U.S. patent number RE37,437 [Application Number 08/544,579] was granted by the patent office on 2001-11-06 for sheet-like diagnostic device.
This patent grant is currently assigned to Dade Behring Marburg, GmbH. Invention is credited to Heinz-Jurgen Friesen, Gerd Grenner, Klaus Habenstein, Helmut Kohl, Hans-Erwin Pauly, Joseph Stark.
United States Patent |
RE37,437 |
Friesen , et al. |
November 6, 2001 |
**Please see images for:
( Certificate of Correction ) ** |
Sheet-like diagnostic device
Abstract
A solid diagnostic device for the quantitative determination of
substances of biological affinity in biological fluids is
described. A process is also described in which the biological
fluid is brought into contact with a specific functional sector of
the device, the fluid migrates through several functional sectors
situated beside one another and containing suitable reagent
components, and one or more substances of biological affinity are
detected in such functional sectors which contain, for each
substance to be detected, at least one combination partner of
biological affinity, attached to a solid phase.
Inventors: |
Friesen; Heinz-Jurgen
(Dugobertshausen, DE), Grenner; Gerd (Binningen,
DE), Pauly; Hans-Erwin (Dautphetal, DE),
Kohl; Helmut (Wetter, DE), Habenstein; Klaus
(Wetter, DE), Stark; Joseph (Ebsdorfergrund,
DE) |
Assignee: |
Dade Behring Marburg, GmbH
(Marburg, DE)
|
Family
ID: |
6252876 |
Appl.
No.: |
08/544,579 |
Filed: |
October 18, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
808563 |
Dec 13, 1985 |
04861711 |
Aug 29, 1989 |
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Foreign Application Priority Data
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Dec 15, 1984 [DE] |
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3445816 |
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Current U.S.
Class: |
436/514;
435/7.92; 435/7.93; 435/7.95; 435/805; 435/806; 435/969; 435/970;
435/971; 435/974; 436/518; 436/528; 436/541; 436/810; 436/814;
436/818 |
Current CPC
Class: |
G01N
33/521 (20130101); G01N 33/525 (20130101); G01N
33/54366 (20130101); G01N 33/558 (20130101); Y10S
436/81 (20130101); Y10S 435/805 (20130101); Y10S
435/962 (20130101); Y10S 435/97 (20130101); Y10S
435/81 (20130101) |
Current International
Class: |
G01N
33/52 (20060101); G01N 33/543 (20060101); G01N
33/558 (20060101); G01N 033/558 () |
Field of
Search: |
;435/7.92,7.93,7.95,805,806,969,970,971,974,287.1,287.2,810
;436/514,518,528,541,810,814,818 ;422/56,57 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1185882 |
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Apr 1985 |
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CA |
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0046004 |
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Feb 1982 |
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EP |
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0088636 A2 |
|
Sep 1983 |
|
EP |
|
0097952 |
|
Jan 1984 |
|
EP |
|
0100619 |
|
Feb 1984 |
|
EP |
|
1440464 |
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Jun 1976 |
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GB |
|
2126224 |
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Mar 1984 |
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GB |
|
48-5925 |
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Jan 1973 |
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JP |
|
Other References
West German Offenlegungsschrift No. 33 29 628, dated Feb. 23, 1984*
.
West German Offenlegungschrift No. 30 43 608, dated Jun. 24, 1982.*
.
West German Patentschrift No. 23 32 760, issued Mar. 4, 1982.*
.
Hawkes et al., "A Dot-Immunobinding Assay for Monoclonal and Other
Antibodies", Analytical Biochemistry, 119(1):142-147 (1982). .
Hashimoto, Thin Layer Chromatography, pp. 1-4 (Hirokwawa Shoten
1962, Tokyo) (with English translation). .
Nakamura, "General Principles of Immunoassays", Immunochemical
Assays and Biosensor Technology for the 1990S, pp. 3-7 (Robert
Nakamura ed., American Society for Microbiology 1992, Washington,
D.C.). .
Suzuki, Tests in Thin Layer Chromatograpy--Hirokawa Chemical Series
No. 3, pp. 18-20, (Hirokawa Shoten 1964, Tokyo) (with English
translation). .
Towbin et al., "Electrophoretic Transfer of Proteins from
Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some
Applications", Proc. Natl. Acad. Sci., 76(9): 4350-4354 (1979).
.
van Raamosdonk et al., "Detection of Antigens and Antibodies by an
Immuno-Peroxidase Method Applied on Thin Longitudinal Sections of
SDS-Polyacrylamide Gels", J. Immunological Methods, 17:337-348
(1977). .
Zuk et al., "Enzyme Immunochromatography--A Quantative Immunoassay
Requiring No Instrumentation", Clin. Chem., 31(7):1144-1150 (1985).
.
Maruzen, Experimental Chemistry Guidebook, pp. 218-219 (Japan
Chemistry Society ed. 1984, Tokyo) (with English translation).
.
Nanzando, Thin Layer Chromatography, pp. 40-41 (Masayuki Ishikawa
et al. ed. 1963, Tokyo) (with English translation). .
