Note: Descriptions are shown in the official language in which they were submitted.
~-~O95/0~22 ~ 7 ~ 6 I ~CT/GB94/01522
TEST DEVICE
The present invention relates to a device for detecting
the presence of analyte in a sample of a body fluid, in
particular a sample other than a serum sample. Such
analytes are typically immunoglobulin molecules but may
also be other proteins.
Immunoglobulin molecules, or fragments thereof, are known
to occur in such non-serum body fluids as saliva and
urine. In particular, the ;mmllnoglobulin molecules of
classes IgA, IgG and IgM predominate in saliva. Saliva
and urine are secreted body fluids and collection of
samples is easily and simply carried out.
In the field of medical diagnostics, there are many tests
and devices known for detecting analytes in samples of a
particular body fluid. These tests include biochemical
assays of the amount and/or presence of particular
proteins or immunoglobulins; so called 'sandwich assays'
for ;mmnnoglobulins and the use of electrophoresis to
identify individual classes of immunoglobulins.
Devices used to detect analytes such as immunoglobulins
in saliva include various probes consisting of a stick
with an absorbent or adsorbent pad of material at one
end. Such probes are disclosed in EP-A-0418739, EP-A-
0442231 and US-A-5103838. The probes of the prior art
additionally include detection systems capable of
identifying antibodies in saliva.
Detection systems used in the past on diagnostic probes
of this type have employed enzyme-linked assays or
particle-agglutination assays to produce visibly
WO95/02U2 PCT/GB94/01522 ~
~ 1~7 o~i 2
detectable results.
However, diagnostic test devices have suffered from an
importan~ disadvantage. All immunodiagnostic tests
relying on antibody-antigen binding are subject to the
occurrence of false -negative results in any one
particular sample. In the classic enzyme-linked
immunosorbent assay, an antigen which will specifically
bind with the antibody of interest is bound to a solid
base. A sample containing the antibody of interest is
added and forms a specific complex with the bound
antigen. Unbound antibody is then washed off and a
labelled antibody is added to detect the complexes formed
on the surface of the test device. This assay, however,
is only reliable when quite large amounts of analyte are
present in the sample. Samples of urine and saliva which
a clinician might wish to assay generally contain levels
of antibody which may be 2 or 3 orders of magnitude lower
than the levels of antibody found in serum or plasma
samples. Antibody concentration in urine and saliva
samples is influenced by several factors, and shows
natural variability. The antibody concentration may be
lowered when urinary output is increased through
ingestion of large volumes of fluid prior to sampling, or
when saliva production has been artificially increased
through stimulation by citric, or other acids.
This variability can give rise to false-negative results,
where the concentration of antibody in the sample is
insufficient to give a positive reaction in an enzyme-
linked assay or similar test procedure. False negative
results can result in the incorrect diagnosis of a
medical condition by a clinician. Where the test is for
the presence of an antibody to a certain pathogenic
~NO95/0~22 PCT/GB94/01522
7~6 1
bacteria or virus or for antibodies associated with
autoimmune diseases or allergic conditions, a false-
negative result due to the use of an inadequate sample
can have devastating consequences for the patient and
cause the subject unnecessary distress. This is
especially so in the case of Human Immunodeficiency Virus
(HIV) infection. Recently, it has been reported that
the United States Food and Drug ~mi ni stration (FDA) has
decided that all test devices in the immunodiagnostic
field must indicate the viability of the sample.
The present invention seeks to solve this problem by
providing a device with an inbuilt means for detecting
false negative results arising from use of an invalid
sample.
In a first aspect of the present invention, there is
provided a device for the detection of an analyte
present in a body fluid of an animal species, the device
comprising a substrate and on the substrate in discrete
locations:
(a) a mobile labelled first specific binding molecule
capable of binding to any characteristic molecule of
the species being tested to form a binary complex;
(b) a means for detecting the analyte; and
(c) an immobilised second specific binding molecule
capable of binding to the binary complex or a
component of it.
The advantage of this device is contained in the final
step of binding the characteristic molecule to a second
W095/02~2 PCT/GB94/01522 -
~7~1 4
immobilised specific binding molecule. This step ensures
that a sufficient quantity of characteristic molecule is
present to give meaningful results and therefore that
particular test can be readily verified. The overall
concentration of characteristic molecule in a test sample
is easily quantitated and if the level is sufficiently
high then the risk of a false negative result is
significantly reduced.
GB-A-2204398 relates to an assay device which also
contains a mobile labelled first specific binding
molecule, a means for detecting analyte and an
immobilised second specific binding molecule. However,
it differs significantly from the device of the present
15 invention because the first specific binding molecule is
specific for the analyte rather than for a characteristic
molecule of the species being tested as is the case with
the present invention. This means that using the device
of GB-A-2204398 it is not possible to determine whether
20 the sample is viable and, thus, to eliminate false
negatives.
In this specification, the term 'binary complex' is used
to define the complex formed by the specific binding
25 molecule bound to a particle on the substrate and a
characteristic molecule which may include analyte
molecules. The analyte may be an antibody.
