
increased by adding a second, particle-bound, (analyte
WO91/04492 2 ~ 3 ~ PCT/AU90/00453
43-
antibody)-binding reagent. The analyte-binding moiety of this
latter reagent is typically an anti-Fc antibody. It may be bound
to the particle either covalently through its own Fc, or by a
particle~binding moiety conjugated covalently or noncovalently
to the analyte binding moiety. If all of the analyte in the
sample is bound by the first reagent, the second reagent, by
binding to the analyte will form additional interparticle
connections.
The sensitivity of assays for analytes with repeating epitopes
is likewise reduced by the formation of multiple bridges between
the indicator particle and the analyte, though with such assays
only a single PBM-ABM reagent is necessary for agglutination.
The method of this invention may be applied to such analytes by
deliberately ignoring any repeating epitopes of the analyte, and
instead identifying and providing binding molecules for two
different nonrepeating epitopes thereof.
Moreover, the present method may provide some
advantage in assaying an analyte for which there is an
epitope which, while repeating, is uncommon, though the
increase in sensitivity will be less pronounced.
In another embodiment of this invention there is provided
an assay ~hich detects antibodies of a particular immunoglobulin
icotype, such as, for example, IgM, or allergen specific IgE
antibodies. This assay also allows the detection of IgG antibodies
to large antigens which would be difficult or impossible to couple
directly to an anti-erythrocyte antibody. The EBMs can be the same
or bind to different sites on the erythrocyte.
One reagent comprises bispecific anti-erythrocyte ~or
other particle) ~ anti IsE antibody, which binds to only one
site on the IgE molecule. The other is the allergen, a
multivalent moiety, added either alone or bound to a carrier
substance such as a latex particle or a protein.
Agglutination occurs when IgE antibodies specific to the
allergen are present in the sample.
Similarly, for detection of a specific IgM antibody,
:
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WO91/OM92 PCT/AU90/00453
-44-
2~7~6
the reagents are a bispecific anti-erythrocyte - anti IgM
conjugate, (which binds to only one site on the IgM molecule)
and a multivalent antigenic reagent added either alone or
bound to a carrier substance such as a latex particle or a
protein. Agglutination occurs when IgM antibodies specific to
the antigen are present in the sample.
Ha~ing now generally described the invention, the same
will be more r~adily understood through reference to the
following examples which is provided by way of illustration,
and are not intended to be limiting of the present invention,
unless specified.
EXAMoeLE 1
PREPARATION QF AN ERYTHROCYTE sINDING MOLECULE (EBM):
ANTI-GLYCOPHORIN ANTIBODY
Mice were immunized with human erythrocytes and mAbs
produced by fusing the spleen cells of immunized animals with
mouse myeloma cells, according to methods well-known in the
art. Hybridoma supexnatants were screened for the presence of
the desired antibody by both spin agglutination assay and
enzyme immunoassay (EIA), wherein glycophorin was bound to
polystyrene in a microtiter plate.
Spin agglutination was performed by a modification of
Wya~t et al. (Aust. J. Med. Lab. sci. ~:48-50 (19 )). 50 ul
of cell culture supernatant was mixed with 50 ul of a 1%
erythrocyte suspension in a microtiter plate. Antibodies
which bound glycophorin, but did not agglutinate, were
selected.
For EIA, microtiter plates were coated with 10 ug/ml human
glycophorin (Sigma Chemical Co., Cat. No. G-73B91), washed and
then incubated with serial dilutions of the mAb. After further
washing, the presence of bound antibody was determined by the
addition of an enzyme-labellad second antibody (specific for
murine immunoglobulin), followed by the addition o~ substra~e for
the en~yme. The antibody titer was determined to be the greatest
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2 ~ 3 ~
- WO91/04492 PCT/AU90/00453
~45-
dilution of hybridoma supernatant which gave an absorption (A420)
reading greater than 0.17 units above background.
Of 384 wells, 40 primary clones were chosen. Each clone
fell into one of three categories: a positive result in the spin
agglutination assay only, binding to glycophorin in EIA only, or
both reactions, as depicted in Table 1.
TAsLE 1
EIA Spin agglutination Number
of clones
Negative Positive 4
Positive Positive 20
Positive Negative 16
Subsequent absorption studies were performed to
confirm that the mAbs recognized a glycophorin domain exposed
on the erythrocyte surface.