Maruzen, Experimental Chemistry Course 2 Fundamental Techniques II,
pp. 215-216 (Japan Chemistry Society ed. 1956, Tokyo) (with English
translation). .
European Patent Application of Fuji Photo Film Co., Ltd.,
Publication No. A2 0 097 952, dated Jan. 11, 1984..
|
Primary Examiner: Chin; Christopher L.
Attorney, Agent or Firm: Finnegan, Henderson, Farabow,
Garrett and Dunner, L.L.P.
Claims
We claim:
1. An analytical device for the detection or determination of .[.a
component.]. .Iadd.components .Iaddend.in a fluid wherein said
.[.component is an analyte with bioaffinity binding properties,.].
.Iadd.components are a plurality of analytes, said analytes
including at least one attachment point of biological affinity,
.Iaddend.comprising a layer of a plurality of substantially planar
zones adjacent one another and in absorbent contact with one
another, said layer including:
a mobile phase application zone (MPAZ),
an intermediate zone (IZ), and
an adsorption zone (AZ),
.[.liquid being.]. .Iadd.wherein said fluid is .Iaddend.capable of
moving by adsorption from said MPAZ through said IZ to said AZ, and
wherein said IZ further comprises a .Iadd.plurality of
.Iaddend.solid phase .[.zone (SPZ).]. .Iadd.zones (SPZs) for the
detection of a plurality of analytes, said SPZs .Iaddend.having at
least one unlabelled reactant, capable of interactions of
biological affinity with at least one analyte.[.;.]. .Iadd., each
of said SPZs being adjacent one another in said layer and each of
said SPZs including said unlabelled reactants fixed thereto, said
unlabelled reactants of each SPZ being specific for a specific
analyte to be detected in each of said SPZs;.Iaddend.
at least one unattached, labelled reactant .[.(conjugate).]. ,
capable of interactions of biological affinity with said at least
one analyte, disposed in an area between .[.said.]. .Iadd.the
.Iaddend.MPAZ and the .[.SPZ.]. .Iadd.SPZs.Iaddend.; and
an analyte application zone disposed at said MPAZ or in between
said MPAZ and said AZ, wherein after application of said at least
one analyte, said at least one analyte is reacted with said
reactants in said layer and is detected in said layer. .[.
2. A device as claimed in claim 1, wherein the MPAZ has the
function of a volume metering element and releases to the
subsequent zones at least sufficient liquid for the liquid,
controlled by capillary forces, to reach the end of the
AZ..]..[.
3. A device as claimed in claim 1, wherein the MPAZ is a plastic
sponge or a particulate layer which is composed of hydrophilic
polymers and which is capable of containing chemicals, buffer
substances or other substances required for certain
tests..]..[.
4. A device as claimed in claim 1, wherein the analyte application
zone retains blood cells..]..[.
5. A device as claimed in claim 1, wherein all or some of the
reagents required for the detection of the labelling are present in
one or more of substantially planar zones of the device..]..[.
6. A device a claimed in claim 1, wherein said at least one
unlabelled reactant is fixed to said SPZ by means of covalent
bonds..]..[.
7. A device as claimed in claim 1, wherein said at least one
unlabelled reactant is fixed to said SPZ by means of
absorption..]..[.
8. A device as claimed in claim 1, wherein said at least one
unlabelled reactant is fixed to said SPZ by means of an interaction
of biological affinity..]..[.
9. A device as claimed in claim 1, further including a plurality of
solid phase zones (SPZs) for the detection of a plurality of
analytes, said analytes including at least one attachment point of
biological affinity each of said SPZs being adjacent one another in
said layer and each of said SPZs including said unlabelled
reactants fixed thereto, said unlabelled reactants of each SPZ
being specific for a specific analyte to be detected in each of
said SPZs..]..[.
10. A device as claimed in claim 1, wherein said layer includes a
chromotographing section in at least a portion of said
substantially planar zones, and further including a sample
application zone laminated onto at least a portion of said
chromatographing section and in adsorptive contact
therewith..]..[.
11. A device as claimed in claim 1, wherein said layer includes a
chromotographing section in at least a portion of said
substantially planar zones, and further including a reagent zone
laminated onto at least a portion of said chromotographing section
and in adsorptive contact therewith, wherein at least some of the
reagents required for the detection of the labelling are present in
said reagent zone..]..[.
12. A process for the detection or determination of a component in
a fluid wherein said component is an analyte with bioaffinity
binding properties by rehydrating or solvating reactants and
reagents by the fluid containing the analyte or by an additional
fluid, said reactants and reagents being present in a dehydrated
state in an analytical device for the detection or determination of
a component in a fluid wherein said component is an analyte with
bioaffinity binding properties, comprising a layer of a plurality
of substantially planar zones adjacent one another and in absorbent
contact with one another, said layer including:
a mobile phase application zone (MPAZ), an intermediate zone (IZ)
and an adsorption zone (AZ), liquid being capable of moving by
adsorption from said MPAZ through said IZ to said AZ, and wherein
said IZ further comprises a solid phase zone (SPZ) having at least
one unlabelled reactant, capable of interactions of biological
affinity with at least one analyte;
at least one unattached, labelled reactant (conjugate), capable of
interactions of biological affinity with said at least one analyte,
disposed in an area between the MPAZ and the SPZ; and
an analyte application zone disposed at said MPA or in between said
MPAZ and said AZ,
said process comprising:
applying a sample to said analyte application zone, reacting the at
least one analyte in the sample in said layer and detecting said at
least one analyte in said layer..]. .[.
