U.S. patent application number 10/633653 was filed with the patent office on 2004-02-05 for ligand binding assay and kit with a separation zone for disturbing analytes.
This patent application is currently assigned to Pharmacia & Upjohn Diagnostics AB.. Invention is credited to Carlsson, Jan, Lonnberg, Maria.
Application Number | 20040023412 10/633653 |
Document ID | / |
Family ID | 20411187 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040023412 |
Kind Code |
A1 |
Carlsson, Jan ; et
al. |
February 5, 2004 |
Ligand binding assay and kit with a separation zone for disturbing
analytes
Abstract
The invention relates to method for determining an analyte by
means of binding reactions, which method comprises: i) applying the
sample to an application zone for sample (ASZ) on a flow matrix in
which transport of components present in the sample can take place
(transport flow), the flow matrix further exhibiting: a) optionally
an application zone (AR*Z) for a binding reactant (Reactant*=R*)
which is analytically detectable; b) a detection zone (DZ), which
is located downstream of ASZ and exhibits an additional binding
reactant (Capturer) firmly anchored to the matrix, and in which a
complex (signal complex) containing the Capturer and the analyte
and/or Reactant* is formed during the reaction, and ii) detecting
the signal complex in the detection zone, the measured signal being
used for determining the analyte. According to the invention, the
flow matrix comprises at least one separation zone (SZ) between ASZ
and DZ, which zone exhibits a structure (ligand) having binding
capability for a component that is transported in the matrix and
which would affect the measurable signal if the component is
transported into DZ. The invention also relates to a test kit
comprising the flow matrix.
Inventors: |
Carlsson, Jan; (Uppsala,
SE) ; Lonnberg, Maria; (Knivsta, SE) |
Correspondence
Address: |
Holly D. Kozlowski
Dinsmore & Shohl LLP
255 E. 5th Street
1900 Chemed Center
Cincinnati
OH
45202
US
|
Assignee: |
Pharmacia & Upjohn Diagnostics
AB.
|
Family ID: |
20411187 |
Appl. No.: |
10/633653 |
Filed: |
August 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10633653 |
Aug 5, 2003 |
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09673882 |
Jan 5, 2001 |
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09673882 |
Jan 5, 2001 |
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PCT/SE99/00722 |
Apr 30, 1999 |
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Current U.S.
Class: |
436/514 |
Current CPC
Class: |
G01N 33/558 20130101;
Y10S 436/81 20130101; G01N 33/54386 20130101; Y10S 436/825
20130101; Y10S 435/97 20130101 |
Class at
Publication: |
436/514 |
International
Class: |
G01N 033/558 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 1998 |
SE |
9801563-9 |
Claims
1. A test kit for determining the binding capability of a structure
(ligand) to an analyte, which kit comprises A. a flow matrix which
in one and the same transport flow comprises: a) an application
zone for the analyte (ASZ), b) a detection zone (DZ) in which there
is a biospecific affinity reactant (Capturer), which is directed
towards an analyte or towards an analyte-related reactant and which
is firmly anchored to the matrix in DZ, B. optionally an
analytically detectable reactant (Reactant*=R*) having biospecific
affinity to either the analyte or the Capturer, characterized in
that the flow matrix comprises a separation zone (SZ) between ASZ
and DZ, which zone exhibits said structure (ligand).
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The invention relates to a method for determining an analyte
in a sample and to a kit for use in the method.
[0002] Starting from the prior art, the method of the invention
comprises the steps:
[0003] i. The sample is applied in a sample application zone (ASZ)
on a flow matrix in which transport of components present in the
sample may take place (transport flow). The flow matrix further
comprises:
[0004] a) optionally an application zone (AR*Z) for a binding
reactant (Reactant*=R*) which is analytically detectable,
[0005] b) a detection zone (DZ) which is located downstream of ASZ
and exhibits another binding reactant (Capturer) firmly anchored to
the matrix and in which a complex (signal complex) containing the
Capturer and the analyte and/or the Reactant* is formed in the
method.
[0006] ii. The flow is allowed to effect the transport of sample
components.
[0007] iii. The signal complex is detected in the detection zone
and the measured signal is used for the determination of the
analyte.
