The present invention relates to a method and a kit for determining the presence of an analyte in a
liquid sample.
Immunoassays for determining the presence of an analyte in a liquid sample are well known. A
common type of immunoassay is a sandwich immunoassay. In such an assay, sample is added to
a test plate coated with a capture antibody. Analyte present in the sample binds the capture
antibody and is retained on the plate. The plate is washed to remove unbound analyte and a
detecting antibody is then added which binds the analyte which is bound to the capture antibody
thereby forming an analyte sandwich between the two antibodies. The analyte sandwich can be
detected, either by virtue of a label attached to the detecting antibody or by the addition of a further,
labelled antibody which binds to the detecting antibody. The amount of detected label is directly
proportional to the concentration of the analyte in the sample.
An alternative approach is a competitive inhibition assay. Again, a capture antibody is provided on
a test plate. Sample and a labelled version of the analyte of interest are added to the test plate.
Labelled analyte competes with analyte present in the sample for binding to the capture antibody.
The plate is washed and the amount of captured labelled analyte present is determined. The
amount of labelled analyte detected is typically inversely proportional to the concentration of
analyte in the sample.
Known assays suffer from various problems. For example, with certain assay formats steric
hindrance may compromise the formation of an immuno-complex between an antibody and an
analyte, for example when such interactions occur at a surface.
In a first aspect, the present invention provides methods of determining the presence of an analyte
present in a liquid sample. The invention provides a method of determining the presence of an
analyte present in a liquid sample, the method comprising :
a) contacting the liquid sample with a predetermined amount of an analogue of the analyte
and an excess of a first binding moiety, wherein the first binding moiety is capable of binding each
of the analyte and the analogue independently, such that a mixture is formed comprising an
analogue-first binding moiety complex, or, when analyte is present in the sample, either (i) an
analogue-first binding moiety complex and an analyte-first binding moiety complex, or (ii) an
analyte-first binding moiety complex and no analogue-first binding moiety complex;
b) contacting the mixture with a second binding moiety, wherein the second binding moiety
is capable of binding the analogue-first binding moiety complex but not the analyte-first binding
moiety complex, and is immobilised or immobilisable on a solid phase;
c) determining the level of a signal indicative of the presence of the analogue-first binding
moiety complex bound to the second binding moiety;
wherein if the level of the signal determined in step c) is lower than the level of a maximum
signal determined when no analyte is present, then analyte is present in the sample.
In step (a) above, the first binding moiety may be capable of binding both the analogue and analyte
simultaneously.
The invention further provides a method of determining the presence of an analyte present in a
liquid sample, the method comprising :
a) contacting the liquid sample with a predetermined amount of an analogue of the analyte
and an excess of a first binding moiety, wherein the first binding moiety is capable of binding each
of the analyte and the analogue independently but not both the analogue and analyte
simultaneously, such that a mixture is formed comprising an analogue-first binding moiety complex,
or, when analyte is present in the sample, either (i) an analogue-first binding moiety complex and
an analyte-first binding moiety complex, or (ii) an analyte-first binding moiety complex and no
analogue-first binding moiety complex;
b) contacting the mixture with a second binding moiety, wherein the second binding moiety
is capable of binding the analogue-first binding moiety complex but not the analyte-first binding
moiety complex, and is immobilised or immobilisable on a solid phase;
c) determining the level of a signal indicative of the presence of the analogue-first binding
moiety complex bound to the second binding moiety;
wherein if the level of the signal determined in step c) is lower than the level of a maximum
signal determined when no analyte is present, then analyte is present in the sample.
The present invention provides a number of advantages over prior art assay formats, which include
formation of a first antibody-target complex in solution phase; operation of a competitive
immunoassay format in solution phase, followed by surface capture and separation of bound from
free label that involves fewer washing steps compared with prior art assay formats. The methods
of the invention may enable the presence of an analyte in a liquid sample to be determined more
quickly than by known methods, for example, the RIA assay and/or Immulite assay. Preferably, the
methods of the invention enable the presence of an analyte in a liquid sample to be determined in
under 24 hours, under 12 hours or under 4 hours.
Once the liquid sample is contacted with the analogue and the first binding moiety, the analogue,
first binding moiety and, where present the analyte, are free to diffuse and interact with one another
in solution according to the well-known principles of Brownian motion. When there is no analyte in
the sample, the first binding moiety can bind only to the analogue, and a mixture comprising
analogue-first binding moiety complex is formed. This mixture is contacted with the second binding
moiety. The second binding moiety binds the analogue (even when it is in complex with the first
binding moiety), and therefore analogue-first binding moiety complex is captured on the second
binding moiety, which is preferably provided in excess. The second binding moiety may be
immobilised, for example, on a surface such as the well of a microtitre plate. Alternatively, the
second binding moiety may be immobilisable, such as by means of an immobilised binding moiety,
such that it becomes immobilised on a surface either before or after it has bound the analogue-first
binding moiety complex. In this case, the method may comprise the additional step of contacting
the second binding moiety with a surface in order to immobilise the second binding moiety on the
surface. Any first binding moiety that has not been captured by the second binding moiety may be
separated from the captured analogue-first binding moiety complex before step (c) is carried out.
This avoids the subsequent detection of any first binding moiety that has not been captured by the
second binding moiety. Any suitable separation method may be used, for example, washing with
buffer, washing with air, magnetic separation, filtration or immunochromatography. The level of a
signal indicative of the presence of the captured analogue-first binding moiety complex is then
determined.
The signal is preferably derived (either directly or indirectly) from a label, which is either directly or
indirectly attached to or associated with the first-binding moiety. Irrespective of how the signal is
produced and how the label is associated with the first binding moiety, the level of signal will be
proportional to the amount of analogue-first binding moiety captured by the second binding moiety.
The first binding moiety may itself comprise a label. Alternatively, the first binding moiety may not
comprise a label, and the method may further comprise contacting the first binding moiety with a
third binding moiety, wherein the third binding moiety binds the first binding moiety (either directly
or indirectly) and comprises a label. Thus, in such an embodiment, the labelled third binding
moiety provides the signal indicative of the presence of the captured analogue-first binding moiety
complex. The third binding moiety may be contacted with the first binding moiety at any stage of
the method before the level of signal is determined. For example, the third binding moiety could be
contacted with the first binding moiety before the liquid sample is contacted with the first binding
moiety, after this step but before the mixture is contacted with the second binding moiety, or after
the mixture is contacted with the second binding moiety but before the level of signal is determined.
The third binding moiety may be contacted with the first binding moiety when the first binding
moiety has formed a complex with the analogue and the complex has been captured by the second
binding moiety. One or more intermediate binding moieties could be used to link the third binding
moiety or label to the first binding moiety. The signal may be produced directly or indirectly by the
label. For example, the label may bring about the production of the signal by reaction with a
reagent. In this case, the method may further include the step of adding a suitable reagent in order
to produce the signal. Suitable labels are discussed in detail below.
The level of signal detected when no analyte is present in the sample is the maximum signal for the
test. This is because there is no analyte to compete with the analogue for binding to the first
binding moiety and thus the maximum amount of analogue-first binding moiety-complex is formed
and captured by the second binding moiety. As such, a maximum signal can be obtained by
carrying out the method of the invention on a sample known not to contain the analyte. This
maximum signal can be compared with tests carried out under the same conditions (e.g.
concentration of first binding moiety, analogue and second binding moiety) on samples with
unknown analyte content, in order to determine whether, and how much, analyte is present.
The first binding moiety is capable of binding to each of the analyte and the analogue
independently but preferably not both the analyte and the analogue simultaneously. Preferably, the
affinity of the first binding moiety for the analyte is substantially the same as the affinity of the first
binding moiety for the analogue. The sample and the analogue may be contacted with the first
binding moiety simultaneously. In this case, when analyte is present in the sample, there is direct
competition between the analogue and the analyte for binding to the first binding moiety.