13. The process as claimed in claim 12, wherein, after the liquid
sample containing the analyte has been fed to the MPAZ or after the
sample has been fed to a sample application zone and a mobile phase
has been fed to the MPAZ, the liquid reaches the end of the AZ,
under the control of capillary forces, and reactions between
reactants contained in the device and the analyte are thereby set
in operation, and, after the labelled reactants which are not
attached to the solid phase have been removed chromatographically,
the amount of the labelling in the solid phase zone, which is a
measure of the analyte concentration in the sample, is
determined..]..[.
14. The process as claimed in claim 11 wherein the reactions taking
place in the device are based on the principals of at least one of
immunological detection reactions, competitive immunometric or
sandwich immunoassay, indirect antibody detection by means of a
labelled antibody and antibody detection by means of a labelled
antigen..]..[.
15. The process as claimed in claim 12, wherein said detecting
includes using a fluorophor as a labelling agent which is detected
or measured directly or is detected or measured after the addition
of a reagent present in the device, or a fluorophor which is
detected or measured directly or after the addition of a further
reagent is formed from the labelling agent by the addition of a
reagent present in the device..]..[.
16. The process as in claim 12, wherein said detecting includes
using a compound which can be excited to give chemiluminescence as
a labelling agent, the chemiluminescence being detectable or
measurable after the addition of a reagent present in the
device..]..[.
17. The process as claimed in claim 12, wherein said detecting
includes using an enzyme as a labelling agent, the activity of
which is determined with the aid of a reagent present in the
device..].
18. An analytical device for the detection or determination of .[.a
component.]. .Iadd.components .Iaddend.in a fluid wherein said
.[.component is an analyte with bioaffinity binding properties.].
.Iadd.components are a plurality of analytes, said analytes
including at least one attachment point of biological
affinity.Iaddend., comprising a layer of a plurality of
substantially planar zones adjacent one another and in
.[.absorbant.]. .Iadd.absorbent .Iaddend.contact with one another,
said layer including:
a mobile phase application zone (MPAZ),
an intermediate zone (IZ), and
an adsorption zone (AZ),
.[.liquid being.]. .Iadd.wherein said fluid .Iaddend.capable of
moving by adsorption from said MPAZ through said IZ to said AZ, and
wherein said IZ further comprises .Iadd.a plurality of
.Iaddend.solid phase .[.zone (SPZ).]. .Iadd.zones (SPZs) for the
detection of a plurality of analytes, each of said SPZs being
.Iaddend.capable of having at least one unlabelled reactant fixed
thereto which is capable of interactions of bioaffinity with at
least one analyte, during analysis said at least one unlabelled
reactant being fixed to at least one second reactant which is fixed
to said solid phase zone.Iadd., each of said SPZs being adjacent
one another in said layer, said unlabelled reactants of each SPZ
being specific for a specific analyte to be detected in each of
said SPZs.Iaddend.;
at least one unattached labelled reactant .[.(conjugate).]. ,
capable of interactions of biological affinity with said at least
one analyte, disposed in an area between said MPAZ and said
.[.SPZ.]. .Iadd.SPZs.Iaddend.; and
an analyte application zone disposed at said MPAZ or in between
said MPAZ and said AZ wherein after application of said at least
one analyte, said at least one analyte is reacted with said
reactants in said layer and is detected in said layer. .[.
19. A device as claimed in claim 18, wherein said at least one
second reactant is fixed to said SPZ by means of covalent
bonds..]..[.
20. A device as claimed in claim 18, wherein said at least one
second reactant is fixed to said SPZ by means of
adsorption..]..[.
21. A device as claimed in claim 18, wherein said at least one
second reactant is fixed to said SPZ by means of an interaction of
biological affinity..]..[.
22. A device as claimed in claim 18, further including a plurality
of solid phase zones (SPZs) for the detection of a plurality of
analytes, mid analytes including at least one attachment point of
biological affinity, each of said SPZs being adjacent one another
in said layer and each of said SPZs including said unlabelled
reactants fixed thereto, said unlabelled reactants of each SPZ
being specific for a specific analyte to be detected in each of
said SPZs..]..[.
23. A device as claimed in claim 18, wherein the MPAZ has the
function of a volume metering element and releases to the
subsequent zones at least sufficient liquid for the liquid,
controlled by capillary forces, to reach the end of the
AZ..]..[.
24. A device as claimed in claim 18, wherein the MPAZ is a plastic
sponge or a particulate layer which is composed of hydrophilic
polymers and which is capable of containing chemicals, buffer
substances or other substances required for certain
tests..]..[.
