U.S. patent application number 10/719049 was filed with the patent office on 2004-06-17 for immunoassay apparatus for diagnosis.
This patent application is currently assigned to CLINICAL DIAGNOSTIC CHEMICALS LIMITED. Invention is credited to Rees, John.
Application Number | 20040115795 10/719049 |
Document ID | / |
Family ID | 10812318 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040115795 |
Kind Code |
A1 |
Rees, John |
June 17, 2004 |
Immunoassay apparatus for diagnosis
Abstract
Immunoassay analytical test apparatus for allergy diagnosis,
which apparatus comprises a zone for receiving a sample containing
an analyte, a zone for receiving a mobile phase (the zone may be
the same as the sample receiving zone, or different thereto), a
detection means for permitting detection of the analyte by
immunoreaction, a first flow path for flow of the analyte in the
mobile phase from the sample receiving zone to the detection means,
and a second flow path permitting flow of a mobile phase to the
detection means.
Inventors: |
Rees, John; (Llandudno,
GB) |
Correspondence
Address: |
David S. Jacobson
GORDON & JACOBSON, P.C.
65 Woods End Road
Stamford
CT
06905-2701
US
|
Assignee: |
CLINICAL DIAGNOSTIC CHEMICALS
LIMITED
|
Family ID: |
10812318 |
Appl. No.: |
10/719049 |
Filed: |
November 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10719049 |
Nov 21, 2003 |
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09440787 |
Nov 15, 1999 |
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6689317 |
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09440787 |
Nov 15, 1999 |
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PCT/GB98/01412 |
May 15, 1998 |
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Current U.S.
Class: |
435/287.2 ;
436/514 |
Current CPC
Class: |
G01N 33/558
20130101 |
Class at
Publication: |
435/287.2 ;
436/514 |
International
Class: |
C12M 001/34; G01N
033/558 |
Foreign Application Data
Date |
Code |
Application Number |
May 15, 1997 |
GB |
9709821.4 |
Claims
What is claimed is:
1. Immunoassay analytical test apparatus, which apparatus
comprises: a) a zone for receiving a sample containing an analyte;
b) a zone for receiving a mobile phase, which zone may be the same
as the sample receiving zone, or different thereto; c) detection
means for permitting detection of said analyte by immunoreaction;
d) a first flow path for flow of said analyte in said mobile phase
from said sample receiving zone to said detection means; and e) a
second flow path permitting flow of said mobile phase to said
detection means.
2. Apparatus according to claim 1, wherein said second flow path
includes labelled immunoreactive material.
3. Apparatus according to claim 1, wherein said first flow path
includes a material selected form the group consisting of: (i)
unlabelled immunoreactive material; (ii) hapten labelled
immunoreactive material; (iii) unlabelled capture material; (iv)
hapten labelled capture material; and (v) detector labelled
material; said material being upstream of said sample receiving
zone.
4. Apparatus according to claim 1, wherein said first flow path
includes a material selected form the group consisting of: (i)
unlabelled immunoreactive material; (ii) hapten labelled
immunoreactive material; (iii) unlabelled capture material; (iv)
hapten labelled capture material; and (v) detector labelled
material; said material being downstream of said sample receiving
zone.
5. Apparatus according to claim 1, wherein said first flow path
includes a material selected form the group consisting of: (i)
unlabelled immunoreactive material; (ii) hapten labelled
immunoreactive material; (iii) unlabelled capture material; (iv)
hapten labelled capture material; and (v) detector labelled
material; said material being upstream and downstream of said
sample receiving zone.
6. Apparatus according to claim 1, wherein said flow paths are such
that said mobile phase is allowed to traverse more than one path
simultaneously, said first flow path being continuous from said
mobile phase receiving zone to said detection means, said second
flow path being continuous from said mobile phase receiving zone to
said detection means.
7. Apparatus according to claim 1, wherein said detection means is
movable from a first position in communication with said first flow
path to a second position in communication with said second flow
path.
8. Apparatus according to claim 7, wherein said detection means is
movable from a first position in contact with said first flow path
to a second position in contact with said second flow path.
9. Apparatus according to claim 7, wherein said detection means is
movable to be in communication with said first and second flow
paths in sequence.
10. Apparatus according to claim 7, wherein said detection means is
movable to be in contact with said first and second flow paths in
sequence.
11. Apparatus according to claim 1, wherein said first flow path
potentiates flow towards said detection means.
12. Apparatus according to claim 11, wherein said first flow path
potentiates flow towards said detection means by a capillary
action.
13. Apparatus according to claim 1, wherein said second flow path
potentiates flow towards said detection means.
14. Apparatus according to claim 13, wherein said second flow path
potentiates flow towards said detection means by a capillary
action.
15. Apparatus according to claim 1, wherein said first flow path is
selected from the group consisting of: a) elongate sheet material;
b) elongate strip material; and c) material absorbent to said
mobile phase.
16. Apparatus according to claim 15, wherein said elongate sheet or
strip comprises a portion contactable with said first flow
path.
17. Apparatus according to claim 1, wherein said second flow path
is defined by a material selected from the group consisting of: a)
elongate sheet material; a) elongate strip material; and b)
material absorbent to said mobile phase.
18. Apparatus according to claim 17, wherein said elongate sheet or
strip comprises a portion contactable with said second flow
path.
19. Apparatus according to claim 1, wherein said analyte is
allergen specific IgE.
20. Apparatus according to claim I, wherein said apparatus further
comprises a sink for collection of excess material exiting the
detection means.
21. Apparatus according to claim 1, wherein said detection means is
manually movable from said first pathway to said second
pathway.
22. Apparatus according to claim 19, wherein said first flow path
comprises a matrix for the removal of non-IgE components.
23. Apparatus according to claim 22, wherein said matrix is
provided between said sample receiving zone and said detection
means.
24. Apparatus according to claim 22, wherein said matrix is
provided in said sample receiving zone.
25. Apparatus according to claim 1, wherein said first flow path
comprises a filter arranged to separate components of said sample,
said filter selected from a group consisting of: i) a blood filter
arranged to permit plasma to pass whilst capturing other blood
constituents; and ii) a matrix for the removal of material other
than said analyte.
26. Apparatus according to claim 25, wherein said filter is
provided between said sample receiving zone and said detection
means.
27. Apparatus according to claim 25, wherein said filter is
provided in said sample receiving zone.
28. Apparatus according to claim 1, wherein material comprising
said first flow path enables transport of said sample along said
first flow path.
29. Apparatus according to claim 1, wherein material comprising
said first flow path enables transport of at least a constituent of
said sample along said first flow path.
30. Apparatus according to claim 1, which includes a store of
mobile phase arranged to be released upstream of said sample.
31. Apparatus according to claim 30, wherein said store is
contactable with at least a part of said first flow path.
32. Apparatus according to claim 30, wherein said store is
contactable with at least a part of said first flow path and at
least a part of said second flow path.
33. Apparatus according to claim 1, further comprising a separate
container which contains said mobile phase, wherein said mobile
phase may be released into said mobile phase receiving zone.
34. Apparatus according to claim 1, wherein said second flow path
is convoluted and includes a zone containing labelled
immunoreactive material capable of reacting with said analyte to
produce labelled analyte, such that unlabelled analyte may reach
and becomes immobilized in said detection zone via said first path
before said labelled immunoreactive material reaches said detection
zone via said second path whereby said labelled immunoreactive
material can react with immobilized analyte.
35. Apparatus according to claim 34, wherein said second flow path
comprises at least a part of said first flow path.
36. Apparatus according to claim 34, wherein said second flow path
comprises substantially the entirety of said first flow path.
37. Apparatus according to claim I which comprises a plurality of
flow paths, wherein said plurality of flow paths are stacked.