Alternatively, the sample may be contacted with the first binding moiety before either the sample or
the first binding moiety is contacted with the analogue. In this case, when analyte is present in the
sample it binds to a proportion of the available first binding moiety. The analogue can then only
bind to first binding moiety that is not occupied by the analyte (if indeed any first binding moiety is
not occupied by analyte). In both embodiments, the analyte is said to compete with the analogue
for binding to the first binding moiety, and analyte-first binding moiety complex is formed in an
amount proportional to the concentration of analyte in the sample. Consequently less analoguefirst
binding moiety complex is formed than when no analyte is present in the sample. If the analyte
is present in a very high concentration, it may completely out-compete the analogue for binding to
the first binding moiety, such that no analogue-first binding moiety is formed. Thus, a mixture is
formed which comprises either (i) an analogue-first binding moiety complex and an analyte-first
binding moiety complex, or (ii) an analyte-first binding moiety complex only.
The mixture is contacted with the second binding moiety (which may or may not be immobilised on
a surface). As there is less analogue-first binding moiety complex present in the mixture (or no
analogue-first binding moiety complex at all) than when analyte is not present in the sample, the
amount of analogue-first binding moiety complex captured by the second binding moiety is also
less than the amount captured when no analyte is present in the sample. The second binding
moiety does not bind the analyte-first binding moiety complex, and therefore, this complex is not
captured by the second binding moiety. The second binding moiety does not have a binding
affinity for the analyte. This means that the maximum amount of second binding moiety is available
to capture analogue-first binding moiety complex, because the second binding moiety only has a
binding affinity for the analogue.
After the second binding moiety has been contacted with the mixture, the level of a signal indicative
of the presence of the analogue-first binding moiety complex bound to the second binding moiety is
determined as described above. Where the second binding moiety is not immobilised on a surface
when it binds the analogue-first binding moiety complex, the method preferably includes the step of
contacting the second binding moiety with a surface such that it becomes immobilised before the
level of signal is determined.
When analyte is present in the sample, the level of signal determined in step (c) will be lower than
the level of signal determined when there is no analyte present in the sample, because less
analogue-first binding moiety complex is formed and captured by the second binding moiety. In
other words, the signal detected is inversely proportional to the concentration of analyte in the
sample.
The method of the invention can also be used to determine the amount or concentration of analyte
present in the sample. To achieve this, the level of signal determined in step (c) is typically
compared to calibration data. Calibration data can be obtained by conducting controls in which the
level of signal produced for a series of known analyte concentrations is determined. The
concentration of analogue, first binding moiety and second binding moiety should be kept constant
for each analyte concentration tested . Similarly, the concentration of each of these used in a test
should be the same as the concentration used to produce the calibration data with which the test
result is to be compared. Thus, the method of the invention may comprise determining the amount
or concentration of the analyte present in the sample by comparing the level of signal determined in
step (c) with calibration data.
As the level of signal detected is inversely proportional to the concentration of analyte, the higher
the level of signal, the lower the concentration of analyte in the sample and conversely, the lower
the level of signal, the higher the concentration of analyte in the sample. This inverse relationship
is particularly useful when the analyte is present at very low levels. This is because the level of
signal that is detected in these circumstances is high and thus easier to detect than in conventional
assays where the signal level is proportional to the concentration of analyte (and thus a very low
signal is produced when the analyte is present in a low concentration). Therefore, the method of
the present invention has the advantage of being highly sensitive to low levels of analyte.
The analyte can be any entity that is capable of being bound by the first binding moiety of the
present invention. For example, the analyte may be or may comprise a peptide, a polypeptide, a
protein, nucleic acid, a polynucleotide, a carbohydrate, an etiological agent, a hormone, a vitamin,
a steroid, a drug (for example a drug of abuse or DOA), an infectious agent or an entity indicative
of an infected state, a microorganism, a bacterium , a virus, a toxin, an organic compound, a
pollutant, a pesticide, a biomarker for a disease, a biomarker for pregnancy or metabolites of or
antibodies to any of the above. The term analyte also includes any antigenic substance, haptens,
antibodies, macromolecules, and combinations thereof. Preferably, the analyte comprises an
epitope recognised by an antibody. In one embodiment, the analyte is human chorionic
gonadotropin (hCG) or a fragment or portion thereof, including intact dimeric hCG, free alpha
subunit, free beta subunit, and clipped, cleaved or truncated forms of intact dimeric hCG, free alpha
subunit, free beta subunit, including glycosylation variants and in particular may include free-beta
core.
Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where
it is routinely detected and quantified by specific immunoassays. Essential in pregnancy and
detectable within a few days of fertilization, hCG has become one of the most frequently assayed
hormones, and simple, one-step, antibody based measurements of hCG are now standard in
pregnancy testing. Furthermore, the role of hCG assays in the diagnosis and management of
malignant trophoblastic disease (choriocarcinoma) is one of the great success stories of oncology.
It is an ideal tumour marker, always present when choriocarcinoma cells exist, and in quantities
directly related to the number of those cells. The correct use of hCG assays in combination with
appropriate therapy has led to a survival rate approaching 100% in this otherwise aggressive
cancer (Gregor, CR et al. , J 201 1, 'Antibody Recognition of a Human Chorionic Gonadotropin
Epitope (hCG beta 6-so) Depends on Local Structure Retained in the Free Peptide', Journal of
Biological Chemistry, vol 286, no. 28, pp. 2501 6-25026).
The analogue of the analyte is related to the analyte in that the first binding moiety is capable of
binding both the analogue and the analyte, preferably with substantially equal affinity. Thus, the
analogue and the analyte may share a degree of structural, sequence, chemical or immunological
similarity, and may comprise a binding epitope. For example, if the analyte comprises an amino
acid or nucleic acid sequence, the analogue may share a degree of sequence identity with the
sequence of the analyte. In some embodiments, the analogue comprises a sequence that is
identical to either the full analyte sequence or a portion of the analyte sequence. In other
embodiments, the analogue comprises a sequence that is at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the
full analyte sequence or a portion of the analyte sequence. The portion of the analyte sequence
that is identical to a portion of the analogue sequence may be from about 5 to about 1000 residues
in length. For example, the portion may be about 5 to about 10 , about 10 to about 15 , about 15 to
about 20, about 20 to about 30, about 30 to about 50, about 50 to about 70, about 70 to about 100,
about 100 to about 150, about 150 to about 250, about 250 to about 500, about 500 to about 750,
or about 750 to about 1000 residues in length.
The analogue-first binding moiety complex can be bound by the second binding moiety.
Preferably, the analogue itself is capable of being bound by the second binding moiety. However,
it may be that the second binding moiety only binds the complex of the analogue and the first
binding moiety. In contrast, the second binding moiety cannot bind the analyte-first binding moiety
complex. Therefore, the analogue may comprise a binding region for the second binding moiety
that is not present in the analyte, or that is present but is not capable of being bound by the second
binding moiety e.g . it is in a position in the analyte that is not accessible to the second binding
moiety. Such a binding region may comprise an amino acid or nucleotide sequence, for example
when the analogue consists exclusively of an amino acid sequence or a nucleic acid sequence.
When the analogue comprises an amino acid sequence, the binding region recognised by the
second binding moiety may be at the N-terminus, at the C-terminus or at any other position in the
amino acid sequence of the analogue. When the analogue comprises a nucleic acid sequence, the
binding region recognised by the second binding moiety may be at the 5' end of the sequence, the
3' end of the sequence or at any other position in the nucleic acid sequence of the analogue.