25. A device as claimed in claim 18, wherein the analyte
application zone retains blood cells..]..[.
26. A device as claimed in claim 18, wherein said layer includes a
chromotographing section in at least a portion of said
substantially planar zones; and further including a sample
application zone laminated onto at least a portion of said
chromatographing section and in adsorptive contact
therewith..]..[.
27. A device as claimed in claim 18, wherein all or some of be
reagents required for the detection of the labelling are present in
one or more of the substantially planar zones of the
device..]..[.
28. A device as claimed in claim 18, wherein said layer includes a
chromotographing section in at least a portion of said
substantially planar zones, and further including a reagent zone
laminated onto at least a portion of said chromotographing section
and in adsorptive contact therewith, wherein at least some of the
reagents required for the detection of the labelling are present in
said reagent zone..]..[.
29. A process for the detection or determination of a component in
a fluid as an analyte with bioaffinity binding properties by
rehydrating or solvating reactants and reagents by the fluid
containing the analyte or by an additional fluid, said reactants
and reagents being present in a dehydrated state in an analytical
device for the detection or determination of the analyte, said
device including a layer of a plurality of substantially planar
zones adjacent one another and in absorbent contact with one
another, said layer including:
a mobile phase application zone (MPAZ), an intermediate zone (IZ)
and an adsorption zone (AZ), liquid being capable of moving by
adsorption from said MPAZ through said IZ to said AZ;
a solid phase zone (SPZ) in said IZ capable of having at least one
unlabelled reactant fixed thereto which is capable of interactions
of bioaffinity with at least one analyte, during analysis said at
least one unlabelled reactant being fixed to at least one second
reactant which is fixed to said solid phase zone;
at least one unattached labelled reactant (conjugate), capable of
interactions of biological affinity with said at least one analyte,
disposed in a zone between the MPAZ and the SPZ; and
an analyte application zone disposed at said MPAZ or in between
said MPAZ and said AZ;
said process comprising:
applying a sample to said analyte application zone, reacting be at
least one analyte in the sample in said layer and detecting said at
least one analyte in said layer..]. .[.
30. The process as claimed in claim 29, wherein, after the liquid
sample containing the analyte has been fed to the MPAZ or after the
sample has been fed to a sample application zone and a mobile phase
has been fed to the MPAZ, the liquid reaches the end of the AZ,
under the control of capillary forces, and reactions between
reactants contained in the device and the analyte are thereby set
in operation, and, after the labelled reactants which are not
attached to the solid phase have been removed chromatographically,
the amount of the labelling in the solid phase zone, which is a
measure of the analyte concentration in the sample, is
determined..]..[.
31. The process as claimed in claim 29, wherein the reactions
taking place in the device are based on the principals of at least
one of immunological detection reactions, competitive immunometric
or sandwich immunoassay, indirect antibody detection by means of a
labelled antibody and antibody detection by means of a labelled
antigen..]..[.
32. The process as claimed in claim 29, wherein said detecting
includes using a fluorophor as a labelling agent which is detected
or measured directly or is detected or measured after the addition
of a reagent present in the device, or a fluorophor which is
detected or measured directly or after the addition of a further
reagent is formed from the labelling agent by the addition of a
reagent present in the device..]..[.
33. The process as in claim 29, wherein said detecting includes
using a compound which can be excited to give chemiluminescence as
a labelling agent, the chemiluminescence being detectable or
measurable after the addition of a reagent present in the
device..]..[.
34. The process as claimed in claim 29, wherein said detecting
includes using an enzyme as a labelling agent, the activity of
which is determined with the aid of a reagent present in the
device..].
Description
The invention relates to a solid diagnostic device which comprises
several functional sectors and is used for the detection and
quantitative determination of substances or analytes in biological
fluids. The invention also relates to a process using this device
in which, after the device has come into contact with the fluid,
the analytes react with specific combination partners having
biological affinity and am detected by means of labelling
reagents.
In methods of diagnosis, the ability to identify and estimate
specific compounds has made it possible to monitor the
administration of medicaments, the quantification of
physiologically active compounds or secondary products thereof and
the diagnosis of infections. In this respect, the immunoassay
methods (RIA, .[.ELSIA.]. .Iadd.ELISA .Iaddend.and the
agglutination test) are of particular importance. The specific
combination reactions utilized in the tests are not limited to
immunological interactions, such as antigen-antibody or
hapten-antibody interactions, but also utilize interactions having
biological affinity, such as lectin-sugar or active
compound-receptor.
Although the existing tests are sensitive and specific, they do not
constitute convenient application forms, because of the long
duration of the test (in most cases several hours or even days) and
the frequent test steps, such as immune reaction, washing steps and
enzymatic reaction. The long test times are not compatible with use
in emergency methods of diagnosis.
Integrated dry chemical test elements, such as are described in the
present invention, simplify the performance of the tests and
shorten the test times.
No sheet-like test element, in which all the components of the
immune reaction of a heterogeneous immunoassay using solid phase
detection, and the functional performance and the "bound-free"
separation, are integrated has been described so far.