38. An immunoassay analytical test method utilizing apparatus
according to claim 1.
39. Immunoassay analytical test apparatus, which apparatus
comprises: a) a zone for receiving a sample containing an analyte;
b) a zone for receiving a mobile phase, which zone may be the same
as the sample receiving zone, or different thereto; c) detection
means for permitting detection of said analyte by immunoreaction;
d) a first flow path for flow of said analyte in said mobile phase
from said sample receiving zone to said detection means; and a) a
second flow path permitting flow of said mobile phase to said
detection means; wherein said detection means is movable from a
first position in communication with said first flow path to a
second position in communication with said second flow path.
40. Apparatus according to claim 39, wherein said detection means
is movable from a first position in contact with said first flow
path to a second position in contact with said second flow
path.
41. Apparatus according to claim 39, wherein said detection means
is movable to be in communication with said first and second flow
paths in sequence.
42. Apparatus according to claim 39, wherein said detection means
is movable to be in contact with said first and second flow paths
in sequence.
43. Apparatus according to claim 39, wherein said second flow path
includes labelled immunoactive material.
44. Apparatus according to claim 39, wherein said first flow path
includes a material selected form the group consisting of: (i)
unlabelled immunoreactive material; (ii) hapten labelled
immunoreactive material; (iii) unlabelled capture material; (iv)
hapten labelled capture material; and (v) detector labelled
material; said material being upstream of said sample receiving
zone.
45. Apparatus according to claim 39, wherein said first flow path
includes a material selected form the group consisting of: (i)
unlabelled immunoreactive material; (ii) hapten labelled
immunoreactive material; (iii) unlabelled capture material; (iv)
hapten labelled capture material; and (v) detector labelled
material; said material being downstream of said sample receiving
zone.
46. Apparatus according to claim 39, wherein said first flow path
includes a material selected form the group consisting of: (i)
unlabelled immunoreactive material; (ii) hapten labelled
immunoreactive material; (iii) unlabelled capture material; (iv)
hapten labelled capture material; and (v) detector labelled
material; said material being upstream and downstream of said
sample receiving zone.
47. Apparatus according to claim 39, wherein said first flow path
potentiates flow towards said detection means.
48. Apparatus according to claim 47, wherein said first flow path
potentiates flow towards said detection means by a capillary
action.
49. Apparatus according to claim 39, wherein said second flow path
potentiates flow towards said detection means.
50. Apparatus according to claim 49, wherein said second flow path
potentiates flow towards said detection means by a capillary
action.
51. Apparatus according to claim 39, wherein said first flow path
is selected from the group consisting of: a) elongate sheet
material; b) elongate strip material; and c) material absorbent to
said mobile phase.
52. Apparatus according to claim 51, wherein said elongate sheet or
strip comprises a portion contactable with said first flow
path.
53. Apparatus according to claim 39, wherein said second flow path
is defined by a material selected from the group consisting of: a)
elongate sheet material; a) elongate strip material; and b)
material absorbent to said mobile phase.
54. Apparatus according to claim 53, wherein said elongate sheet or
strip comprises a portion contactable with said second flow
path.
55. Apparatus according to claim 39, wherein said analyte is
allergen specific IgE.
56. Apparatus according to claim 39, wherein said apparatus further
comprises a sink for collection of excess material exiting the
detection means.
57. Apparatus according to claim 39, wherein said detection means
is manually movable from said first pathway to said second
pathway.
58. Apparatus according to claim 55, wherein said first flow path
comprises a matrix for the removal of non-IgE components.
59. Apparatus according to claim 58, wherein said matrix is
provided between said sample receiving zone and said detection
means.
60. Apparatus according to claim 58, wherein said matrix is
provided in said sample receiving zone.
61. Apparatus according to claim 39, wherein said first flow path
comprises a filter arranged to separate components of said sample,
said filter selected from a group consisting of: i) a blood filter
arranged to permit plasma to pass whilst capturing other blood
constituents; and ii) a matrix for the removal of material other
than said analyte.
62. Apparatus according to claim 61, wherein said filter is
provided between said sample receiving zone and said detection
means.
63. Apparatus according to claim 61, wherein said filter is
provided in said sample receiving zone.
64. Apparatus according to claim 39, wherein material comprising
said first flow path enables transport of said sample along said
first flow path.
65. Apparatus according to claim 39, wherein material comprising
said first flow path enables transport of at least a constituent of
said sample along said first flow path.
66. Apparatus according to claim 39, which includes a store of
mobile phase arranged to be released upstream of said sample.
67. Apparatus according to claim 66, wherein said store is
contactable with at least a part of said first flow path.
68. Apparatus according to claim 66, wherein said store is
contactable with at least a part of said first flow path and at
least a part of said second flow path.
69. Apparatus according to claim 39, further comprising a separate
container which contains said mobile phase, wherein said mobile
phase may be released into said mobile phase receiving zone.
70. Apparatus according to claim 39, wherein said second flow path
is convoluted and includes a zone containing labelled
immunoreactive material capable of reacting with said analyte to
produce labelled analyte, such that unlabelled analyte may reach
and becomes immobilized in said detection zone via said first path
before said labelled immunoreactive material reaches said detection
zone via said second path whereby said labelled immunoreactive
material can react with immobilized analyte.
71. Apparatus according to claim 70, wherein said second flow path
comprises at least a part of said first flow path.
72. Apparatus according to claim 70, wherein said second flow path
comprises substantially the entirety of said first flow path.
73. Apparatus according to claim 39 which comprises a plurality of
flow paths, wherein said plurality of flow paths are stacked.
74. An immunoassay analytical test method utilizing apparatus
according to claim 39.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation in part of
international application PCT/GB98/01412 filed on 15th May 1998 by
the same applicant as the present invention.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to apparatus for the diagnosis
of allergies. In particular, the invention relates to immunoassay
apparatus that does not require sophisticated laboratory equipment
or technical expertise and is suitable for use in the home or the
doctor's surgery.
[0003] A number of kits using immunoassay technology (mainly for
pregnancy testing or fertility prediction) are widely available for
use in the home or in a doctor's surgery. The format of the
immunoassay technique in such kits is broadly similar, utilizing
test strips containing immobilized immunoreactant and requiring the
user to provide and apply a sample of a predetermined body fluid.
Some devices that use urine as the source of the analyte require no
further intervention than the application of the sample to the
device. This is an ideal situation where urine not only contains
the analyte but also acts as the fluid component of a mobile phase
which initiates the chemical reaction within the device.
[0004] However, other samples may not be readily available in such
copious volumes. For applications where urine is not appropriate, a
blood sample is usually required, although other body fluids may
also be of use, for example, saliva or tears. Due to practical and
ethical reasons, devices that are designed for use in the home and
that require a blood sample must work with a capillary sample not
usually larger than a few hundred microliters in volume.
[0005] For most devices, because of physical constraints such as
flow properties, this volume is insufficient to initiate the
chemical reaction and permit it to proceed. Additional fluid
components must then be added to the kit in order to provide a
mobile phase. This mobile phase can be contained within a
compartment inside the apparatus, the release of which is initiated
either by physical intervention by the user, or by a chemical
interaction of the sample with a physical barrier separating the
mobile phase from the immunoreactants. The mobile phase could also
be added from a separate container to a receptacle on the device
itself. Of course the more steps that are involved with the
operation of the kit, the more chance there is for error by the
user and less is the likelihood of obtaining a correct result.
[0006] For some medical conditions, it is important to obtain a
rapid test result. For example, a result within minutes of sample
application may be required with a test to be used in emergency
situations, whereas for other conditions a result within minutes
may not be necessary.