Where the analogue comprises an amino acid sequence, it is preferably expressed recombinantly
to include an amino acid sequence (a binding region) that can be bound by the second binding
moiety and that is not present in the analyte, or is present in the analyte but is not accessible to the
second binding moiety. Alternatively, the analogue may be synthesized using standard peptide
synthesis techniques. An advantage of expressing the analogue recombinantly or synthesising the
analogue de novo to include an amino acid binding region recognised by the second binding
moiety, is that only one of these binding regions may be incorporated into each analogue molecule.
If the binding region recognised by the second binding moiety is added to the analogue after the
analogue has been synthesised, there is a possibility that multiple binding regions are incorporated
into the analogue. The presence of multiple binding regions in the analogue may disrupt or inhibit
the method of the invention. For example, multiple binding regions may prevent the analogue from
competing with the analyte to bind the first binding moiety, or may prevent the analogue from
binding to the second binding moiety when in complex with the first binding moiety.
N-terminal or C-terminal sequences may be of the type generally referred to as tags. Non-limiting
examples include, in general, affinity tags, solubilisation tags, chromatography tags and epitope
tags. Non-limiting examples of specific tags include FLAG-tag, MYC-tag , HA-tag, GST-tag, Streptag
and a poly-histidine tag. The skilled person will be aware of many other general classes of tags
and more specific examples of each class, and will be able to choose an appropriate second
binding moiety accordingly. In some embodiments, the binding region may be a known epitope
from an antigen or may be synthetic. If synthetic it may be designed specifically for the analogue in
view of the analyte. An epitope for which a known antibody or antibody fragment is specific may
form part of the analogue.
In one embodiment, the analyte is hCG and the analogue comprises an amino acid sequence that
is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID
NO: 1, or SEQ ID NO: 2 :
SEQ ID NO 1
HHHHHHH NELSHFLE GSSSS KEPLRPRCRP INATLAVEKE GCPVCITVNT TICAGYCPTM
TRVLQGVLPA LPQVVCNYRD VRFESI RLPG CPRGVNPVVS YAVALSCQCA LCRRSTTDCG
GPKDH PLTCD DPRFQDSSSS KAPPPSLPSP SRLPG PSDTP ILPQ
SEQ ID NO 2 :
NELSHFLEGS SSSKEPLRPR CRPINATLAV EKEGCPVCIT VNTTICAGYC PTMTRVLQGV
LPALPQVVCN YRDVRFE SIR LPGCPRGVN P VVSYAVALSC QCALCRRSTT DCGGPKDH PL
TCDDPRFQDS SSSKAPPPSL PSPSRLPG PS DTPILPQ
The underlined region of both sequences represents a unique epitope region, referred to herein as
"ST068". The underlined region is bound by the second binding moiety and is not present in the
analyte i.e. hCG.
The double underlined region of SEQ ID NOs 1 and 2 , i.e. the sequence: SIRLPGCPRGVNP
VVSYA, represents a binding domain known as beta-3 that is found in hCG . Thus, the analogue
may comprise (and the first binding moiety may bind) a sequence comprising or consisting of the
double underlined portion of SEQ ID NO: 1, i.e. residues 85 to 102 of SEQ ID NO: 1 or the double
underlined portion of SEQ ID NO: 2 , i.e. residues 78-95 of SEQ ID NO: 2 . The analogue may
comprise (and the second binding moiety may bind) a sequence comprising or consisting of
residues 8-1 5 of SEQ ID NO: 1 or residues 1-8 of SEQ ID NO: 2 , i.e. the sequence: NELSHFLE,
which may be referred to as a TAG epitope. The analogue may comprise a sequence having at
least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence:
SIRLPGCPRGVN PVVSYA, and/or may comprise a sequence having at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% or 100% identity to the sequence: NELSHFLE. The
analogue may include a polyhistidine tag e.g . residues 1-7 of SEQ ID NO: 1.
In other embodiments, in particular, those in which analyte is hCG, the analogue comprises at least
157 amino acids, and may comprise at least amino acids 1-8 and 84-95 of SEQ ID NO: 2 .
Alternatively, the analogue may comprise at least 164 amino acids, and may comprise at least
amino acids 1- 15 and 95-1 02 of SEQ ID NO: 1. In one embodiment, the analogue has 50%
identity to SEQ ID NO: 1, and comprises at least amino acids 1- 15 and 7 1-83 of SEQ ID NO: 1.
In another embodiment, the analogue comprises NELSH FLEXmSIRLPGZnPVVSYA (SEQ ID NO:
3), wherein X is a linker having a length "m" and Z is a linker having a length "n". X and Z may be
the same or may be different and may each independently be, for example, an amino acid
sequence, polyethylene glycol (PEG), VA (see structure below) or beta-alanine.
VA linker:
A function of linker X is to reduce steric interference between the TAG epitope and the hCG
sequence. To this end a distance of at least 1.5nm ( 15 angstroms) should be sufficient. This is
approximately equivalent to five amino acids or two AEEAc linkers (short alternatives to PEG) or an
appropriate PEG linker (e.g . with two repeat units) .
A function of linker Z is to encourage an antiparallel beta turn so that the two flanking sequences of
hcG-beta3 align in a conformational epitope that can be bound by the first binding moiety. The
affinity of the first binding moiety for this conformational epitope is important in the context of the
assay and can be optimised by adjusting the properties of the linker, for example its length and
amino acid content.
When X and/or Z is an amino acid sequence, "m" and "n" may each be from about 3 to about 100
residues in length, about 5 to about 90 residues in length, about 10 to about 80 residues in length ,
or about 20 to about 70 residues in length. For example, "m" and/ or "n" may be 3 , 4 or 5 residues
in length. In the embodiment where "m" and/or "n" is 3 residues in length , the sequence PPN may
be used. The term amino acid used herein includes any molecule with an amino group and a
carboxylic acid group including naturally occurring amino acids, non-naturally occurring amino
acids and amino acids derivatives, such as, for example, AEEAc and amino-PEG-acids.
The amount of analogue required will depend on various factors. Typically, the analogue is
provided in a low, limiting concentration. This may be around 4-8 picograms/ml but the
concentration required will vary from batch to batch and an appropriate concentration can be
determined by the skilled person.
The binding moiety (first, second and/or third) may be naturally derived or wholly or partially
synthetic. The binding moiety and its target, (the analogue and the analyte are the targets of the
first binding moiety, the analogue-first binding moiety complex is the target of the second binding
moiety and the first binding moiety is the target of the third binding moiety where present) together
form a pair of binding partners. The members of the pair have the property of binding specifically
to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin ,
hormone-hormone receptor, complementary nucleotide sequences, complementary peptide
sequences, receptor-ligand, enzyme cofactor-enzyme, enzyme inhibitor-enzyme, enzymesubstrate,
carbohydrate-lectin, polymeric acid-base, dye-protein binder, peptide-specific protein
binder (e.g . , ribonuclease, S-peptide and ribonuclease S-protein), and the like. While the present
invention is generally concerned with antigen-antibody type reactions and preferred binding
moieties of the invention are antibodies or fragments thereof, other embodiments are also
envisaged employing other types of binding moieties and binding pairs.
The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active
portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that
specifically binds an antigen, whether natural or partly or wholly synthetic. The term also covers
any polypeptide or protein having a binding domain which is, or is homologous to, an antibody
binding domain. These can be derived from natural sources, or they may be partly or wholly
synthetically produced. Examples of antibodies are the immunoglobulin isotypes (e.g. , IgG, IgE,
IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding
domain such as Fab, F(ab')2, scFv, Fv, dAb, Fd ; and diabodies. Antibodies may be polyclonal or
monoclonal. A monoclonal antibody may be referred to herein as "mab".