Whereas in the test strip assembly the immune reaction steps and
the separation of bound and free phases are operated in the
heterogeneous test by directed streams of liquid, in test element
assemblies operating by means of thin layers laminated over one
another (film technology), processes controlled by diffusion and
directed by the concentration gradient are possible driving forces.
A fluorescence labelling is used in German Offenlegungsschrift No.
3,329,728 (Japanese Patent No. P144,341/82) and EP A No. 0,097,952
(Japanese Patent No. 114,359/82). The labelling has a low molecular
weight and hence promotes processes controlled by diffusion.
However, the test has to be carried out at an elevated temperature.
In the first of these two cases both the free phase and also the
bound phase are evaluated. In film technology the absorption of
solvent is effected either by hydrating swellable components or by
filling capillary cavities. In the case Of assemblies having layers
laminated over one another only the top layer and the bottom layer
are accessible to detection without major difficulties.
After the reaction steps have taken place it is difficult to react
reagents with components in intermediately placed layers. In the
test strip assembly having zones situated one behind another, such
as is used in the present invention, in principle each zone is
readily accessible both from above and also from below, for a
determination and also for the addition of reagents which may
perhaps be required.
The invention relates to a sheet-like diagnostic device which
contains all the reagent components and which contains not only all
the components required for the functional sequence, but also the
functional sequences themselves in an integrated form, and by means
of which it is possible to detect an analyte having properties of
biological affinity, in such a way that a solution of the analyte
is brought into contact with a functional region of the device
designed for this purpose, and the analyte as detected via a
signal-producing system in a single functional region, a solid
phase zone.
A second analyte, or further analytes, as constituents of the same
solution can be detected at the same time by means of the device,
if these analytes possess properties of biological affinity
different from the hit analyte. They are also detected in the same
manner as the first analyte in a single functional region, a solid
phase zone appropriate for them. The functional regions for the
detection of the second or further analytes are situated on the
sheet-like device in front of or behind the functional region for
the detection of the first analyte. The device can also contain
several solid phase zones which are appropriate for an analyte and
different measurement ranges of this analyte. The device contains
all reactants and reagents in a dehydrated form.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a summary table illustrating test assemblies according to
certain preferred embodiments of the present invention; and
FIG. 2 is a summary table illustrating test assemblies according to
certain preferred embodiments of the present invention.
DESCRIPTION OF PREFERRED EMBODIMENTS
The sheet-like diagnostic device comprises one or several strips
arranged behind one another, of material which have a capacity for
absorbing aqueous solutions. The strips are fixed on a solid
support. They contain the reagent components required for the
particular diagnostic agent and thus become functional sectors or
functional regions. The functional sector situated at one end of
the strip-shaped device (solvent application zone) is brought into
contact with the analyte solution by being dipped into the latter
or by the application of the latter. The solution migrates through
all the functional regions. The absorptive capacity of the
supporting materials of which the strips are composed causes a flow
of liquid which stops at the other end of the strip-shaped device.
The analyte can also be applied in the middle region of the device,
and a flow of liquid from one end of the device to the other can
then be induced.
The sample does not have to be applied directly to the
chromatographing section of the device. It can also be applied to
an absorptive material which is situated on the device and has the
function of removing blood cells from the sample. After being
filtered the sample then reaches the device. In the course of this
filtration process the addition of reagents can be effected at the
same time by dissolving the latter out of components present in the
filter in a dry state. Interfering factors can be eliminated from
the solution by means of such components. Thus, for instance, the
ascorbic acid present in a sample, which interferes in the use of
oxidases and peroxidases as labelling agents, can be rendered
harmless by means of a suitable oxidizing agent. Furthermore, the
filter can also have the function of an adsorbent which removes
interfering factors from the sample by adsorption. The filtration,
adsorption and reagent admixing function for conditioning the
sample for the test can also be taken over by the mobile phase
application zone or a zone situated behind the latter.
The distribution of the solvent in the individual functional
regions depends on the adsorptive capacity and the dimensions of
the materials used.
`The solvent application zone can have be function of a volume
metering element, as described in German Patent Nos. 3,043,608 and
2,332,760, and U.S. Pat. Nos. 3,464,560, 3,600,306, 3,667,607,
3,902,847, 4,144,306 and 4,258,001. It can contain, in dry form,
the various reagents required for the function of the test element.
The solvent application zone can be a piece of fabric paper which
is located at one end of the test element and which becomes
completely saturated with a definite volume of liquid merely by
being dipped into a solution, for example a solution of the sample,
or by being briefly flushed with tap water, and then releases the
liquid to the succeeding zones more slowly and in a controlled
manner. The solvent application zone has dimensions such that it
takes up sufficient liquid to permit the latter to migrate to the
other end of the device, the end of the absorption zone.
Between the solvent application zone and the absorption zone there
are located the functional regions in which are contained reaction
components for the performance of the test and in which all the
reaction stages of the performance of the test take place. Part of
the reaction components for the performance of the test can also be
homed in be sample application zone. The absorption zone has the
function of absorbing excess and freely mobile reagent components
and reaction products of the .[.single.].