The term 'mobile', when used in connection with the first
specific binding molecule, indicates that the first
specific binding molecule is not immobilised on the
substrate.
The term 'characteristic molecule' refers to a molecule
`~095/02U2 PCT/GB94/01522
5 ~16 7~61
which is typically found in the type of sample to be
tested and which will therefore be present in detectable
concentration if the sample is viable. For example, in
saliva samples, it is usual to find detectable quantities
of IgG, and IgG would therefore be a suitable
characteristic molecule for saliva samples. The first
specific binding molecule would, in that case, bind
specifically to all antibodies of the IgG isotype.
The device of the present invention is suitable for the
detection of an analyte in any body fluid but it is
especially useful for overcoming the problems of
detecting analytes in saliva and urine samples where the
concentrations of analytes tend to vary. It is also
lS useful for other body fluids such as blood.
The analyte to be tested by the device of the present
invention may be an antibody to a bacterial, viral,
fungal or protozoal pathogen for example Helicobacter
pylori, the Human Immunodeficiency viruses (HIV l and 2),
a Salmonella spp., a Streptococcus species, polio virus,
hepatitis A, hepatitis B surface antigen or hepatitis B
core antigen, Candida spp., Aspergillus spp., Entamoeba
spp. or Giardia spp. The analyte may also be an antibody
to a tissue or species-specific antibody associated with
autoimmune diseases, or allergic conditions.
Alternatively, the analyte may be an antigen, for example
a pathogen protein or a hormone, blood group
characterising protein or any other protein or
glycoprotein which may require detection. Nucleic acids
can also be detected using the device of the present
invention.
W095/02822 PCT/GB94/01522 ~
21~7 ~ 6
The analyte should also form a portion of the
characteristic molecule, or be a subset of characteristic
molecules, which bind to the first specific binding
molecule to form binary complex.
Thus, when the analyte is an antibody of a particular
isotype, for example IgG, IgM, IgE or IgA, the
characteristic molecule will be any antibody of that
isotype and the first specific binding molecule will be
selected so that it binds to all antibodies of that
isotype. Similarly, if the analyte is a glycoprotein,
the first specific binding molecule may be a lectin which
binds to all glycoproteins present in the sample and
labels them and if the analyte is a nucleic acid, a
suitable first specific binding molecule would be a
labelled low specificity nucleic acid probe.
Because the analyte is a subset of the characteristic
molecule, a portion of the labelled binary complex will
contain the analyte and this is a particularly
advantageous feature of the invention as it eliminates
the need for more than one detectable label.
In a preferred embodiment the device provided by this
invention is designed for use in diagnosing medical
conditions suffered by humans.
As mentioned above, the first specific binding molecule
will often be an antibody which will preferably be of the
IgG or IgM isotype. Other isotypes of antibody may be
similarly so used if required. The first specific
binding molecule may be of polyclonal or monoclonal
origin, and may be either an intact molecule or a
specific binding-site containing fragment derived by
-~095/02822 PCT/GB94/01522
7 ~7061
enzymatic or chemical cleavage, or obtained through the
application of recombinant DNA technology or peptide
synthesis.
The first specific binding molecule may be labelled with
any convenient label. The label may, in particular,
allow the specific binding molecule to be visualised, or
otherwise detected, when immobilised. Particles
constitute preferred labels for this purpose. Particles
coupled to the first specific binding molecule on the
test device may be of any known type, for example, latex,
colloidal metal, liposomes or polystyrene. Chromophores,
fluorophores, and/or other labels may alternatively or
additionally be used.
The means for detecting the analyte on the test device
may suitably comprise one or more discrete areas
comprising an immobilised binding molecule specific for
the analyte. Because the analyte forms a part of or is
a subset of the characteristic binding molecule, it will
already be bound to the detectably labelled first
specific binding molecule and will therefore be
detectable once it has been immobilised in the detection
zone.
It is particularly useful to provide several analyte
detection areas on the device since this allows
quantitation of the analyte. Thus, if the analyte is
present only in small amounts, it will interact with the
immobilised binding molecule in the first detection area,
but there will be no surplus analyte which can pass to
the further detection areas. If more analyte is present,
some surplus may pass to the next detection area and so
on so that the amount of analyte present will be
W095/0~2 PCTIGB94/01522 ~
c~
indicated by the number of detection areas in which it is
detected.
Alternatively, two or more immobilised binding molecules
specific for different analytes may be deposited on
discrete areas of the test device, permitting the
simultaneous detection of the presence of two or more
different analytes in the same sample.
When the analyte is one of the antibodies discussed
above, the immobilised binding molecule in the detection
area will generally be an immobilised pathogen protein,
carbohydrate or other fragment, or a human tissue-derived
protein, carbohydrate or other fragment, or a non-human
species-derived protein, carbohydrate or other fragment.
The immobilised binding molecule may also be an anti-
idiotypic antibody.