[0007] Currently available apparatus is often non-sequential in
nature, meaning that the reaction involves a single step. However,
one of the main problems with non-sequential immunometric test kits
is the occurrence of a phenomenon called a "high dose hook effect".
The high dose hook effect is evident when high levels of antigen in
the system saturate the assay. This is caused by free analyte being
left in the sample after reaction of all the available labelled
immunoreactant and the inadequate binding capacity of the
immunoadsorbent for the amount of complexed and free analyte in the
system. This unlabelled analyte can then compete downstream for the
immobilised immunoreactant. In some cases, when there is a large
amount of analyte in the sample, false negative results can be
obtained merely because of saturation of the labelled
immunoreactant and immunoadsorbent. These false negative, or
abnormally low, results may influence decisions on treatment made
by the user or clinician. This will not occur if sufficient
immunoadsorbent is present. However, there are limitations to the
binding capacity of an immunoadsorbent, and it is not generally
practical to increase the amount of labelled immunoreactant in the
system to overcome the high dose hook effect, as this will tend to
increase the non-specific binding of the labelled immunoreactant,
thereby reducing the analytical sensitivity of the assay, giving
rise to an elevated reference (blank) reading. In conventional
laboratory immunoassays, this problem is overcome by carrying out a
sequential assay, with discrete wash steps between each addition of
sample and immunoreactant. Sequential immunometric assays can be
easily run in a laboratory. For example, a sample which contains
analyte may be allowed to react with an immobilized immunoreactant
specific for the analyte in question. After a predetermined
incubation period, unbound analyte in the sample may be washed
away, usually by a combination of decantation and washing. Then the
labelled immunoreactants are added.
[0008] A further method to circumvent the high dose hook effect,
and to avoid the sequential format, would be to use a competitive
assay format. However, this is not ideal. For example, with such
assays, the precision is highly dependent on the region of the dose
response curve being examined. In contrast to immunometric assays,
the response in competitive assays is inversely proportional to the
dose--that is, a lower signal is obtained with higher
concentrations of analyte. In general, this is not a preferred
attribute for a home testing kit where the absence of a signal
confers a positive result. A preferred way to avoid the high dose
hook effect while maintaining an acceptable assay format is a
sequential immunometric assay approach. However, the requirement
for separate addition of reagents by the user and intervening
discrete wash steps, would make such a testing device too
cumbersome for home or office use.
[0009] EP 0314499 A discloses a test device for carrying out an
immunoassay and making a color change result visible to the user.
The device comprises a first "fast" flow path and a second "slow"
flow path of porous or fibrous liquid conductive material. The ends
of both tracks are contacted with a sample liquid store (mobile
phase containing the analyte). The first path for flow of the
analyte contains an enzyme and the second path contains a labelling
substrate corresponding to the enzyme. Both paths lead to a
detection means.
[0010] EP 0186799 A discloses an analytical device comprising a
zone for receiving sample containing an analyte, a zone for
receiving the mobile phase and a detection zone. The document
teaches the possibility of having two parallel flow paths which
start from the mobile phase reservoir and lead the detection zone,
wherein the main path transports the sample containing the analyte
and the second path entrains reagents for color detection.
[0011] EP 0590695 A discloses a liquid transfer device for use in
assay procedures, comprising a sheet of porous material for
capillary liquid flow therethrough. The sheet is formed to define
two liquid flow channels leading from two channel ends to a site in
a common channel portion, where the two channels merge. The two
channels are operable to deliver liquid to said common site in a
sequentially timed manner following simultaneous application of
such liquid to the channel ends.
OBJECTS OF THE INVENTION
[0012] The present invention aims to overcome the problems
associated with non-sequential immunometric assays by providing a
self diagnosis apparatus which can use a sequential immunoassay
method, but which does not require sophisticated laboratory
equipment or technical expertise.
[0013] It is an object of the present invention to provide a self
diagnosis apparatus utilising immunoassay technology which is
particularly useful for the measurement of antigen-specific human
immunoglobulin E (IgE), as in allergy diagnosis, but which may also
be used for other analytes and isotypes.
SUMMARY OF THE INVENTION
[0014] Immunoassay analytical test apparatus for allergy diagnosis
according to the present invention comprises:
[0015] (a) a zone for receiving a sample containing an analyte;
[0016] (b) a zone for receiving a mobile phase, which zone may be
the same as the sample receiving zone, or different thereto;
[0017] (c) a detection means for permitting detection of said
analyte by immunoreaction;
[0018] (d) a first flow path for flow of said analyte in said
mobile phase from said sample receiving zone to said detection
means; and
[0019] (e) a second flow path permitting flow of said mobile phase
to said detection means.
[0020] It is a preferred feature of the present invention that the
second flow path includes therein labelled immunoreactive material
capable of reacting with the analyte to produce labelled
anylate.
[0021] It is a preferred feature of the present invention that the
first flow path includes unlabelled or hapten labelled (such as
biotinylated) immunoreactive material being placed upstream and/or
downstream of the sample receiving zone. It is a further preferred
feature that the first flow path includes both unlabelled or hapten
labelled capture material and detector labelled material. The
materials may be dried onto the flow path substrate upstream and/or
downstream of the sample receiving zone. As the mobile phase
containing the analyte flows towards the detection means the dried
material may be solubilized by the mobile phase.
[0022] The problems associated with non-sequential immunometric
assays may be alleviated by permitting the analyte to reach the
detection means, where it is immunoextracted by an immunoadsorbent,
before a substantial amount of labelled immunoreactive material
reaches the detection means.
[0023] According to a first embodiment of the present invention
this may be achieved by providing detection means which may be
separable from the flow paths, thereby allowing the detection means
to be moved from the flow path comprising the analyte to the flow
path comprising labelled immunoreactive material only after
sufficient immunoadsorption of the analyte in the detection zone
has taken place.
[0024] Therefore, according to a first embodiment of the present
invention the test apparatus comprises:
[0025] (a) a zone for receiving a sample containing an analyte;
[0026] (b) a zone for receiving a mobile phase;
[0027] (c) a first flow path for flow of an analyte in said mobile
phase from a sample receiving zone to a detection means;
[0028] (d) a second flow path for flow of a mobile phase from a
mobile phase receiving zone to detection means, said flow path
having therein a zone containing labelled immunoreactive material
capable of reacting with said analyte to produce labelled analyte;
and
[0029] wherein the detection means is movable from a first position
in communication with said first flow path to a second position in
communication with said second flow path.
[0030] It is a preferred feature of the first embodiment of the
present invention that the detection means is movable to be in
communication with the first and second flow paths in sequence. It
is further preferred that there is contact between the detection
means and the first and second flow paths.
[0031] The flow paths according to the present invention comprise
elongate sheet or strip material having a portion connectable with
said first and/or said second flow path. The flow paths are
designed to potentiate flow toward the detection means by
permitting movement of mobile phase in a longitudinal
direction.
[0032] According to a second embodiment of the present invention
the arrival of the labelled immunoreactive material to the
detection means may be delayed by having a longer flow path for the
labelled immunoreactive material relative to the flow path for the
analyte.
[0033] Therefore, according to a second embodiment of the present
invention, the test apparatus comprises:
[0034] (a) a zone for receiving a sample containing an analyte;
[0035] (b) a zone for receiving a mobile phase, which zone may be
the same as the sample receiving zone, or different thereto;
[0036] (c) a detection means for permitting detection of said
analyte by immunoreaction; and
[0037] (d) first and second flow paths for flow of said analyte in
said mobile phase from said sample receiving zone to said detection
means, said second flow path having therein a zone containing
labelled immunoreactive material capable of reacting with said
analyte to produce labelled analyte, such that unlabelled analyte
reaches said detection zone via said first path before said
labelled immunoreactive material reaches said detection zone via
said second path whereby said labelled immunoreactive material can
react with immobilized analyte.