It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA
technology to produce other antibodies or chimeric molecules which retain the specificity of the
original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin
variable region, or the complementary determining regions (CDRs), of an antibody to the constant
regions, or constant regions plus framework regions, of a different immunoglobulin. See, for
instance, EP-A-1 841 87, GB 2 188638A or EP-A-239400.
As antibodies can be made or modified in a number of ways, the term antibody should be
construed as covering any specific binding member or substance having a binding domain with the
required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents
and homologues of antibodies, humanised antibodies, including any polypeptide comprising an
immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric
molecules comprising an immunoglobulin binding domain, or equivalent, fused to another
polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in
EP-A-01 20694 and EP-A-01 25023. A humanised antibody may be a modified antibody having the
variable regions of a non-human, e.g. murine, antibody and the constant region of a human
antibody. Methods for making humanised antibodies are described in, for example, US Patent No.
5225539.
It has been shown that fragments of a whole antibody can perform the function of binding antigens.
Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1
domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment
consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al. ,
Nature 341 :544-546 ( 1 989)) which consists of a VH domain ; (v) isolated CDR regions; (vi) F(ab')2
fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules
(scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two
domains to associate to form an antigen binding site (Bird et al., Science 242 :423-426 ( 1988) ;
Huston et al., PNAS USA 85 :5879-5883 ( 1 988)) ; (viii) bispecific single chain Fv dimers
(PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene
fusion (WO94/1 3804; P. Hollinger et al. , Proc. Natl. Acad. Sci. USA 90: 6444-6448 ( 1 993)).
Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a
binding region of an immunoglobulin light chain and a second domain comprising a binding region
of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but
unable to associate with each other to form an antigen binding site: antigen binding sites are
formed by the association of the first domain of one polypeptide within the multimer with the second
domain of another polypeptide within the multimer (W094/1 3804).
Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which
can be manufactured in a variety of ways (Hollinger & Winter, Current Opinion Biotechnol. 4 :446-
449 ( 1 993)), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific
antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather
than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only
variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of
bispecific antibodies include the single chain "Janusins" described in Traunecker et al. , EMBO
Journal 10 :3655-3659 ( 1 991 ) .
Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they
can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such
as antibody fragments) of appropriate binding specificities can be readily selected using phage
display (WO94/1 3804) from libraries. If one arm of the diabody is to be kept constant, for instance,
with a specificity directed against antigen X, then a library can be made where the other arm is
varied and an antibody of appropriate specificity selected.
An "antigen binding domain" is the part of an antibody which comprises the area which specifically
binds to part or all of an antigen. Where an antigen is large, an antibody may only bind to a
particular part of the antigen, which part is termed an epitope. An antigen binding domain may be
provided by one or more antibody variable domains. An antigen binding domain may comprise an
antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
"Specific" is generally used to refer to the situation in which the binding moiety will not show any
significant binding to molecules other than its target(s), and, e.g. , has less than about 30%,
preferably 20%, 10%, 1%, 0.5%, 0.3%, 0.2% or 0 .1% cross-reactivity with any other molecule. The
term is also applicable where e.g. an antigen binding domain is specific for a particular epitope
which is carried by a number of antigens, in which case, the binding moiety carrying the antigen
binding domain will be able to bind to the various antigens carrying the epitope.
The first, second and third binding moieties do not necessarily have to be the same type of binding
moiety, although they may be in some embodiments.
The first binding moiety may be an hCG p3-loop-specific monoclonal antibody, for example the
antibody "8G5" (see Gregor et al. , J Biol Chem. 201 1 Jul 15;286(28) :2501 6-26). The first binding
moiety may be used at a concentration of about 1 ng/ml and may be an antibody-HRP conjugate.
The second binding moiety may be immobilised or immobilisable on a surface selected from the
group comprising a microtiter plate, a nitrocellulose matrix, a glass fibre matrix, a particle, a
membrane, a peg, a slide, a bibulous membrane, a surface of a microfluidic device, a
chromatographic material. The particle may be, for example, a magnetic particle, a latex particle, a
glass particle, a magnetically susceptible particle, or a colloidal metal particle
As mentioned, the signal is preferably derived (either directly or indirectly) from a label, which is
either directly or indirectly attached to or associated with the first-binding moiety. "Label" refers to
any substance which is capable of producing a signal that is detectable by visual or instrumental
means. Various labels useful in the present invention include labels which produce signals through
either chemical or physical means, such as being optically detectable. Such labels include
enzymes and substrates, chromogens, catalysts, fluorescent compounds, chemiluminescent
compounds, electroactive species, dye molecules, radioactive labels and particle labels. The first
binding moiety itself may be inherently capable of producing a detectable signal. The label may be
covalently attached to the first binding moiety. In particular, the label may be chosen from one that
is optically detectable. When the label comprises an enzyme, the method may include a step of
contacting the enzyme with a substrate in order to produce the signal. In one embodiment, the
label is horseradish peroxidase (HRP).
The label may comprise a particle such as gold, silver, colloidal non-metallic particles such as
selenium or tellurium, dyed or coloured particles such as a polymer particle incorporating a dye, or
a dye sol. The dye may be of any suitable colour, for example blue. The dye may be fluorescent.
Dye sols may be prepared from commercially- available hydrophobic dyestuffs such as Foron Blue
SRP (Sandoz) and Resolin Blue BBLS (Bayer). Suitable polymer labels may be chosen from a
range of synthetic polymers, such as polystyrene, polyvinyltoluene, polystyrene-acrylic acid and
polyacrolein. The monomers used are normally water-insoluble, and are emulsified in aqueous
surfactant so that monomer micelles are formed, which are then induced to polymerise by the
addition of initiator to the emulsion. Substantially spherical polymer particles are produced. An ideal
size range for such polymer particles is from about 0.05 to about 0.5um. According to an exemplary
embodiment, the label is a blue polymeric particle or a gold particle.
As already mentioned, the second binding moiety may be immobilised on a surface or may not be
immobilised at the beginning of the method. In the latter case, the second binding moiety may be
contacted with the analogue-first binding moiety complex either before or after it has been
contacted with a surface and immobilised .
The liquid sample can be derived from any source, such as an industrial, environmental,
agricultural, or biological source. The sample may be derived from or consist of a physiological
source including blood, serum , plasma, interstitial liquid, saliva, sputum, ocular lens liquid, sweat,
urine, milk, ascites liquid, mucous, synovial liquid, peritoneal liquid, transdermal exudates,
pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal liquid, semen,
cervical mucus, vaginal or urethral secretions and amniotic liquid. The liquid sample may be
derived from a semi-solid or solid source by dilution, treatment or extraction into an aqueous liquid.
Under certain circumstances, the sample may be expected to contain a very high concentration of
analyte. This may be the case, for example, if the sample has been obtained from a subject having
an advanced disease state. In such circumstances, the sample may be diluted before it is
contacted with the first binding moiety and analogue to bring the analyte concentration into an
appropriate range for the assay.
In a second aspect, the invention provides test kits for detecting an analyte of interest. The
invention provides a test kit for detecting an analyte of interest comprising
i) an analyte analogue;
ii) either (a) a labelled first binding moiety or (b) an unlabelled first binding moiety and a
labelled third binding moiety; and
iii) an immobilisable second binding moiety
wherein the first binding moiety is capable of binding each of the analogue and the analyte of
interest independently, the second binding moiety is capable of binding a complex of the analogue
and the first binding moiety but is not capable of binding the analyte or a complex of the first
binding moiety and the analyte, and the third binding moiety is capable of binding the first binding
moiety or the analogue-first binding moiety complex.
The first binding moiety may be capable of binding both the analogue and analyte simultaneously.