When the analyte is a protein, the immobilised binding
molecule may be a monoclonal or polyclonal antibody which
binds specifically to the protein or some other binding
molecule specific for the particular protien to be
detected. For nucleic acids, a suitable immobilised
binding molecule would be a high specificity nucleic acid
probe.
Binary complexes which contain a characteristic molecule
other than the analyte will not be immobilised or
otherwise removed at location (b), where the analyte is
detected and will pass to location (c), which functions
as a verification station. A second specific binding
molecule immobilises the complexes, which are then
detectable by means of the label. The second specific
binding molecule may specifically bind any part of the
~_NO95/0~22 PCTIGB94/01522
9 216706~
complex. It is preferred for the second specific binding
molecule to bind the characteristic molecule; so, for
example, if the characteristic molecule is a human
antibody isotype, the second specific binding molecule
may again be anti-human antibody such as anti-human IgG.
Other suitable binding molecules are as discussed above
in relation to the first specific binding molecule and
will again be chosen depending on the analyte and the
characteristic molecule.
The device may further comprise a sample receiving
portion of adsorbent material continuous with or separate
from the substrate. This flexibility of construction
permits maximum utility of operation depending on how the
sample is to be applied.
The substrate material of the test device may be composed
of paper or membrane. The substrate material may also
optionally be a carbohydrate and may preferably be a
nitrocellulose material.
It is preferred that the device be constructed so that
body fluid (or components of it) can flow to locations
(a), (b) or (c) successively, for example by capillary
action. Other means of delivering body fluid to the
locations are within the scope of the invention.
In a preferred aspect of the invention, as mentioned
above, sample is wic~ed along the membrane or paper
substrate by capillary action. No additional solvent is
required although particular sample diluents may increase
the speed at which a result is obtained, or enhance the
signal obtained with a positive test result.
W095/0~22 PCT/GB94/01522 ~
In a second aspect of the present invention there is
provided a method for the detection of analytes present
in the body fluid of an animal, the method comprising:
(i) adsorbing a sample onto an adsorbent
substrate;
(ii) bringing the sample into contact with a
mobile labelled specific binding molecule
such that characteristic molecules of the
tested species present in the sample bind
to the specific binding molecule to form
a binary complex;
15- (iii) bringing the binary complex comprising in
contact with an immobilised analyte-
specific binding molecule such that binary
complex comprising analyte binds to the
analyte-specific binding molecule to give
a detectable reaction product; and
(iv) bringing binary complex not comprising
analyte into contact with an immobilised
specific binding molecule which reacts
with the binary complex to form a visibly
detectable product.
The detectable products in steps (iii) and (iv) are
preferably visibly detectable, as they are when the label
referred to in step (ii) is a particle.
Preferred features of the second aspect of the invention
are as for the first aspect, mutatis mutandis.
~~~095/0~ PCT/GB94/01522
~1 67o6I
The invention will now be further described by way of
example with reference to the following drawing in which:
FIGURE 1 shows a perspective view of a device in
accordance with the invention.
Referring now to Figure 1, a sampling device 10 comprises
a substrate 12 formed from an adsorbent material,
typically nitrocellulose paper, and, attached to the
substrate, a sample receiving portion 14. On the
substrate is positioned a first area 16 in which is
contained an anti-human IgG antibody coupled to a
particle. The particles may be of any known type, for
example latex, colloidal metal, liposomes or polystyrene.
Sample detection areas 18, 20 contain antibodies specific
for the analyte immobilised on the nitrocellulose
substrate 12. A particularly preferred analyte which
this device may be used to detect is antibody to H.
pylori and, therefore, in that case, the detection areas
18, 20 would contain an immobilised antigen specific for
H . pyl ori antibody.
The final area on the device is the verification area 22
which contains immobilised anti-human IgG. This may be
the same as the anti-human IgG which is bound to the
particles but this is not necessarily so.
In use, a sample is placed on the sample receiving
portion 14 and, because of the adsorbent nature of the
substrate 12, the sample wicks along the substrate until
it reaches the first area 16. At this point, any human
IgG present in the sample binds to the anti-IgG coupled
to the particles to form a binary complex. The binary
complex continues to wic~ along the substrate 12 until it
WO95/02822 PCT/GB94/01522~-
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encounters the first sample detection area 18. In this
area, binary complex containing analyte binds to the
immobilised antibody to form a discrete visible
clustering on the sample detection area 18. Unbound
binary complex continues to pass along the substrate
until it reaches the second sample detection area 20
where any rem~ini~g analyte-containing binary complex
will be immobilised to form a second discrete visible
clustering. Finally, remaining binary complex passes
along the substrate until it reaches the verification
area 22, at which point it will bind to the anti-IgG in
this area and will form a further discrete visible
clustering. This visible signal in the verification area
is an indication that the initial sample contained a
sufficient amount of human IgG for the sample to be
viable and any positive result to be relied upon.
Clearly, therefore, the device of the present invention
is simple to use and provides a quick and easy method of
verifying that the sample is a viable one.