[0038] According to the second embodiment of the invention, it is
preferred that the flow paths are such that the mobile phase is
allowed to traverse more than one route simultaneously. The first
and second flow paths are preferably both continuous from the
mobile phase receiving zone to the detection means. The most direct
route of the mobile phase is through the sample receiving zone and
into the immunoadsorbent. However, simultaneously, the mobile phase
can also travel along a different conducting element. Within this
alternate route the mobile phase passes through a zone of labelled
immunoreactant which is free to move in the conducting element when
in the moist state only. This alternate path of mobile phase
carrying the labelled immunoreactants also leads to the
immunoadsorbent.
[0039] The immunoassay apparatus according to the present invention
preferably comprises an external body and an internal body. The
external body typically includes a receptacle for the addition of a
sample containing the analyte, and a compartment where the result
of the test can be observed.
[0040] Furthermore, the external body of the apparatus may comprise
a lancet to enable the user of the device to provide a blood
sample. The lancet may be integrally molded to the external body of
the apparatus, or it may be provided separately.
[0041] Furthermore, a desiccant tablet may also be included in the
apparatus and the entire unit hermetically sealed to prevent
deterioration through humidity changes. The apparatus is
particularly suited for use in the home, at a patient's bedside or
in a doctor's office.
[0042] A further embodiment of the device is the inclusion of a
recess into the external body of the apparatus to which a drop of
blood is added. It is preferred that this recess leads directly to
the surface of the first flow path. The recess may be arranged to
prevent the user touching the surface of the flow path during the
course of the test.
[0043] All the components necessary to obtain a result, except for
the sample containing the analyte to be detected, may be provided
as a kit and therefore the use of the apparatus according to the
invention is not limited to the laboratory.
[0044] To operate the apparatus according to the present invention,
a user provides a sample containing the analyte, for example, a
sample of blood. A capillary blood sample can be taken using a
lancet (which is preferably incorporated into the apparatus) to
prick a finger. The blood or other sample is then allowed to be
taken up by a sample receiving zone. The sample receiving zone
preferably comprises a suitable blood filter to aid separation of
plasma from the whole blood sample. Mobile phase may then be
applied upstream of the sample.
[0045] Aliquots of mobile phase may be applied indirectly to the
mobile phase receiving zone from a separate container holding
mobile phase, for example, from one or more dropper bottles. The
mobile phase, in this case, would be applied onto an adsorbent
solid phase which is a continuation of the body of the testing
apparatus, or it may be applied in the form of a well with a
connecting chamber to the sample application zone and other
conducting elements. Alternately, the mobile phase, which typically
comprises physiological saline and antimicrobial preservatives, may
be released into the apparatus directly from an integrated
reservoir. In this case, the mobile phase may be contained within a
blister or the like, permitting release of the mobile phase during
operation of the apparatus. The mobile phase may be released by
applying pressure or, for example, by suitable piercing means, to
the surface of the blister causing the blister to be punctured. The
piercing means may be incorporated into the apparatus. Once
punctured, pressure on the top of the blister causes deformation of
the blister thereby squeezing out the mobile phase from the
blister. Alternately, the direct release of mobile phase may be by
chemical intervention. The chemical intervention may be initiated
by compounds in the sample acting to "digest" a membrane, thereby
releasing the contents of a reservoir for the mobile phase. No
further intervention is required by the user except to read the
result of the test, a positive result being indicated by color in
the detection zone, after a predetermined incubation time.
[0046] As previously mentioned, the present invention is
particularly suitable for use at home or in the doctor's surgery.
It is therefore impractical to obtain a blood sample greater than a
few hundred .mu.l in volume, a preferred volume being less than 50
.mu.l. This volume of whole blood is roughly 50% cellular which
normally leaves less than 20 .mu.l of liquid sample available for
analysis. Allergy tests have to be highly sensitive to detect
minute amounts of IgE present in the circulating blood. It is
therefore advantageous to extract as much of the plasma into the
test system as possible. A system that uses the sample as a source
of mobile phase for the labelled immunoreactant and subsequent wash
steps of a sequential immunoassay is therefore impractical for the
minute volumes available in this instance. Here a separate mobile
phase is added from a reservoir that pushes the plasma out of the
sample laterally through the filter.
[0047] Advantageously, it is this lateral separation rather than
transverse flow of the capillary blood sample through a suitable
filtration means (which may advantageously comprise the material of
the first flowpath) which results in the efficient extraction of
plasma components free of red cells. The mobile phase pushes the
plasma component out of the sample causing the plasma to travel
laterally along the length of the flow path (rather than causing
separation through the depth of a filter in a conventional manner
which would cause a significant fraction of the plasma to be
retained). The sample, free of cells, is drawn laterally through
the flow path by the addition of a mobile phase, which may also
contain labelled reactants, towards the immunoadsorbent. This is in
contrast to single step devices in which the immobilized reactants
nearest to the application zone are exposed to more of the analyte
than immobilized reactants more distal to the application zone.
[0048] In a preferred embodiment of the present invention, the
apparatus additionally comprises a blood filter. A sample of whole
blood (typically approximately 50 .mu.l) may be applied to the
surface of a suitable cell filter, for example, a CytoSep filter
(Ahlstrom filtration) or a glass fiber filter such as GF/A
(Whatman), or GF51 (Schleicher and Schuell) which may be
pre-treated with detergents, anticoagulants and other reagents
common in immunoassay technology. The receptacle housing such a
filter protects the user from the chemical and allergenic contents
of the apparatus. The filter may contain a chemical anticoagulant
such as oxalate or fluoride, a chelating agent (such as EDTA) or an
anticoagulant such as heparin. Some cells may also be removed by
specific binding agents immobilized close to the sample application
zone, such as polyfunctional group reactive lectins. The cells
could also be lysed by physical or chemical shock, such as by
change in the pH or ionic environment, freezing, heating,
desiccation or the action of specific biological lysing agents or
organic compounds. However, for some analytes, removal of the cells
will not be necessary for the successful use of the apparatus
according to the invention.
[0049] Once a sample of blood or the like has been fully adsorbed,
the plasma may be separated from the cells by allowing the free end
of the filter to come into contact with a solution containing
anti-human IgE, which may be hapten or directly labelled, together
with preservatives and stabilizers (common to the practice of
immunoassay development used to minimise non-specific
interactions). However, the use of an anti-human IgG may be useful
when diagnosing food allergies that are not dependent on IgE
mechanisms. If an indirect label is added at the above step, then a
second reagent reactive with labelled groups on the anti-IgE or
anti-IgG antibody can be added by simply exposing the free end of
the filter to a reagent containing the labelled second antibody.
The latter method thereby increases the sensitivity of the
detection method. After a predetermined time, the immunoadsorbent
will display a fingerprint of the allergic profile of the patient
as a series of bands which can be attributed to the existence of
allergen specific IgE or IgG antibodies, hence suggesting the
possibility of IgE/IgG mediated allergic reactions against those
allergens tested.
[0050] Referring specifically to the embodiment described with
movable detection means, the mobile phase may be absorbed
simultaneously along two separate paths leading from the mobile
phase receiving zone. The sample receiving zone may comprise a
blood filter. This will allow lateral separation of the plasma from
the cells and subsequent flow of the plasma into the detection
means. After a fixed time, typically 10 minutes, the plasma
component of the blood sample will have been extracted from the
blood sample and passed through to the detection means, followed by
a mobile phase which has the effect of removing free unreacted
analyte in the plasma from the detection means.