.Iadd.signal.Iaddend.-producing system.
The absorbent supporting materials in the form of one or more
strips, as constituents of the various functional regions, can,
according to choice, be composed of cellulose, of chemical
derivatives of cellulose or of plastics having a porous or fibrous
structure and adequately hydrophilic properties, or of particles
such as cellulose or silica gel embedded in a synthetic membrane,
and also of natural products which are .[.hydrophillic.].
.Iadd.hydrophilic .Iaddend.but have been rendered insoluble in
water. A combination of strips composed of different materials can
be used. Suitable absorbent materials are selected on the basis of
the requirements set for the particular diagnostic device.
Reactants with immunological binding properties such as antigens,
haptens or antibodies are incorporated in various embodiments of
the device. In the event that glycoproteins or oligosaccharides
which attach themselves to lectins are to be detected, one reactant
having biological affinity can be the specific lectin, while the
second reactant having immunological affinity can be an antibody
which is directed against a point of attachment on the analyte
other than that of the lectin. In the event that microbial active
compounds are to be detected, .[.One.]. .Iadd.one
.Iaddend.combination partner can be the receptor substance for the
active compound, while the second combination partner can be an
antibody which is directed against another point of attachment on
the active compound.
We combination partner having biological affinity becomes attached
during the progress of the reaction, or has already been attached
to the supporting material in the functional region designed for
the detection of the analyte (solid phase zone). It is also called
the solid phase combination partner. The other combination
partner(s) are present in the supporting materials. They are
provided with a labelling.
Amongst the various known possibilities of labelling, enzyme
labelling is preferred. It requires chromogenic substrate systems
or substrate systems which produce fluorescence or
chemiluminescence. Chemiluminescence labelling represents a further
example of a labelling which is only measured after the addition of
a reagent. It is possible to measure either the chemiluminescence
itself or a fluorescence excited by the latter. In most cases
fluorescence labelling is measured without the addition of a
reagent being required. However, as in the use of certain rare
earth chelates, it can also be desirable to produce the fluorophore
to be measured only as the result of adding a reagent, or to add a
second fluorophore which becomes excited by the first or which
excites the first fluorophore. The fluorescence can be measured at
one point, as a function of time or as fluorescence
polarization.
A reagent required for detection can be induced to react with the
immune complex to be detected in various ways, after the separation
stage. Part of the signal-producing system can be located in the
.[.sold.]. .Iadd.solid .Iaddend.phase zone. After the solid phase
has been adequately washed, a reagent required to detect the
labelling can be released at a retarded rate in various embodiments
in the heterogeneous immunoassay with detection in the bound phase.
The following are possible examples:
The application of reagents by means of a stream of liquid arranged
parallel to the main stream of liquid, but flowing more slowly and
starting from the mobile phase reservoir and entering in front of
the zone containing the labelled component. The parallel stream of
liquid can be controlled by using an absorbent medium which
chromatographs more slowly, for example a paper which
chromatographs suitably slowly or a paper which is impregnated in
places with "components temporarily blocking the way", such as, for
example, polymers which impart a high viscosity on passing into
solution (for example polyvinyl alcohols or dextrans).
After the solid phase has been adequately washed (=completion of
chromatography), the application of reagents can be effected by
pressing down an element which is a solid constituent of the test
element. The "pressing down" can be effected mechanically or by
removing distance pieces by the action of a stream of liquid. For
example, the mechanical pressing down of an element containing the
reagents can be effected by pressing down a flap or a piece of
paper supported by distance pieces. The lowering of an element
containing the reagents by the action of the stream of liquid can
be effected, for example, by laminating over one another the solid
phase, a water-soluble polymer and the reagent carrier (for example
a suitably impregnated piece of paper).
A retarded introduction of reagents into the liquid stream can be
effected using a microencapsulated reagent which only emerges from
the encapsulation after the solid phase has been adequately washed,
or by coating the reagent adhering in the matrix with components
which dissolve slowly.
One possible means presented for the special case of enzyme
labelling is as follows: when a peroxidase labelling is used, a
glucose oxidase zone can be placed in from of the solid phase zone.
Glucose and also the chromogen are then incorporated into the
liquid stream, which can result in color formation behind the
glucose oxidase. Appreciable color formation is only observed if,
at an appropriately high concentration of peroxidase, sufficient
H.sub.2 O.sub.2 is formed by the oxidase. This formance of the
peroxide sets in slowly, reaches an optimum concentration and
finally reaches a high concentration which results in inhibition of
the enzyme and this automatic cessation of the color formation.
This coloration can be moderated if an H.sub.2 O.sub.2 -acceptor,
for example a thioether as a mild reducing agent, or the enzyme
catalase is incorporated in the oxidase zone or in front of the
latter.
In this example a reagent for detecting the labelling is produced
by a delay circuit, making use of an enzyme. The color formation in
the solid phase zone only begins after this zone has been
adequately washed free from nonspecifically bound labelling by the
strewn of liquid.