The invention further provides a test kit for detecting an analyte of interest comprising
i) an analyte analogue;
ii) either (a) a labelled first binding moiety or (b) an unlabelled first binding moiety and a
labelled third binding moiety; and
iii) an immobilisable second binding moiety
wherein the first binding moiety is capable of binding each of the analogue and the analyte of
interest independently but not both the analogue and analyte simultaneously, the second binding
moiety is capable of binding a complex of the analogue and the first binding moiety but is not
capable of binding the analyte or a complex of the first binding moiety and the analyte, and the third
binding moiety is capable of binding the first binding moiety or the analogue-first binding moiety
complex.
The analyte, analogue, and first, second and third binding moieties may be as defined in relation to
the first aspect of the invention.
In a third aspect, the invention provides a polypeptide that comprises or consists of an amino acid
sequence that is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the
sequence of SEQ ID NO: 1, or SEQ ID NO: 2 , or a polypeptide that comprises or consists of the
amino acid sequence NELSHFLEX nSIRLPGZ nPVVSYA (SEQ ID NO: 3), wherein X and Z are
linkers having a length "m" and "n" respectively. X, Z, "m" and "n" are as defined in the first aspect
of the invention.
The polypeptide may comprise a sequence having at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% or 100% identity to the sequence: SIRLPGCPRGVN PVVSYA, and/or may
comprise a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or
100% identity to the sequence: NELSHFLE. The polypeptide may comprise at least 164 amino
acids, and may comprise at least amino acids 1- 15 and 95-1 02 of SEQ ID NO: 1. The polypeptide
may comprise at least 157 amino acids, and may comprise at least amino acids 1-8 and 84-95 of
SEQ ID NO: 2 . In one embodiment, the polypeptide has 50% identity to SEQ ID NO: 1, and
comprises at least amino acids 1- 15 and 7 1-83 of SEQ ID 1.
The polypeptides of the present invention may be provided in isolated or recombinant form, and
may be fused to other moieties. The polypeptides may be provided in substantially pure form, that
is to say free, to a substantial extent, from other proteins. Thus, a polypeptide may be provided in
a composition in which it is the predominant component present (i.e. it is present at a level of at
least 50%; preferably at least 75%, at least 90%, or at least 95%; when determined on a
weight/weight basis excluding solvents or carriers).
The fourth aspect of the invention provides nucleic acid encoding a polypeptide of the third aspect.
As used herein with respect to nucleic acid molecules, isolated or recombinant means any of a)
amplified in vitro by, for example, polymerase chain reaction (PCR), b) recombinantly produced by
cloning, c) purified by, for example, gel separation, or d) synthesised , such as by chemical
synthesis.
The nucleic acid, for example DNA and RNA, may be synthesised using methods known in the art,
such as using conventional chemical approaches or polymerase chain reaction (PCR) amplification
or other methods of amplification.
In a fifth aspect, the invention provides an expression vector comprising nucleic acid encoding a
polypeptide of the third aspect.
A variety of host-expression vector systems may be utilised to express a polypeptide of the
invention. Such host-expression systems represent vehicles by which the coding sequences of
interest may be produced and subsequently purified, but also represent cells which may, when
transformed, transduced or transfected with the appropriate nucleotide coding sequences, express
the polypeptide of the invention in situ. These include but are not limited to microorganisms such
as bacteria (e.g . , E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing polypeptide coding sequences; yeast (e.g.,
Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing
polypeptide coding sequences; insect cell systems infected with recombinant virus expression
vectors (e.g. , baculovirus) containing the polypeptide coding sequences; plant cell systems infected
with recombinant virus expression vectors (e.g. , cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. , Ti plasmid)
containing polypeptide coding sequences; or mammalian cell systems (e.g. , COS, CHO, BHK, 293,
3T3 cells) harbouring recombinant expression constructs containing promoters derived from the
genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. , the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors may be advantageously selected depending
upon the use intended for the polypeptide being expressed. For example, when a large quantity of
such a polypeptide is to be produced, vectors which direct the expression of high levels of fusion
protein products that are readily purified may be desirable. Such vectors include, but are not
limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2 :1791 ); pIN vectors
(Inouye & Inouye, 1985, Nucleic Acids Res. 13 :31 0 1-31 09; Van Heeke & Schuster, 1989, J. Biol.
Chem. 24 :5503-5509) ; and the like. pGEX vectors may also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion
proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a
matrix comprising glutathione-agarose beads followed by elution in the presence of free
glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage
sites so that the cloned target gene product can be released from the GST moiety.
In an insect system , Autographa californica nuclear polyhedrosis virus (AcN PV) is used as a vector
to express foreign genes. The virus grows in Spodoptera frugiperda cells. The polypeptide coding
sequence may be cloned individually into non-essential regions (for example, the polyhedrin gene)
of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin
promoter). In mammalian host cells, a number of viral-based expression systems (e.g. , an
adenovirus expression system) may be utilised.
As discussed above, a host cell strain may be chosen which modulates the expression of the
inserted sequences, or modifies and processes the gene product in the specific fashion desired.
Such modifications (e.g. , glycosylation) and processing (e.g. , cleavage) of protein products may be
important for the function of the protein.
For long-term, high-yield production of polypeptides of the invention, stable expression is preferred.
For example, cells lines that stably express the polypeptide can be produced by transfecting the
cells with an expression vector comprising the nucleotide sequence of the polypeptide and the
nucleotide sequence of a selectable (e.g . , neomycin or hygromycin), and selecting for expression
of the selectable marker. Such engineered cell lines may be particularly useful in screening and
evaluation of compounds that interact directly or indirectly with the polypeptide.
The expression levels of the polypeptide of the invention can be increased by vector amplification
(for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for
the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New
York, 1987)). When a marker in the vector system expressing the polypeptide is amplifiable,
increase in the level of inhibitor present in culture of host cell will increase the number of copies of
the marker gene. Since the amplified region is associated with the polypeptide gene, production of
the polypeptide will also increase (Crouse et al. , 1983, Mol. Cell. Biol. 3:257).
Once the polypeptide of the invention has been expressed recombinantly, it may be purified by any
method known in the art for purification of a polypeptide, for example, by chromatography (e.g. , ion
exchange chromatography, affinity chromatography such as with protein A, poly-histidine or
specific antigen , and sizing column chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins.
Alternatively, any fusion protein may be readily purified by utilising an antibody specific for the
fusion protein being expressed. For example, a system described by Janknecht et al. allows for
the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et
al. , 1991 , Proc. Natl. Acad. Sci. USA 88:8972-897). In this system , the gene of interest is
subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is
translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves
as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant
vaccinia virus are loaded onto Ni + nitriloacetic acid-agarose columns and histidine-tagged proteins
are selectively eluted with imidazole-containing buffers.
In one embodiment of the invention, in a first reaction phase, a labelled antibody is incubated in the
presence of a limiting quantity of a modified form ("tagged-target" or "analogue") of the intended
target molecule (analyte) in order to form an analogue-labelled antibody complex. A limiting
quantity of analogue is applied to the mixture, to avoid the analogue out competing the naturally
occurring analyte present in the sample for binding to the antibody. Thus, due to the configuration
of the assay, the greater the amount of analyte present in the sample under investigation, a lesser
amount of the analogue will be captured by the first binding moeity. The assay is thus considered a
negative read immunoassay, since the detectable signal derived from the labelled antibody is
inversely proportional to the concentration of analyte of interest present in the sample.
In a second phase the analogue-labelled antibody complexes are applied to a solid phase on which
is immobilised a capture species specific for a unique tag on the analogue molecule in a reaction
chamber. The solid phase is loaded with an excess of capture molecule to ensure efficient capture
of any analogue-labelled antibody complexes present in solution. Following capture of analoguelabelled
antibody complexes on the solid phase capture molecule, any unbound labelled antibodies
(i.e. those which are complexed with the target analyte) are removed from the reaction chamber by
a washing step, leaving only the analogue-labelled antibody complexes that have been captured on
the solid phase. Thereafter, the presence of label is quantified using a suitable method, in
accordance with the label that has been used.