The hybridoma cells producing mAbs having the desired
reactivities were grown in vivo by intraperitoneal injection
into mice according to methods well known in the art. The
ascites fluids, containing the secreted mAbs, were clarified
by centrifugation and screened.
The results of the screening assays of ascites fluids
containing various mAbs are shown in Table ~, below:
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, .. . .
WO9l/04492 PCT~AU90/00453
TABLE 2
Glycophorin Reactivity of Monoclonal Antibodies
Ascites Fluid Titer
Clone Spin Glycophoxin EIA Red Cell
Agglutination Absorption
RAT lD3/167 512000 <1000 Posi~ive
RAT 3D6/5 6400 1024000 Positive
RAT lC3/86 <1000 1024000 Positive
RAT 3B1/172 256000 2000 Positive
RAT 3D3/22 4000 1024000 Positive
RAT 3D5/61 128000 1024000 ~ositive
RAT lA2/187 <1000 256000 Posi~ive
RAT 2A2/187 <1000 128000 Positive
RAT lA3/129 <1000 12800 Weak
RAT lC4/5 <1000 128000 Positive
RAT 4C3/13 <1000 128000 Positive
RAT 3B1/70 <1000 517000 Positive
RAT lC3/86 was selec~ed and deposited under the
Budapest Treaty at the American Type Culture Collection
(12301 Parklawn Drive, Rockville MD, 20852) on 7 September
1988. The cell line has the designation G 26.4.IC3/86, and
received the ATCC accession number HB9893.
MAbs were purified to homogeneity from ascites fluids
by chromatography on hydroxyapatite (Stanker et al., J.
Immunol. Meth. 76:157 (1985)).
EX~MPLE 2
Preparation of chimeric antibodies ~Anti-Glyco~horin/Anti-
uman chorionic ~onadotropin,, h~G) and use ln Ass,a~__or hCG
Monoclonal antibodies reacting with both the beta subunit of
hCG and wi~h the complete molecule were prepared by methods
, : . , ~ .
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wo 91/0449~ 2 $ ~ ~ ~ 3 ~ PCT/AU90/00453
~7~
well known in the art. Hybridoma supernatants were screened
for the presence of desired antlbody by enzyme immunoassay
wherein the beta subunit of hCG, the alpha subunit and intact
hCG were bound to polystyrene in a microtitre plate
essentially as described in Example l.
The results of screening assays of ascites fluids containing
various MAbs are shown in Table 3.
ENDPOINT TITRES
alpha subunit beta subunit whole molecule
HCG 2A1/67 <1000 10280000 512000
HCG 2C1/4 <1000 <1000 64000
HCG 3D2/34 <1000 128000 128000
HCG lA3/61 <1000 512000 512000
HCG 2B5/34 <1000 1000 128000
HCG 2B4/98 <1000 <1000 256000
MAb's were purified to homogeneity from ascitic fluids
by chromatography on hydroxylapatite (Stanker et al J.
Immunol. Meth 76 157 1985).
Monoclonal antibodies RAT lC3/86 (anti-human red blood
cell) and HCG-2A1/67 and HCG-lA3/63 (anti-human hCG) were
digested with pepsin essentially as described by Hackman, et
al., 1981, Immunology, 15, 429-436, and purified by
chromatography on a TSK-3000 SW column. 2mg RAT lC3/86 was
digested for 45 minutes with 1% w/w pepsin in a buffer
containing OolM acetic acid, 70mM sodium chloride pH 3.5.
Meanwhile, 2mg HCG 2Al/67 and 2mg HCG lA3/63 were digested
with 1% w/w pepsin for 2 hours in the same bufferO The
reactions were terminated by the addition of 1.5M Tris to
raise the pH to 8. The F~ab)2 fragments were purified by gel
.. . . . .
' ' :: -: . ' : .,
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WO91/0~92 PCT/AU90/00453 ~
3 ~
filtration chromatography on a TSK-3000 SW column.