There are several possible means of preparing the solid phase zone.
The components fixed there can be attached by chemical covalent
bonds or adsorptively to an absorptive support which is a part of
the test element. These components can also be attached to a
dispersion of particles which remain fixed at the place of
application after they have been applied to an absorbent support.
For example, suspensions of cells carrying specific receptors on
their surface, such as, for instance, Staphylococcus aureus Cowan I
cells, or latex particles carrying combination partners of
biological affinity attached to their surface, are suitable for
being fixed in a paper matrix. The components of the test strip
which are attached to pipettable supports and also the unattached
components of the device can be dried onto the absorbent matrix of
the element by air drying; freeze-drying stages are not absolutely
necessary.
A few test performance will be illustrated as examples of
embodiments which can be regarded as independent of the labelling
used. For the sake of simplicity, they are only described for the
detection of a single analyte by means of the diagnostic
device.
The following two embodiments, which conform to the principle of
competitive immunoassay, will be described for the case where the
analyte has only a single combination point of biological affinity
or only one combination point of biological affinity out of several
is utilized:
The solid phase combination partner is attached by covalent bonds
or adsorptively to the supporting material of the solid phase
functional region. The solution of analyte renders mobile a
predetermined amount of labelled analyte contained in the
diagnostic agent. The two components migrate into the functional
sector containing the solid phase combination partner and compete
for combination with the solid phase combination partner. If the
proportion of analyte is high compared with the labelled analyte,
little labelled analyte will be attached. if it is low, a great
deal of labelled analyte will be attached.
The solid phase combination partner is housed as an unattached
component in a functional region in front of the solid phase
functional region. The oncoming front of solvent transports it into
the solid phase functional region, where it becomes attached This
solid phase attachment is produced by combination systems of
biological affinity which are independent of the combination system
of the analyte. A combination partner which is conjugated with
biotin attaches itself to avidin attached to the support. An
immunoglobulin, such as IgG, as a combination partner, is fixed via
its Fc component to support-attached protein A of S. aureus, or is
attached by solid phase antibody of another species, non
idiotypically directed to said immunoglobulin.
As previously described, the analyte and the labelled analyte
compete, as constituents of the diagnostic agent for the
attachments to the solid phase combination partner during the
processing period. This competition reaction takes place partly
with the dissolved solid phase combination partner and partly with
the solid phase combination partner which has already been attached
to the solid phase.
If two combination points of differing specificity are present in
an analyte, several embodiments, conforming to the principle of
sandwich immunoassay, of the diagnostic agent are conceivable. Two
of these will also be illustrated below:
If the solid phase combination partner is attached by covalent
bonds or adsorptively to the supporting material of the solid phase
functional region, the analyte forms, with the labelled combination
partner, a binary complex which migrates together with the solvent
into the solid phase functional region and reacts there with the
solid phase combination partner, with the formation of a ternary
complex, attached to the solid phase, which can be detected via the
labelling of the first combination partner. The excess labelled
combination partner is removed by the solvent into the subsequent
functional region, the absorption zone.
If the solid phase combination partner is present in a non-attached
form in the diagnostic agent and is rendered mobile by the solvent,
the two reactants of the analyte of biological affinity are housed
in the functional regions in such a way that the analyte reacts
simultaneously or successively with both reactants and the
resulting ternary complex then migrates into the solid phase
functional region, where, as already described above, it becomes
attached to the solid phase via a second system of biological
affinity which is independent of that of the analyte.
In order to illustrate the embodiments .[.descried.].
.Iadd.described .Iaddend.above and further embodiments which
conform to the immunometric test principle, the principle of
indirect antibody detection or the ELA (enzyme-labelled-antigen)
principle of immunoassay, FIGS. 1 and 2 illustrate in an exemplary
manner the distribution of the components of the agent in the
functional regions and, after the performance of the reaction, the
composition of the solid phase complex, the amount of which is a
measure of the concentration of analytes in the sample.
It has been found that a completely integrated test strip operating
in accordance with the principle of heterogeneous immunoassay by
means of solid phase detection is not only feasible in principle,
but can, in addition, also be evaluated within a period of less
than one hour, the quantification and the sensitivity of
conventional RIAs or ELISAs being achieved. The detection of trace
components in the range of 10.sup.-12 mol/liter has been made
possible at reaction times of less than 30 minutes, at room
temperature, the amounts of sample required being 10.sup.-16 mol,
corresponding, for example, to approx. 1 pg. The arrangements
described also enable tests of lower sensitivity requirements to be
carried out, however. Standard curves over two to three decades
were obtained when evaluation was carried out with the Sanoquell
reflectometer (made by Quelle). The chromatography time for the
test element, including complete color development, is not more
than 16 minutes. Evaluation can also be carried out visually. With
HCG as analyte, the start of the range of determination in an
example using a glucose oxidase attached to a solid phase and a
peroxidase labelling was 0.3 ng/ml (corresponding to 3
U/liter).