The label on the antibody may be selected from an enzyme, a fluor, a radionuclide, a colloidal sol,
a chromophore, luminescent compound. Depending on the enzyme selected the reaction product
of the enzyme may either be determined optically (absorbance or fluorescence or visually) or
electrochemically.
The invention also provides the following :
1. A recombinant peptide having at least 50% homology to SEQ ID NO: 1.
2 . A recombinant peptide having at least 75% homology to SEQ ID NO 1.
3 . A recombinant peptide having at least 50% homology to SEQ ID NO 2 .
4 . A recombinant peptide having at least 75% homology to SEQ ID NO 2 .
5 . A recombinant peptide comprising at least 157 amino acids, consisting of at least amino
acids 1-8 and 84-95 of SEQ ID NO 2 .
6 . A recombinant peptide comprising at least 164 amino acids, consisting of at least amino
acids 1- 15 and 95-1 02 of SEQ ID NO 1.
7 . A recombinant peptide comprising the sequence of SEQ ID NO 3 , wherein X and Z each
represents a linker.
8 . Use of the peptide according to SEQ ID NO 1 in the preparation of an immunoassay,
comprising ;
a first antibody having a binding specificity for the region comprising amino acids
95-1 02;
a second antibody having binding specificity for the region comprising amino acids 8-1 5 ;
wherein the second antibody is immobilised on a solid phase and wherein the first
antibody is conjugated to a detectable label.
The immunoassay of paragraph 8 wherein the detectable label is selected from the group
comprising an enzyme, a fluorophore, a radionuclide, a colloidal sol, a chromophore,
luminescent compound.
The immunoassay of paragraph 8 wherein the second antibody is immobilised to a solid
phase selected from the group comprising a microtiter plate, a magnetic particle, a
membrane, a peg, a latex particle, a slide, a bibulous membrane, a surface of a
microfluidic device, a chromatographic material.
A method of performing an immunoassay, comprising ;
contacting a sample suspected of containing a target of interest with a first-labelled
antibody, and a recombinant peptide according to one of SEQ ID NO 1, SEQ ID NO 2 or
SEQ ID NO 3 to initiate a competitive immunoassay for binding of the first labelled antibody
to the target to form a first antibody-recombinant peptide complex;
applying the first labelled antibody-target complex to the second antibody, to form
a second antibody-first labelled antibody-recombinant peptide complex;
separating unbound first antibody from the second antibody-first antibodyrecombinant
peptide complex mixture; and
detecting the presence of first labelled antibody on the recombinant peptide
captured by the second antibody.
The method of paragraph 11 wherein the step of forming a first labelled antibody target
complex occurs in bulk solution.
The method of paragraph 11 wherein the step of forming the second antibody-first labelled
antibody recombinant peptide complex occurs at a surface, and wherein the second
antibody is immobilised on the surface.
The method of paragraph 11 wherein the detectable label is selected from the group
comprising an enzyme, a fluor, a radionuclide, a colloidal sol, a chromophore, luminescent
compound.
The method of paragraph 13 wherein the second antibody is immobilised on the surface of
a particle, a membrane, a microtiter plate, a slide, a nitrocellulose matrix, a glass fibre
matrix.
The method of paragraph 15 wherein the particle is a magnetically susceptible particle, a
colloidal metal particle, a latex particle, or a glass particle.
The method of paragraph 11 wherein the step of separating the unbound first labelled
antibody from the second antibody first labelled antibody target complex comprises
washing with buffer, washing with air, removal of the complex from solution using a
magnet, filtration or immunochromatography.
18 . A recombinant peptide having at least 50% homology to SEQ ID NO 1, consisting of at
least amino acids 1- 15 and 7 1-83 of SEQ ID NO 1.
The invention may be further defined in the following set of numbered clauses:
1. A method of determining the presence of an analyte present in a liquid sample, the method
comprising :
a) contacting the liquid sample with a predetermined amount of an analogue of the analyte
and an excess of a first binding moiety, wherein the first binding moiety is capable of binding each
of the analyte and the analogue independently but not both the analogue and analyte
simultaneously, such that a mixture is formed comprising an analogue-first binding moiety complex,
or, when analyte is present in the sample, either (i) an analogue-first binding moiety complex and
an analyte-first binding moiety complex, or (ii) an analyte-first binding moiety complex and no
analogue-first binding moiety complex;
b) contacting the mixture with a second binding moiety, wherein the second binding moiety
is capable of binding the analogue-first binding moiety complex but not the analyte-first binding
moiety complex, and is immobilised or immobilisable on a solid phase;
c) determining the level of a signal indicative of the presence of the analogue-first binding
moiety complex bound to the second binding moiety;
wherein if the level of the signal determined in step c) is lower than the level of a maximum
signal determined when no analyte is present, then analyte is present in the sample.
2 . The method of clause 1, wherein the sample is contacted with the first binding moiety prior
to being contacted with the analogue.
3 . The method of clause 1 or clause 2 , further comprising a step of determining the amount or
concentration of analyte present in the sample by comparing the level of signal determined in step
c) with calibration data.
4 . The method of any preceding clause, wherein the analyte is hCG or a fragment or portion
thereof.
5 . The method of any preceding clause, wherein the signal is derived from a label that is
either directly or indirectly associated with the first-binding moiety.
6 . The method of clause 5 , wherein the label is selected from the group consisting of an
enzyme, a fluorophore, a radionuclide, a colloidal sol, a chromophore and a luminescent
compound.
7 . The method of any preceding clause, wherein the analyte comprises an amino acid
sequence and the analogue includes an amino acid sequence that is identical to either the full
analyte sequence or a portion of the analyte sequence.
8 . The method of any preceding clause, wherein the analogue comprises a binding region
that is not present in the analyte.
9 . The method of any preceding clause, wherein the analogue comprises an amino acid
sequence and is expressed recombinantly.
10 . The method of any preceding clause, wherein the analogue comprises the amino acid
sequence NELSHFLEX mSIRLPGZ nPVVSYA (SEQ ID NO: 3), wherein X and Z are linkers having a
length "m" and "n" respectively.
11. The method of any preceding clause, wherein the analogue comprises (i) at least 164
amino acids, and at least amino acids 1- 15 and 95-1 02 of SEQ ID NO: 1, or (ii) at least 157 amino
acids, and at least amino acids 1-8 and 84-95 of SEQ ID NO: 2 .
12 . The method of any preceding clause, wherein the analogue comprises residues 8-1 5 of
SEQ ID NO: 1 and/or residues 85 to 102 of SEQ ID NO: 1.
13 . The method of any preceding clause, wherein the analogue comprises an amino acid
sequence that is 90%, 95%, or 100% identical to the sequence of SEQ ID NO: 1, or SEQ ID NO: 2 .
14 . The method of any preceding clause, wherein the first binding moiety and/or the second
binding moiety is an antibody.
15 . The method of any preceding clause, wherein the first binding moiety is an hCG b3-Ioor-
specific monoclonal antibody.
16 . The method of any preceding clause, wherein the sample is derived from or consists of a
physiological source including blood, serum , plasma, interstitial liquid, saliva, sputum , ocular lens
liquid, sweat, urine, milk, ascites liquid, mucous, synovial liquid, peritoneal liquid, transdermal
exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal liquid,
semen, cervical mucus, vaginal or urethral secretions or amniotic liquid.