[0008] The invention is primarily directed to the flow matrix which
may be of the same type as those previously used in, for example,
immunochromatography, see below.
[0009] Suitable binding reactants are those which participate in
so-called affinity reactions, especially biospecific affinity
reactions, and covalent binding reactions, especially exchange
reactions between free thiol and reactive disulphide and other
reactions between soft electrophiles and soft nucleophiles. Common
biospecific affinity reactions are immunochemical, i.e. between
antibody and antigen or hapten. Other types of bioaffine reactions
are hybridization between complementary nucleic acids (including
oligonucleotides), reaction between lectin and carbohydrate
structure, between Ig(Fc)-structure and Ig(Fc)-binding protein,
such as protein A or protein G, etc. The bioaffine reactions
include the reaction between a biomolecule and a synthetically
prepared ligand/capturer.
[0010] For the type of method in question, one talks about
non-competitive methods, for example sandwich technique, and
competitive methods. Sandwich technique usually means that an
analytically detectable complex is formed in which the analyte
binds to two bioaffine counterparts, one of which is analytically
detectable and the other is Capturer. In common competitive
variants, the analyte and an analytically detectable analyte
analogue will compete for a limiting amount of bioaffine
counterpart. As examples of two competitive variants may be
mentioned those that use: a) competition between analyte and
analyte analogue, which is labelled, for a limiting amount of
ligand in the form of a firmly anchored Capturer, and b)
competition between analyte and analyte analogue in the form of
firmly anchored Capturer for a limiting amount of soluble and
analytically detectable bioaffine counterpart.
[0011] For further information on previously used methodology
within the technical field of the invention it is referred to U.S.
Pat. No. 4,861,711 (Behringwerke), WO 88/08534 (Unilever), U.S.
Pat. Nos. 5,120,643 and 4,740,468 (Abbott), EP-A-284,232 and U.S.
Pat. No. 4,855,940 (Becton Dickinson) and WO 96/22532 (Pharmacia
AB).
HETEROFORMS
[0012] Compounds which can compete for the binding to a counterpart
via one of the above mentioned binding reactions. Heteroforms may
be isoforms of proteins, e.g. isoenzymes etc. Within the term
heteroforms are included inter alia different forms of bioaffine
complexes which "resemble" each other by meeting the above
definition. Examples are immunocomplexes where the antigen is the
same but the antibody is of different class/subclass. See further
under the title "Analyte" below.
[0013] Determination of whether two compounds are heteroforms to
each other may be made in so-called inhibition tests.
PROBLEMS TO BE SOLVED BY THE INVENTION
[0014] The components of a sample that may affect or influence the
signal that is to be detected in DZ can be divided into two main
groups: a) the analyte and b) components which directly or
indirectly disturb the detection. Directly disturbing components
are those which interfere with the signal as such, for example
fluorescent components in serum in case the complex is to be
detected by fluorescence. Examples of indirectly disturbing
components are heteroforms with regard to Capturer and/or an added
bioaffine reactant R (for example R*). Other indirectly disturbing
components, for example heterophilic antibodies, may be present in
the original sample and interfere With the formation of the signal
complex in DZ. In certain embodiments of the invention, ligands
that are released from the separation zone of the invention may act
disturbingly (see Example 1).
[0015] Problems with disturbing components in samples have often
meant that for analytes that are present in low concentrations, the
separation of disturbing components and the detection have been
performed in different systems.
[0016] An example where after ion-exchange separation, analysis has
been carried out either by immunological systems or by on-line
measurement of an absorbing group (460 nm), is in the measurement
of carbohydrate deficient transferrins (CDT=CD-transferrin=asialo-,
monosialo- and disialo-transferrin). When CDT is present at a
relatively high concentration (10.sup.-9 M), both detection
alternatives have been possible but at lower concentrations of
analyte, immunological measurement is required. The ion-exchange
chromatography separation is controlled from an advanced and costly
equipment, which requires specially educated personnel. Also the
traditional immunological tests are expensive and require
well-educated personnel.
[0017] The technique for immunological on-line measurement after a
chromatographic separation step has been described by Afeyan et al.