[0051] The blood filter may additionally contains additives for the
specific removal of non-IgE isotypes from the plasma. As the plasma
sample from a positively testing subject may contain antibodies
directed against the allergens in the detector zone, other than
allergen specific IgE (which condition may be quite marked in
patients with food allergies) there will be a tendency for the IgG
antibodies, for example, in the plasma sample, to swamp out the
binding of the IgE antibodies against the allergen. This could
result in a false negative result being reported for the plasma
sample due to inhibition of the test by non-IgE antibody isotypes.
Here much of the plasma, and hence analyte, passes through the
blood filter that aids removal of the non-IgE antibodies. The means
for removal of non-IgE components may comprise ion-exchange
material that allows IgE antibodies to pass through unaffected but
binds to the non-IgE isotypes preventing their entry into the
detection means.
[0052] The matrix or filter for the removal of non-IgE components
may be provided separate from the blood filter. The blood filter
and/or the matrix for removal of non-IgE components are preferably
located between the sample application zone and detection
means.
[0053] An important embodiment of the apparatus according to the
invention which increases the specificity of the diagnostic test is
that the sample, for example for measuring allergen specific IgE,
may be adsorbed free of non-IgE isotypes by passing the sample
through a matrix containing an adsorbent reactive with human IgG,
IgM or IgA e.g. lectins, protein A or anti-human antibodies, other
than the specific antibodies used, thereby facilitating the
adsorption of non-specific isotypes (for measurements of IgE, IgD,
and IgA or IgG subclasses). Incorporation of an adsorption matrix
between the sample application zone and the immunoadsorbent will
yield more specific and sensitive allergy assays by excluding
interference with non-IgE antibodies. Alternately, the matrix may
be provided in the sample application zone within the blood filter.
Alternately, the matrix is provided separate to the blood filter.
It is also possible to immobilize antibodies to provide a zone in
the detection means prior to the allergen impregnated means. For
example, the presence of large quantities of non-IgE anti-allergen
antibodies in the sample will inhibit the binding of IgE antibodies
by competition for the allergosorbent, giving the possibility of a
false negative result which may be particularly important for the
diagnosis of food allergy mediated by IgE, or where patients have
undergone immunotherapy with allergen extracts (which may result in
the elevation of non IgE immunoglobulin isotypes). It would
therefore be expected that an assay using the apparatus according
to the invention would classify some patients as reacting
positively to certain allergens, while other commercial allergy
assays may have classified as negative or as a lower allergy class.
This may be an important feature when objective measurements of IgE
in the absence of other isotypes are desired, or when specific
isotypes of antibody are measured without interference from other
isotypes. A further implication of this embodiment is the removal
of immune complexes of allergen, IgE and IgG because of the
polyclonal nature of the immune response to a number of epitopes on
the allergen surface. Removal of such complexes could decrease the
number of asymptomatic specific IgE positive results, which is
currently found with current classical allergy tests for the
measurement of specific IgE. The absorption technique could also be
extended to the removal of crossreactive antigens in the case of
immunoassays for other analytes, or for the removal of interfering
components (such as immune complexes as in certain disease
conditions) and also where biologically active components or
chemically interfering materials (such as proteases and other
binding substances) need to be removed.
[0054] The mobile phase also simultaneously passes along the second
flow path through the labelled immunoreactive material to the
detection zone. During the initial ten minute separation of the
plasma from the whole blood sample, the mobile phase also serves to
rehydrate dried developer reagent, such as a labelled anti-human
IgE antibody, in the second flow path.
[0055] After a specified period of time, typically ten minutes, the
second phase of reaction is initiated by the physical movement of
the detection zone from the first flow path to the second flow
path. This process may be carried out manually or by other means.
Now the active flow path is from the reservoir or mobile phase
receiving zone through the rehydrated labelled antibodies into the
detection zone. An important feature of the breaking of the flow of
the first flow path is that there is little chance for the red
cells from the blood sample to diffuse into the detection zone and
thereby obscure the result on the detection filter.
[0056] The labelled immunoreactants pass through the detection zone
as a bolus which serves two purposes:
[0057] (i) the concentration of the labelled immunoreactants in the
detection means is increased hence increasing the reaction rate;
and
[0058] (ii) the bolus is followed by a zone of mobile phase which
serves to clear the detection zone of remaining labelled
immunoreactants thereby reducing any nonspecific binding and
increasing the signal to background readings.
[0059] At the end of the detection means lies a sink which may or
may not be always in contact with the detection means except during
the second phase of the test whereby the detection means and the
second flow path are in contact. This area serves as a sink for
unreacted material and can thereby accumulate unreacted (excess)
labelled immunoreactants thereby changing the visual appearance of
the sink either directly in the case of particulate labels or
indirectly through reaction of the labelled immunoreactants by a
further developer. This enables the excess labelled immunoreactants
to serve as a means of determining when the test is complete.
[0060] The movement of the detection means is a key feature. The
detection means is clipped into the second flow path and
importantly holds the filter at an angle to the surface of the
detection means. The first flow path and second flow path are also
inclined at a similar angle such as when the detection means is
pulled from the first flow path, the detection means is forced
against the second flow path, ensuring a good contact to allow
continuity of the flow of the mobile phase and reactants it
carries. The detection means may also contain a window through
which the result of the test can be observed.
[0061] According to another feature of the present invention, an
allergosorbent may be used. Due to the high sensitivity required
for the detection of small amounts of allergen specific IgE
compared to allergen specific IgG, and the fact that the IgE may be
directed to only a small fraction of the allergosorbent, the
properties of the substrate for binding the allergens should be
highly efficient. The flow rate is also important, because too
rapid a flow rate would result in too low a sensitivity. A plastics
backed nitrocellulose membrane (SSLU, Schleicher and Schuell) with
a pore size of 3 to 5 .mu.m, or a Whatman 3 mm, has been found to
be suitable for the determination of allergen specific IgE.
[0062] The apparatus according to the invention may be used for the
detection of multiple allergen specific IgE rather than IgE towards
a single allergen. For example, in the case of the embodiment
according to the present invention with non-movable detection
means, the rate of flow through the allergosorbent will be
proportional to the length of the respective flow path. Hence the
larger the panel of allergens tested, the longer will be the
development time for a result to be observed.
[0063] The allergens may be applied to the nitrocellulose with a
pen capable of dispensing aqueous allergen extracts at 1 .mu.l per
linear cm at concentrations ranging between 1 to 10 mg/ml in 50 mM
Tris buffered saline pH7.4 (the protein concentration depending on
the source of extract used). The nitrocellulose may then be allowed
to dry at room temperature for 30 minutes. The excess reactive
groups on the nitrocellulose may then be blocked for 1 hour with a
Tris buffered saline solution containing 0.05% v/v Tween-20. A
further wash with the above buffer may be performed for 15 minutes
prior to allowing the membrane to dry at room temperature for 3
hours and then storing the desiccated material at 2 to 8.degree.
C.
[0064] It is preferred that the detection means comprises an
immunoadsorbent. Many current immunoassay devices operate by the
sample flowing through a zone of labelled immunoreactants
downstream from the sample application zone, which immunoreactants
bind specifically with the analyte if found to be present in the
sample. The label constitutes the means by which the specific
analyte is made detectable to the user. This labelled
immunoreactant can be direct, for example, when bound to dyed latex
particles, gold colloids or dye sols. The labelled immunoreactant
may also be indirect, such as when the label is a biological enzyme
that requires treatment with a substrate and chromogen prior to
detection of the label, or when silver enhancer reagents are
required before previously undetectable gold labels are rendered
visible. The labelled immunoreactant may be free to move along with
the mobile phase. The labelled immunoreactant-analyte complex, if
present, moves downstream according to the flow initiated by the
reservoir of mobile phase, towards an immunoadsorbent layer.