17 . A test kit for detecting an analyte of interest comprising
i) an analyte analogue;
ii) either (a) a labelled first binding moiety or (b) an unlabelled first binding moiety and a
labelled third binding moiety; and
iii) an immobilisable second binding moiety
wherein the first binding moiety is capable of binding each of the analogue and the analyte of
interest independently but not both the analogue and analyte simultaneously, the second binding
moiety is capable of binding a complex of the analogue and the first binding moiety but is not
capable of binding the analyte or a complex of the first binding moiety and the analyte, and the third
binding moiety is capable of binding the first binding moiety or the analogue-first binding moiety
complex.
18 . A polypeptide comprising or consisting of an amino acid sequence that is 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, or SEQ ID
NO: 2 , or a polypeptide that comprises or consists of the sequence
NELSHFLEX mSIRLPGZ nPVVSYA (SEQ ID NO: 3), wherein X and Z are linkers having a length "m"
and "n" respectively.
19 . Nucleic acid encoding a polypeptide according to clause 18 .
20. An expression vector comprising nucleic acid according to clause 19 .
Preferred features of each aspect of the invention are as for each of the other aspects mutatis
mutandis. The prior art documents mentioned herein are incorporated to the fullest extent
permitted by law.
Description of the Figures
Figure 1 represents a schematic description of a hybrid competitive immunoassay according to an
embodiment of the invention, and indicates possible cross reaction that might occur between the
target analyte and another species present or potentially present in the sample. In an exemplary
embodiment where the target analyte is hCG, the cross reacting species is LH.
Figure 2 represents an inhibition curve, indicating the degree with which a selected monoclonal
antibody for an hCG assay, mo , that has been raised against the conserved p3-hCG loop region,
cross reacts with LH. The data indicate cross reactivity with LH is 0.27% compared with hCG ,
based on the quantity of ligand needed to saturate 50% of the antibody.
Figure 3 provides further characterising data that demonstrate the specificity of the monoclonal
antibody, mod , for binding to phCG. The graph represents number of counts on the y-axis due to
11 25 radiolabeled mod that has bound tagged hCG , and the x-axis indicates the level of dilution
from 1000 h to 5 12000 h . The data represent a series of curves that demonstrate the ability of the
mod antibody to distinguish between phCG and either ahCG or LH. The buffer control , 569 free
alpha and LH all follow a similar profile, with high counts at low dilution, declining as sample is
diluted. However, when the antibody and tagged hCG are incubated with either free phCG,
"nicked" phCG, intact hCG , "nicked" hCG or "core" hCG (unlabelled target), the signals are
significantly suppressed, even at low dilution, because the unlabelled target has successfully
outcompeted the tagged-hCG for binding to the antibody.
Figure 4 represents a comparison of recombinant mo Fab ( monomer) and Fab2 (dimer).
Figure 5 shows a comparison between the serum TAG assay and the RIA assay based on samples
with low to high hCG concentrations. The x-axis displays the hCG concentration (IU/L) measured
via the TAG assay. The y-axis displays the hCG concentration ( IU/L) measured via the RIA assay.
Figure 6 shows a comparison between the serum TAG assay and the Immulite assay based on
samples with low to high hCG concentrations. The x-axis displays the hCG concentration ( IU/L)
measured via the TAG assay. The y-axis displays the hCG concentration ( IU/L) measured via the
Immulite assay.
Figure 7 shows a comparison between the serum TAG assay and the RIA assay based on samples
with mid to high hCG concentrations. The x-axis displays the hCG concentration ( IU/L) measured
via the TAG assay. The y-axis displays the hCG concentration ( IU/L) measured via the RIA assay.
Figure 8 shows a comparison between the serum TAG assay and the Immulite assay based on
samples with mid to high hCG concentrations. The x-axis displays the hCG concentration (IU/L)
measured via the Immulite assay. The y-axis displays the hCG concentration (IU/L) measured via
the TAG assay.
Example 1 - Protocol for serum TAG assay
Reagents
Standard diluent Serasub (SS)
Sample pre-diluent Normal sheep serum (NSS)
mo 10ul stock ( 1 :1 000) in 4ml 3% BSA/1 % NSS/PBS
TAG 10ul stock ( 1 :1000) in 4ml 3% BSA/1 % NSS/PBS
Microtitre plate pre-coated wiith anti TAG
Sample preparation
0-1 00 IU/L - dilute 1:2 in SS.
100-1 000 - dilute 1:5 in NSS then further in SS as required.
> 1000 - dilute 1:50 in NSS then further in SS as required.
Assay
• Prepare standard curve by serial diluting top hCG standard ( 100 IU/L) six times in SS to
generate 50, 25, 12.5, 6.25, 3 .125 and 1.56 IU/L standards. Include SS alone to give a
zero standard.
• Dilute serum samples as appropriate (detailed above).
• Using a multichannel pipette, add 35m I of mo solution to wells of the microtitre plate.
• Add 35m I of standards and samples.
• Using a multichannel pipette, add 35m I of TAG solution to all wells.
• Shake plate for 1 hour at room temperature.
• Remove the content of the wells by tipping and blotting onto tissue paper.
• Wash 6x with 0 .1% Tween/PBS.
• Add 00m I femto substrate and read on BMG Fluostar luminometer.
• Calculate results using BMG Omega Mars data analysis software.
Example 2 - Protocol for urine TAG assay
Reagents
Standard diluent - Urisub (US)
Sample diluent - 10% Normal sheep serum (NSS)/US
mod —10ul stock ( 1 1000) in 4ml 3% BSA/1 % NSS/20%FCS/PBS
TAG - 2.3ul stock ( 1 :1 000) in 4ml 3% BSA/1 % NSS/PBS
Microtitre plate - pre-coated with anti TAG
Sample preparation
0-1 00 IU/L Neat
> 100 IU/L - dilute in 10% NSS/US
Assay
• Prepare standard curve by serial diluting top hCG standard ( 100 IU/L) six times in US to
generate 50, 25, 12.5, 6.25, 3 .125 and 1.56 IU/L standards. Include SS alone to give a
zero standard.
• Dilute urine samples as appropriate (detailed above).
• Using a multichannel pipette, add 35m I of mo solution to wells of the microtitre plate.
• Add 35m I of standards and samples.
• Using a multichannel pipette, add 35m I of TAG solution to all wells.
• Shake plate then incubate for 2 hours at 37°C.
• Remove the content of the wells by tipping and blotting onto tissue paper.
• Wash 6x with 0 .1% Tween/PBS.
• Add 00m I femto substrate and read on BMG Fluostar luminometer (see settings data).
• Calculate results using BMG Omega Mars data analysis software (see analysis data).
Example 3 - Comparison of mo Fab variants
Recombinant mod Fab (monomer) and Fab2 (dimer) were produced in E. coli by conventional
methods known in the art by the skilled person . Each Fab variant (monomeric variant and dimeric
variant) was purified and conjugated to horseradish peroxidase (HRP) using Lightning Link HRP
kits (Innova Bioscience) according to the manufacturer's instructions. The molecular weight of each
Fab variant was taken into account for the conjugation process so that the monomeric Fab variant
and dimeric Fab2 variant were subjected to comparable levels of labelling with HRP.
Following labelling with HRP and initial activity testing, the labelled Fab variants were assessed in
a competition assay using TAG and hCG in order to derive a standard curve following the general
method provided in Example 1.
The results of the assays are given in Table 1 and Figure 4 .
Table 1. Averaged data showing assay output and % Signal relative to negative hCG standard
Table 1 and Figure 4 demonstrate that the Fab2 dimer variant has a significant, approximately two
fold, increase in chemiluminescent output relative to the Fab monomer variant under comparable
assay conditions. In addition, the Fab2 dimer variant has a greater sensitivity to hCG concentration
in the sample over the hCG concentration range 1.56-25 IU/L relative to the Fab monomer variant
as indicated by the more acute decrease in % signal across this range (see Figure 4).