Reduction of the F(ab~2, and subsequent blocking of
the Fab fragment, was carried out as described by ~rennan,
et al., 1985, Science 229, 81-83. A 3mg/ml F(ab)2 preparation
was treated wi~h 1 mM mercaptoethylamine, in the presence of
lOmM sodium arsenite, for 16 hours at 25 C. The Fab fragments
were stabilized by reaction with 5,5'-dithiobis, (2-
nitrobenzoic acid) (Ellman's reagent) for 3 hours at 25 C.
The Fab fragment was then purified by gel filtration
chromatography on a TSK-3000 SW column.
The thiol forms of HCG 2A1/67 and HCG lA3/63 were
regenerated by reaction with lOmM mercapthelylamine for 30
minutes at 25 C. Excess reagent was removed by gel filtration
chromatography on a TSK-3000 SW column. Separately the thiol
form of each of the HCG antibodies and the Ellman's reagent-
treated RAT-lC3/86 were incubated ~or 16 hours at 25 C as
described by Brennan et al. The chimeric antibodies thus
formed were finally purified by gel filtration chromatography
on a TSK-3000 SW column.
EX~MPLE 3
"TWO DROP" AGGLUTINATION ASSAY FOR HCG
Reagent 1: 10 ug/ml of Conjugate A (a hybrid antibody
consisting of "half" erythrocyte-binding antibody, RAT lC3/86,
and "half" ~CG-sp~cific antibody A) in PBS, pH 7.4, containing
1 mg/ml BSA and 0.010% sodium azidP.
Reagent 2: 10 ug/ml of Conjugate B (a hybrid antibody
consisting of "half" erythrocyte-binding antibody, RAT lC3/86,
and '~half" HCG specific antibody B) in PBS, pH 7.4, containing
1 mg/ml BSA and O.01% sodium azide.
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Method
The method requires two small drops of blood, one ofwhich is first mixed with conjugate l, the other with
conjugate 2. (These two drops are thus the "first and second
sample portions" as previously described.) After an incubation
period, the two drops are then mixed together.
Two 5 ul samples of whole blood were placed on a slide about l
cm apart. l0 ul of reagent l was added to one and mixed with a
small stirring rod (e.g., a toothpick) to Eorm a small circle.
l0 ul of reagent B was added to the second sample and mixed in
a similar manner.
The sensitivity of the test will be lower should the two
samples be mixed accidentally before the conjugates have bound to
the erythrocytes.
The agglutination plate was then rocked gently for 30
seconds and the two pools combined by mixing with a toothpick.
The mixture was then rocked gently for a further 2 minutes and
the extent of agglutination determined.
This method can, o~ course, be automated or adapted to a
single use device. Upon comparison of "two drop" and "one drop"
assays at various dilutions, it was apparent that in each case
the visible agglutination response of each sample assayed by the
"two drop assay" of the invention was much stronger than for the
corresponding sample assayed by the "one drop assay".
EXAMPLE 4
"ONE DROP" PARTICLE CAR~IER ASSAY FOR HCG
Reagent: l0 ug/ml of conjugate A (as in Example 3), a 0.5~
suspension of latex particles (polystyrene, 0.8 um diameter)
coated with HCG-specific antibody B, l mg/ml BSA in PBS
containing 0.01% azide.
j Method
The method only requires one drop of blood and provides
similar sensitivity to the assay of Example 3. To l0 ul of blood,
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WO91/04492 PCT/AU90/00453 _
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25 ul of test reagent was added and mixed with a small stirring
rod, rocked gently for a further 2 minutes, and the presance or
absence of agglutination was noted.
EXAMoeLE 5
PREPAR~TION OF MELITTIN AS AN ALTERNATIVE EBM
A peptide from bee venom, mellitin (amino acid sequence:
CVLTTGLPALISWIKRKRQQ), was used as an alternative to the
erythrocyte-binding mAb. This peptide binds to the
erythrocyte surface without lysing the cell (deGrado W.F. et
al., J. Amer. Chem. Soc. 103: 679-81 (1981)). The peptide was
synthesized by the Merrifield procedure (Hodges et al., Anal.
Biochem. 65:241 (1975)).
One advantage of using mellitin as the EBM is that f
mellitin and a peptide-type ABM may be synthesized as a single
unit, having both erythrocyte and analyte binding activity in the
single peptide.
Having now fully described this invention, it will be
appreciated by those skilled ln the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the inventions
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential
features hereinbefore set forth as follows in the scope of the
appended claims.
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