[0065] Here, for example, a further immunoreactant, again specific
for the analyte (but to a different reactive group on the analyte)
can bind to the labelled immunoreactant-analyte complex, downstream
from the labelling zone. The presence of analyte in the sample can
therefore be visualized by the accumulation of labelled complex at
the site of the immunoadsorbent. In a further feature of the
apparatus, depending on the nature of the labelled immunoreactants,
the mobile phase may be water-based (e.g. a saline solution), it
may contain a substrate and/or chromogen for an enzyme label, or it
may contain a silver enhancer reagent (such as one commercially
available suitable for the visualization of histological sections
with colloidal gold reagents from Sigma-Aldrich Company) or one of
the components of such a reagent when gold-labelled immunoreactants
are used for detection (or indeed other detection agents).
[0066] The assay can be rendered more sensitive by the lateral flow
of a silver enhancer solution. This allows the use of smaller gold
colloids giving better penetration and resolution of the membrane
but then enhanced by the deposition of metallic silver on the
surface of the colloidal gold. This should be useful to the
clinical laboratory where applications involving minute sample
volumes are required or where ultra-sensitive detection systems are
required for the simultaneous determination of multiple analytes
from a minute sample. The gold conjugates may also be silver-coated
prior to their inclusion within the apparatus according to the
invention.
[0067] Such conjugates may be prepared by incubating a titered
aliquot of gold labelled antibody (dialysed or suitably diluted to
remove ions such as chloride), preferably 5 nm or less, with a
reactive solution of silver enhancer for 5 to 10 minutes, followed
by centrifugation and three washes with equal volumes of distilled
water, to remove excess silver enhancer.
[0068] A positive result in such immunoassays using such pre-silver
stained gold conjugates with silver-stained gold conjugate is
represented by an intense black coloration at the binding site,
thereby providing sensitivities many orders of magnitude greater
than that of the original gold colloid. These silver-stained gold
conjugates can be readily prepared and are stable in both liquid
and dried form. Smaller gold conjugates of 5 nm or less are most
appropriate for silver staining. The intensity of silver staining
can be modulated by the concentration of reactants, removal of
interfering salts, temperature (a reduction in temperature can be
used to control the rate of reaction) and the type of labelling
system used. These silver stained gold conjugates may also be of
use where a passive immunoassay format is used, such as in
classical protein and nucleic acid blotting.
[0069] In a further embodiment of the invention, there may be
included an onboard control that indicates when the assay has been
complete or successful. This control (or a further control) can
also be used as a reference to the test result obtained with the
specific analyte. Thus, by comparing the intensity of the reference
with the test result, an indication as to whether the test result
was positive or negative will be obtained. Hence, by means of a
further apparatus that measures the intensity of the response, a
quantitative measure could be achieved by comparing the activity of
the reference with the test result.
[0070] Alternately, the device may also contain an electronic means
of detecting the result, the termination of the test and audible or
visible step-by-step instructions for performing the test.
Inclusion of these electronic features would allow greater
confidence of the user in performing the test correctly and reduce
the requirement for the reading of instruction leaflets prior to
use. For example, regions of the device could contain sensors that
detect moistness of the flow paths thus determining the step the
test is at during the processing of the sample. Such sensors could
be linked to visual or audible displays of the processing step and
indicate the next step. Such sensors in the detection zone could
include densitometric measurements, that determine the amount of
labelled antibody specifically bound by analyte in the detection
zone in order to produce a quantitative result or a means of
indicating when the detection means should be pulled to the
developer position such as an audible warning or visual instruction
or signal. The electronic sensor could also indicate when
sufficient blood has been added to the sample receiving zone by,
for example, the determination of the change in conductivity of the
filters between the dry and wet state or when the ionic composition
of the filter changes due to the addition of polar solvents or
sample or by measuring a change in optical density such as that
resulting from the addition of a colored sample (as is the case
with a blood drop) by means of a photocell for example.
[0071] A further embodiment could include means for measuring the
amount of sample added. This again could be done through electronic
means as described above or more simply through the use of an
integral capillary tube or similar that would "draw up" a fixed
volume of sample into the device for the analysis.
[0072] The detector could also take the form of an electronic
device where the physical properties of the solid phase to which
the allergens are absorbed is changed by the binding of the
labelled anti-human IgE antibodies to these zones when in the
presence of the analyte ie allergen specific IgE.
[0073] The present invention therefore helps to eliminate common
pitfalls in immunoassay technology associated with known
apparatus.
[0074] Sequential assay also allows amplification of the assay
system by the use of multiple additions of antibodies e.g. in the
case of a sensitive allergy test, the first antibody may be a
biotinylated anti-human IgE which is washed through by a
gold-labelled anti-biotin second antibody which is readily visible
to the naked eye when concentrated, in the form of a discrete
reaction zone at the immunoadsorbent site.
[0075] The intermission of discrete wash steps common to routine
sequential assays as performed in clinical laboratories is
unnecessary for this format of the assay, because the capillary
nature of the filters and membranes, as the first antibody is
washed out with little reaction and mixing by the second antibody.
The same can be said about the sample which is pushed out of the
filter by the influx of the primary antibody.
[0076] A principal embodiment of the apparatus according to the
invention is the circumvention of the requirement for discrete wash
steps while still retaining a sequential immunometric assay format
and is especially applicable for apparatus to be used in the home
or doctor's office. This is achieved by the construction and, in
the case of the embodiment with non-movable detection means,
geometry of the conducting elements transporting the mobile phase.
In contrast to known apparatus, it is a feature of the apparatus
according to the invention that the sample can be applied to a
region close to the immunoadsorbent. Mobile phase can then be
applied upstream of the sample either by release from a compartment
within the device or added from a separate container supplied as
part of the apparatus. This circumvents the necessity for the
mobile phase to enter the labelled immunoreactant zone before
reaching the sample.
[0077] Therefore, due to a possible difference in flow
characteristics of the two paths mentioned above, it is evident
that arrival of the labelled immunoreactants much later at the
immunoadsorbent than the initial analyte from the sample
application point will effectively result in a sequential
immunometric assay. This procedure allows the sequential format
described, to take place without the requirement of a previous
interaction with a labelled immunoreactant and without discrete
wash steps or separate multiple applications of reagents by the
user.
[0078] In a further embodiment of the invention, the number of
paths need not be limited to two. A further increase in the number
of paths of varying flow characteristics provides more flexibility
with the testing system, allowing more complex chemistries to take
place or facilitate enhanced washing steps if required, or chemical
modifications steps or reaction with different labelled
immunoreactants in turn on the solid phase. Nevertheless, the only
requirement for the user, irrespective of the number of flow paths,
is to provide a sample at the application point and then to
initiate the flow of the mobile phase either from within the device
or via an external container.
[0079] The most rapid path need not necessarily lead to the sample.
The present invention could also be applicable in carrying out the
immobilization of immunoreactants during operation of the
apparatus, rather than immobilized immunoreactants being prepared
by the manufacturer.
[0080] A sequential format is particularly useful when monoclonal
antibodies are unavailable. In general, polyclonal antibodies can
potentially give greater sensitivity in immunoassays and can be
more stable when labelled than monoclonal antibodies. It is
important when using a non-sequential assay device, as described
previously, that the labelled antibody is specific for one epitope
while the immobilized immunoreagent is specific for a spatially
distinct distant epitope. However, in some cases this would
preclude the use of polyclonal antibodies because of the
possibility of reaction with the epitopes recognized by the
immobilized immunoreactant on the immunoadsorbent, thereby
effectively masking the epitopes to the immunoadsorbent, reducing
binding and hence reducing the signal.