Example 4 - Clinical studies comparing the TAG assay with Immulite and RIA assays
Studies were performed to compare the ability of the TAG assay employing the Fab2 dimer variant
as described herein and known assays, the Immulite assay and RIA assay, to detect hCG in
clinical samples.
Clinical serum samples were analysed for the presence and concentration of hCG by each assay
method. The TAG assay was performed as described in Example 1. Immulite assays (Siemens)
were performed according to the manufacturer's instructions. RIA assays were performed
according to protocol instructions provided by the National Health Service, UK. In short, the assay
uses a purified polyclonal antibody and iodinated hCG wherein sample is diluted and mixed with
antibody and labelled hCG for 24 hours prior to measurement.
The results of the assays is given in Tables 2 and 3 and Figures 5-8.
Table 2 . hCG detection in clinical serum samples via the TAG assay, Immulite assay or RIA assay
method whereby the hCG concentration range across the samples was from low to high hCG
concentrations.
22 175265 194800 164721
23 2461 5 22330 25561
24 1545 1067 1425
25 3 <4 < 1
26 2 <4 < 1
27 12 9 13
28 1405 1063 1402
29 8 9 11
30 1 <4 < 1
3 1 33 32 39
32 2 <4 < 1
33 9 6 10
34 10 9 13
Table 3 . hCG detection in clinical serum samples via the TAG assay, Immulite assay or RIA assay
method whereby the hCG concentration range across the samples was from mid to high hCG
concentrations.
19546 19670 15863
22642 14285 2 1467
27591 25800 14364
156336 106600 86333
376837 273500 148267
28086 18550 15583
66289 46560 33289
114379 591 60 11431 5
28862 16770 2691 8
Figures 5-8 demonstrate that the detection and measurement of hCG concentration in clinical
serum samples via TAG assay correlates strongly with the known Immulite and RIA assays with R2
values greater than 0.9 in all cases. Some variation between assays methods exists due to
differences in antibody recognition of the target epitopes on hCG , however all assays can
accurately detect hCG in clinical sera.
Furthermore, further clinical studies have demonstrated that the key benefit of the TAG assay is its
ability to report specifically with good sensitivity. It has been shown to detect very low levels of hCG
and detect increases in patient samples before the known RIA and Immulite assays were able to
do so which is invaluable in monitoring patients undergoing therapy, particularly when the levels of
hCG are very low. In addition to the superior sensitivity of the TAG assay over known assay
methods, the TAG assay is laboratory based and can be completed in a much shorter period of
time (4 hours) than the known assays using simple laboratory facilities and equipment.

Claims
1. A method of determining the presence of an analyte present in a liquid sample, the method
comprising :
a) contacting the liquid sample with a predetermined amount of an analogue of the analyte
and an excess of a first binding moiety, wherein the first binding moiety is capable of binding each
of the analyte and the analogue independently, such that a mixture is formed comprising an
analogue-first binding moiety complex, or, when analyte is present in the sample, either (i) an
analogue-first binding moiety complex and an analyte-first binding moiety complex, or (ii) an
analyte-first binding moiety complex and no analogue-first binding moiety complex;
b) contacting the mixture with a second binding moiety, wherein the second binding moiety
is capable of binding the analogue-first binding moiety complex but not the analyte-first binding
moiety complex, and is immobilised or immobilisable on a solid phase;
c) determining the level of a signal indicative of the presence of the analogue-first binding
moiety complex bound to the second binding moiety;
wherein if the level of the signal determined in step c) is lower than the level of a maximum
signal determined when no analyte is present, then analyte is present in the sample.
2 . A method of determining the presence of an analyte present in a liquid sample, the method
comprising :
a) contacting the liquid sample with a predetermined amount of an analogue of the analyte
and an excess of a first binding moiety, wherein the first binding moiety is capable of binding each
of the analyte and the analogue independently but not both the analogue and analyte
simultaneously, such that a mixture is formed comprising an analogue-first binding moiety complex,
or, when analyte is present in the sample, either (i) an analogue-first binding moiety complex and
an analyte-first binding moiety complex, or (ii) an analyte-first binding moiety complex and no
analogue-first binding moiety complex;
b) contacting the mixture with a second binding moiety, wherein the second binding moiety
is capable of binding the analogue-first binding moiety complex but not the analyte-first binding
moiety complex, and is immobilised or immobilisable on a solid phase;
c) determining the level of a signal indicative of the presence of the analogue-first binding
moiety complex bound to the second binding moiety;
wherein if the level of the signal determined in step c) is lower than the level of a maximum
signal determined when no analyte is present, then analyte is present in the sample.
3 . The method of claim 1 or claim 2 , wherein the sample is contacted with the first binding
moiety prior to being contacted with the analogue.
4 . The method of any one of claims 1 to 3 , further comprising a step of determining the
amount or concentration of analyte present in the sample by comparing the level of signal
determined in step c) with calibration data.
5 . The method of any preceding claim , wherein the analyte is hCG or a fragment or portion
thereof.
6 . The method of any preceding claim , wherein the signal is derived from a label that is either
directly or indirectly associated with the first-binding moiety.
7 . The method of claim 6 , wherein the label is selected from the group consisting of an
enzyme, a fluorophore, a radionuclide, a colloidal sol, a chromophore and a luminescent
compound.
8 . The method of any preceding claim , wherein the analyte comprises an amino acid
sequence and the analogue includes an amino acid sequence that is identical to either the full
analyte sequence or a portion of the analyte sequence.
9 . The method of any preceding claim , wherein the analogue comprises a binding region that
is not present in the analyte.
10 . The method of any preceding claim , wherein the analogue comprises an amino acid
sequence and is expressed recombinantly.
11. The method of any preceding claim , wherein the first binding moiety and/or the second
binding moiety is an antibody.
12 . The method of any preceding claim , wherein the first binding moiety is a Fab or F(ab)2.
13 . The method of any preceding claim , wherein the first binding moiety binds specifically to
hCG.
14 . The method of any one of claims 1 to 11, wherein the first binding moiety is an hCG b3-
loop-specific monoclonal antibody.
15 . The method of any preceding claim , wherein the sample is derived from or consists of a
physiological source including blood, serum , plasma, interstitial liquid, saliva, sputum , ocular lens
liquid, sweat, urine, milk, ascites liquid, mucous, synovial liquid, peritoneal liquid, transdermal
exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal liquid,
semen, cervical mucus, vaginal or urethral secretions or amniotic liquid.
16 . A test kit for detecting an analyte of interest comprising
i) an analyte analogue;
ii) either (a) a labelled first binding moiety or (b) an unlabelled first binding moiety and a
labelled third binding moiety; and
iii) an immobilisable second binding moiety
wherein the first binding moiety is capable of binding each of the analogue and the analyte of
interest independently, the second binding moiety is capable of binding a complex of the analogue
and the first binding moiety but is not capable of binding the analyte or a complex of the first
binding moiety and the analyte, and the third binding moiety is capable of binding the first binding
moiety or the analogue-first binding moiety complex.
17 . A test kit for detecting an analyte of interest comprising
i) an analyte analogue;
ii) either (a) a labelled first binding moiety or (b) an unlabelled first binding moiety and a
labelled third binding moiety; and
iii) an immobilisable second binding moiety
wherein the first binding moiety is capable of binding each of the analogue and the analyte of
interest independently but not both the analogue and analyte simultaneously, the second binding
moiety is capable of binding a complex of the analogue and the first binding moiety but is not
capable of binding the analyte or a complex of the first binding moiety and the analyte, and the third
binding moiety is capable of binding the first binding moiety or the analogue-first binding moiety complex.