In the example following, the application of the principle of the
competitive double antibody test is presented as a concrete
embodiment. In this test configuration, four components have to be
reacted successively for the determination reaction and the
separation stage, the reaction times and the concentrations of the
reactant being critical values. The example is not to be regarded
as limiting in any way, but merely serves to illustrate the subject
of the invention further.
EXAMPLE
Completely integrated enzyme-immunochemical device for the
detection of HCG by means of a built-in chromogen substrate
system.
1.1. Reagents
1.1.1. HCG-peroxidase conjugate
HCG having a specific activity of approx. 3000 U/mg was obtained
from Organon. Peroxidase from horseradish was obtained from
Bochringer Mannheim (catalog no. 413,470). The hetero-bifunctional
reagent N-.lambda.-maleimidobutyryloxysuccinimide (GMBS) was
obtained from Behring Diagnostics and was reacted with the HCG as
described by Tranimori et al., 1983, in J. Imm. Meth. 62, 123-131.
2-iminothiolane hydrochloride (Sigma, catalog no. I 6256) was
reacted with peroxidase as described by King et al., 1978, in
Biochemistry 17, 1499-1506. A conjugate was prepared from the
GMBS-HCG and the iminothiolane-peroxidase as described by Tanimori
et al. The crude conjugate was purified by gel chromatography over
Ultrogel ACA 44 (LKB). The fraction in which about 1-2 peroxidase
molecules were coupled per HCG molecule was used for the test. The
conjugate was diluted with Enzygnost IgE incubation medium made by
Behringwerke, order no. OS D, designated briefly as incubation
medium in the following text.
1.1.2. Antibodies
Antibodies against HCG were obtained by immunizing rabbits, and
antibodies against rabbit-IgG were obtained by immunizing goats.
The IgG fractions were isolated from serum by ammonium sulfate
precipitation and anion exchange chromatography, and were purified
further by immunadsorption. The methods used are described in the
book "Immunologische Arbeitsmethoden" (Immunological working
methods), Helmut Friemel, Editor, 1984, Gustav Fischer Verlag,
Stuttgart. The anti-HCG antibody was finally diluted in the
conjugate dilution buffer indicated above.
1.1.3. Glucose oxidase
Glucose oxidase from Aspergillus niger was obtained as a solution
containing 300 U/mg (Serva, catalog No. 22,737). The glucose
oxidase was finally diluted with incubation medium.
1.1.4. Glucose and Tetramethylbenzidine
.alpha.-D-glucose and tetramethylbenzidine hydrochloride were
obtained from Serva, catalog no. 22,720 and 35,926,
respectively.
1.2. Preparation of the Device
The sheet-like functional regions were prepared as follows:
The mobile phase application zone was prepared by cutting, to
dimensions of 20.times.6 mm, a fabric sponge cloth made by Kalle;
this is a synthetic sponge of regenerated cellulose which has been
compressed in a dry state. It was impregnated with a solution of 50
mg of glucose and 0.75 mg of tetramethylbenzidine hydrochloride per
ml of water, and was dried in a stream of air.
The conjugate, the anti-HCG antibody and glucose oxidase (5 .mu.l
of each at 25 .mu.l/ml, 100 .mu.l/ml and 0.1 mg/ml, respectively)
were applied behind one another, at uniform distance, to a
45.times.5 mm, piece of MN no. 1 paper (Macherey & Nagel), and
were dried in the air.
A piece measuring 5.times.5 mm of Schleicher & Schull No. 597
paper was coated in a covalent manner with anti-rabbit IgG-antibody
as the solid phase zone. This was effected by coupling the antibody
with the paper, which had been activated with cyanogen bromide, as
described by Clarke et al., 1979, Meth.Enzymology, volume 68,
441-442.
A 20.times.5 mm. piece of Schleicher & Schull No. 2668/8 paper
was used as the absorption zone.
The four pieces of paper, with a 0.5-1 mm overlap behind one
another, were fixed on a plastic ribbon by means of double-sided
adhesive tape (Tesaband made by Beiersdorf), so that a test strip 5
mm wide was formed.
1.3. Performance of the Test
The test was carried out in each case by applying 200 .mu.l of an
HCG dilution in incubation medium to the fabric.
1.4 Results
The chromatographic development of the test element and the
self-actuating color development were complete after 15 minutes at
room temperature, and evaluation could be carried out either
visually or by means of a reflectometer.
The following values were obtained when evaluating the solid phase
zone (No. 597 paper) with the Sanoquell blood glucose evaluation
apparatus made by Quelle:
Measured values HCG concentration (mg of glucose per dl of
(U/liter) blood) 0.3 107 3 117 30 95 300 70 3000 0
The following values were obtained with the same test strips using
the Rapimat urine test strip evaluation apparatus made by
Behringwerke:
HCG concentration Measured values (U/liter) (BIT) 0.3 76 3 76 30 94
300 119 3000 39
The test strip assembly shown here can also be achieved if the
glucose oxidase and the anti-HCG antibody are located in the same
zone. The test strip, which is correspondingly shorter, then
renders the result after approx. 10 minutes.
* * * * *