[0081] A sequential assay format eliminates these problems and is
particularly useful when the analyte is an immunoglobulin or when
only polyclonal antisera are available. The immunoadsorbent is
usually immobilized allergen extract. However, specific allergen
assays may also be conducted by using an immobilized anti-human IgE
antibody and labelled allergens. The multiple simultaneous
detection of IgE and IgG antibodies directed against an allergen
could also be performed by adapting this method using labelled
allergens. In this method, for example, the detector may comprise
two zones:
[0082] (a) a zone containing an immobilized anti-IgE antibody;
and
[0083] (b) an immobilized IgG binding reagent such as protein A or
anti-IgG antibody.
[0084] Unlike many immunometric assays where the immunoadsorbent is
a preparation of purified antibody, for allergen extracts they are
usually complex mixtures of a multitude of proteins. For example,
house-dust mite extracts contain many antigens and allergens each
reacting differentially with blood samples from different
individuals. Here, previous reaction of the IgE present in a
capillary blood sample with labelled anti-human IgE antibodies, as
would occur using conventional devices for home/office testing, can
possibly inhibit interactions with the allergosorbent solid phase
due to the orientation of the anti-IgE/IgE complex interfering with
the binding of such complex to the immunoadsorbent. A particular
feature of most laboratory based allergy tests is that they adopt a
sequential assay format, thus circumventing this problem of steric
hindrance by allowing the IgE in the patient sample to bind to the
allergen solid phase first. Then, a polyclonal labelled anti-human
IgE antibody preparation can be used in excess to ensure
quantitative estimation of the specific IgE bound.
[0085] A major advantage of the use of the sequential format in a
diagnostic device is in the case of celiac disease. Here the
presence of Immunoglobulin A (IgA) antibodies against the cereal
protein gliadin in the blood and some other body fluids, for
example saliva, is an indication of celiac disease. However, the
fraction of gliadin specific IgA compared to total IgA in blood
samples from individuals with celiac disease can be very small.
This means that in order to detect gliadin specific IgA, a
sensitive detection system is required together with a large sample
volume, if the signal obtained with a sample containing gliadin
specific IgA antibodies is to be distinguished from blood samples
that are not. The increase in the sample size, corresponds to an
increase of total IgA respectively, which has the result that
existing non-sequential assays could be too insensitive, as there
is a possibility that due to the increased sample volume and total
IgA levels, that the high levels of non-gliadin specific IgA could
potentially swamp out the detection of the gliadin specific IgA by
saturation of the labelled anti-human IgA antibodies.
[0086] Thus the use of a sequential assay, of first allowing the
human anti-gliadin specific IgA to bind to immobilized gliadin
immunoadsorbent, then reacting in a separate stage thereafter with
the labelled anti-human IgA, will result in a more specific,
sensitive and reduced possibility of false negative assays which
may have been caused by saturation of the assay system with the
non-gliadin specific IgA found in the blood sample. It is also
possible that the mobile phase could be used to cause a slow or
gradual release of labelled reactants from a matrix which would act
in concert with the sequential assay format described above to
provide an even clearer separation of immunoreactants during
operation of the device. For example, in the case of specific IgA
against gliadin, the assay would be relatively insensitive if
non-specific IgA was not removed prior to detection with an
anti-IgA label, which in conventional laboratory based immunoassays
is circumvented by a sequential assay format which incorporates a
wash step to remove non-specific immunoreactants prior to the
addition of labelled antibody. However in a diagnostic apparatus
designed for use in the home or the office, discrete wash steps are
inconvenient and cumbersome and a possible source of error.
[0087] The result depends on a number of factors including; the
manual intensity of washing performed by the operator, the reagents
used for the wash step may differ, especially if tap water is
advised e.g. contaminants, microbial, particulates, force and
temperature.
[0088] To circumvent the requirement for a wash step, the apparatus
should contain where necessary means for the removal of
non-specific reactants. It is therefore a further embodiment of the
apparatus described here that, if necessary and depending on the
analyte to be measured, means for the slow release or desorption of
the labelled immunoreactants should be an integral part of the
operation. In practice, such a slow release mechanism could take
the form of a mechanical barrier, for example, a gel or
encapsulating material, or chemical means such as adsorption to a
charged matrix such as an ion-exchange material or affinity
adsorbed by a specific property of binding of the labelled reactant
for an immobilized ligand and desorbed by a suitable change in
ionic strength, hydrogen ion concentration, or addition of
compounds that antagonize ligand-reactant binding. The slow release
in the latter case may be mediated by the immobilized labelled
analyte being surrounded by an environment that promoted binding
where the mobile phase antagonizes this binding. Essentially this
sets up a gradient through the matrix housing the adsorbed labelled
reactant, until eventually at a particular gradient strength, the
adsorbed material will begin to be released. Prior to the release
of the labelled reactant, the passage of the mobile phase through
the sample adsorption zone will allow the specific binding of the
human antibody specific for the immobilized ligand in the detection
zone without the interference from the labelled reactants with
non-specific analytes.
[0089] Translocation of the mobile phase including the
immunoreactants may be achieved by movement along chambers or
channels formed by the body of the apparatus itself, specific
capillary tubing, channels and troughs or from compartments with
flow limiting orifices or movement along wicks by capillary action.
Such wicks may be of synthetic or natural materials. The choice of
material suitable for the translocation must be such that passive
adsorption of the immunoreactants onto these conducting elements
does not occur; the material can be treated to prevent deposition
of the immunoreactants on the surface of the conducting elements.
If required, the materials used for the conducting elements can
either be treated before the device is assembled and/or during
operation of the device. For example, blocking components such as
irrelevant proteins, polymers, detergents and other routinely used
blocking reagents common to the practice of immunoassay design
should not be present. The capillary elements main purpose is to
provide a flow path for the mobile phase and to connect sections of
the apparatus that contain immunoreactants or detecting agents that
need to be separate prior to conducting the test, and to allow
reagents to be added to the immunoadsorbent at different times.
Viscosity modifiers can be used to adjust the rate of translocation
together with the flow characteristics of the conducting elements.
Regions of the conducting elements may also be modified to perform
chemical/immunological interactions with the mobile phase and its
contents at that particular region of the apparatus. The choice of
material for the conducting elements may therefore be of great
importance for the successful operation of the apparatus.
[0090] An important embodiment by which a sequential assay may be
performed by a single addition of mobile phase at the start of the
operation of the apparatus is to have a number of separate
conducting elements. In such a system, conducting elements emerge
from a single reservoir which may or may not contain chemically or
immunologically active constituents as is required by the
particular analyte to be detected. By allowing a single reservoir
to feed a number of conducting elements, this allows different
reagents to be applied to the test zone at various positions and at
controlled intervals of time. That is, differentially varying the
flow characteristics of each conducting element will result in
different flow rates and hence arrival of various reagents at
different times to the immunoadsorbent. The flow of the mobile
phase and reactants through a particular conducting element could
also be modulated by the composition of the conducting element
matrix and also by the addition of flow modifiers to each of the
conducting elements to change the viscosity. An important
embodiment of this invention is the circumvention of the
requirement of detection agent modulators. For example, reducing
agents may be used to prevent premature chromogen formation when
using horseradish peroxidase labelled immunoreactants.
[0091] The apparatus according to the invention may permit an
unlimited number of additions of reagents even though they are all
initiated from a single reservoir of mobile phase. This will
greatly enhance the potential usefulness of such single step
apparatus as the problem of sequential addition of reagents can be
overcome. The multiple conducting element system circumvents the
requirement for discrete wash steps in such apparatus, allowing the
measurement of a wider dynamic range of analytes and
concentrations, while eliminating the possibility of high dose hook
effects which can occur in some non-sequential immunometric assays,
that do not have a wash step incorporated to remove unbound analyte
to the solid phase immobilized immunoreactant.
[0092] The multiple conducting elements can be arranged as a sheet
in two dimensions or as a three dimensional structure where more
than one "sheet" of conducting elements can be separated by
"insulating" layers of material forming a sandwich of conducting
elements. For some analytes, the optimal configuration for the
apparatus may be a combination of the two formats especially if
more than one analyte is to be detected at a time. Either way, such
a design would allow a complex series of chemical and
immunoreactions to occur separated in time and distance but
ultimately brought together in the correct sequence at the
detection zone to give the required result.
BRIEF DESCRIPTION OF THE DRAWINGS
[0093] An immunoassay analytical test apparatus for allergy
diagnosis according to the present invention is shown schematically
in FIGS. 1 to 4 of the accompanying drawings, which are by way of
example only. In the drawings:
[0094] FIG. 1 shows the outward appearance of an immunoassay
apparatus;
[0095] FIG. 2 shows an immunoassay apparatus according to the
second embodiment of the present invention, having therein a longer
flow path for the labelled immunoreactive material relative to the
flow path for the analyte;
[0096] FIG. 3 shows the apparatus according to the first embodiment
of the present invention, having therein a movable detection zone
which is shown in connection with the first flow path; and
[0097] FIG. 4 shows the apparatus of FIG. 3 with the detection zone
in connection with the second flow path.
[0098] FIG. 1 shows the outward appearance of the apparatus (FIG.
1) includes a receptacle for receiving the mobile phase (1), a zone
for the addition of the users sample containing the analyte (2),
which may also be incorporated in (1), both (1) and (2) being near
the surface of the apparatus housing (3). The detection zone (4) is
situated within the housing (3) and may be protected by a
transparent window. The detection zone contains the immunoadsorbent
on which the result is observed.
[0099] An important embodiment of the apparatus of FIG. 2 is the
construction of the conducting elements. In principle, the
apparatus consists of conducting elements of various path lengths
and flow characteristics. The most rapid and direct path for the
mobile phase arises at the point of the reservoir application (1)
where the mobile phase is added and leading to the sample
application zone (2) situated on or near the immunoadsorbent. The
geometry of the solid phase, together with the materials used to
construct the reservoir (1), sink (8), conducting elements (6) and
immunoadsorbent (4) are designed to potentiate flow towards the
sink (8). The sample application zone leads to the immobilized
reactant on the immunoadsorbent (5) via a filter 9 for removing
non-IgE components. The labelled antibody zone (7), is freely
mobile within the conducting elements when mobile phase from the
reservoir is present and may lead directly to the immunoadsorbent
or pass through the sample zone as indicated in FIG. 2. The time
taken for the labelled reactants to reach the immunoadsorbent is
longer than for the sample. This configuration is simple to
manufacture from available materials and will result in a
sequential assay format essentially due to the difference in
arrival times of the sample and labelled antibody or further
reagents at the immobilized immunoreactant zone.
[0100] Referring to FIG. 3, a further embodiment of the present
invention is represented by a reservoir (1) comprising the mobile
phase, a first flow path comprising a sample application zone (2),
a filter (9) for removal of non-IgE components and a second flow
path comprising a labelled antibody zone (7). There is further
provided a movable detection zone (4), comprising a sink (8). The
detection zone (4) is shown in connection with the first flow
path.
[0101] Referring to FIG. 4, the apparatus of FIG. 3 is shown with
the detection zone (4) in the second flow path.
[0102] There need not be limitation to two conducting elements as
illustrated but there may be a multitude whenever a more complex
chemistry of reactants is desired. This arrangement can also be
simply extrapolated to form a three dimensional structure where the
conducting elements of various physical properties are stacked one
upon another prior to adjoining the immunoadsorbent.
EXAMPLES
[0103] The invention will now be illustrated in more detail with
reference to the following worked examples.
Example 1
[0104] A piece of Schleicer and Schuell plastic backed
nitrocellulose (SSLU) 5 mm.times.60 mm was prepared as described
above. Three lines of allergen were imprinted within the
nitrocellulose 10 mm apart representing Timothy Grass/Cocksfoot
Mixed allergens (2 mg/ml), Cat allergens (10 mg/ml, Bayer) and
Dermatophagoides pteronyssinus extract (20 mg/ml, Smithkline
Beecham). A piece of GF/A glass fiber filter paper (Whatman, 5
mm.times.35 mm) was attached to the proximal end of the
nitro-cellulose and a piece of CHR17 (Whatman, 5 mm.times.40 mm) to
the distal end by solvent adhesive to a plastics support, with a 5
mm overlap onto the nitro-cellulose surface. A 25 .mu.l sample of
three serum pools RAST grade 3 to 4 positive for either grass
pollen, cat or mite allergens or a normal goat serum pool were
spiked with an equal volume of fresh human normal donor red blood
cells, and added as a 50 .mu.l aliquot to the centre of the GF/A
filter. Once absorbed, the free end of the filter was placed in
contact with 200 .mu.l of 50 mM Tris buffered saline pH7.4
containing 0.5 .mu.g of biotinylated Goat anti-human IgE (Vector
Laboratories) and allowed to absorb for 1 hour. The free end of the
filter was then placed in contact with 300 .mu.l of the above
buffer containing 30 .mu.l of gold (40 nm) labelled Goat
anti-biotin (British Biocell International) and allowed to absorb.
After 30 minutes positive results were observed by the presence of
pink/red lines corresponding to the representative allergens from
each of the serum pools, except for the normal goat serum which
served as a negative control where no lines were visible for any of
the allergens tested.
Example 2
[0105] A piece of Schleicer and Schuell nitro-cellulose (5.times.30
mm) was impregnated with mite extract as described above. A piece
of GF51 (Schleicher and Schuell, 5.times.25 mm) was attached to the
nitro-cellulose with a 5 mm overlap and both components fixed by
adhesive to a plastics support. A 25 .mu.l aliquot of RAST grade 4
positive serum from a house dust mite positive patient pool was
then added to the GF51 to a region previously impregnated with 0.5
.mu.g of biotinylated goat anti-human IgE in 10 .mu.l of Tris
buffered saline. A 100 .mu.l aliquot of gold(40 nm) labelled goat
anti-biotin in Tris buffer containing 1% w/v bovine serum albumin
was then added dropwise to the GF51 filter. A positive band
corresponding to reactivity of the serum with the mite extract was
observed within 30 minutes.
[0106] The reactants, except for the mobile phase, are probably
best stored within the apparatus in a solid form to enhance
stability, as it is desirable for the apparatus to be stored at
ambient conditions. Biological components are notorious for
degradation in solution whereas storage in a dried state or
chemically coupled to some matrix confers some protection to their
innate lability. Another problem with biological materials
(especially proteins) is their tendency to adsorb non-specifically
to matrix surfaces such as glass, paper and plastics. These
interactions have to be minimized if the reactants are to be of any
use in the apparatus. One method is to pretreat the matrix holding
the reactants with a non-specific blocking reagent as described
above. Another method may be to chemically bind them to the solid
phase but to include a releasing agent that releases the reactant
from the solid phase into the mobile phase. One way to add the
reactants is in a liquid form onto the non-adsorbent matrix and
then to remove the liquid thereby drying as a film. The reactants
could also be added in the form of a gel (such as agarose or
gelatin), or simply dried into the matrix with a detergent. The
components of the device also contain preservatives to help prevent
deterioration of the product by microbial organisms and to help
protect against adverse environmental conditions, for example,
temperature light and humidity.
* * * * *