(Nature 358 (1992) 603-604) and Irth et al. (Anal. Chem. 14 (1995)
355-361). Its difficulties have been summarized by Krull et al.
(LC-GC 15(7) (1997) 620-629).
[0018] Transport of whole cells into DZ may interfere with the
signal from the detection complex. It is previously known to use
flow matrices where the cells are captured mechanically (through
filtration) in a denser pre-zone (Oudheusden et al., Ann. Clin.
Biochem. 28 (1991) 55-59).
[0019] EP-A-696,735 discloses a chromatographic immunoanalytical
system where, in order to extend the measuring range for the
analyte, a predetermined amount of analyte-binding antibody has
been immobilized in the sample application zone so that a certain
amount of analyte is retained therein.
[0020] EP-A-702,233 discloses a chromatographic immunoanalytical
system where, in a similar manner to that described in
EP-A-696,735, a dilution effect of the sample is achieved by
capturing a certain amount of analyte before it reacts with
labelled reactant which is then detected in the detection zone.
[0021] WO 97/35205 discloses a chromatographic membrane for
immunoanalysis having (i) a zone for the detection of labelled
analyte-binding reactant which has not bound to the analyte, and
(ii) a zone for the detection of the complex between
analyte-binding reactant and the analyte. The relative amounts of
unbound analyte-binding reactant and analyte: reactant complex
gives a measure of the amount of analyte in the sample.
[0022] WO 94/06012 discloses an analytical test apparatus having a
negative control zone placed before the analyte detection zone. The
negative control zone has the function to indicate the presence in
the sample of components that affect the analyte detection so that
it becomes unreliable.
OBJECTS OF THE INVENTION
[0023] A first main object of the invention is to create a simple
and rapid method that facilitates the determination of an analyte
in the presence of disturbing components. A particular object is to
avoid problems with disturbing components that are soluble or
suspendable in liquid media of interest.
[0024] A second main object of the invention is more rapid and
simpler determinations of individual heteroforms or combinations
thereof, especially heteroforms, that exhibit peptide, carbohydrate
or lipid structures, including various types of biologically active
compounds. Among lipids are included steroids and other fat-soluble
substances.
[0025] A third main object of the invention is to facilitate the
measurement of analytes in the concentration range <10.sup.-7 M,
particularly <10.sup.-9 M, especially for samples containing
disturbing heteroforms of the analyte.
[0026] A fourth main object of the invention is to simplify the
determination of individual heteroforms or combinations thereof in
samples originating from biological materials.
[0027] A fifth main object of the invention is to provide more
rapid and simpler evaluations of libraries of compounds, for
example chemical libraries, such as combinatorial libraries.
[0028] A subobject of the above mentioned four main objects is to
improve the possibilities of making determinations in field
environment (usually semi-quantitatively) as well as in advanced
laboratories (with the possibility of accurate quantification).
THE INVENTION
[0029] The above mentioned objects may be achieved with the method
mentioned in the introductory part herein, if the flow matrix
contains one or more separation zones (SZ) between ASZ and DZ,
which should permit at least one component, capable of influencing
the signal from the signal complex in DZ, to be retarded/separated.
This should take place in SZ by means of the ligand interactions
mentioned below, which can be reversible or irreversible. The
component may be either a disturbing component or the analyte. If
the component is not an analyte, the retardation means that the
component (or components) migrates more slowly than the analyte
through SZ or is bound irreversibly to SZ and thereby is prevented
from reaching DZ such that the detection of analyte in DZ
essentially will not be disturbed by the component (or components)
in question. Usually, this means that there should be a sufficient
amount of ligand for substantially all of the disturbing component
or components in the sample to be affected. "Substantially all"
depends on the relative concentrations of the component(s), but
usually means that at least 90%, preferably at least about 95%, and
more preferably at least 99% of the disturbing component(s) are
retarded or captured in the separation zone. The component may be
the analyte if it is desired to study the capability of one or more
ligands to bind the analyte. In this case such a ligand is
immobilized in the separation zone.
[0030] The choice of retarding structure/ligand in the separation
zone is determined by the components that are retarded. The
retardation may be based on various more or less specific
interactions between the ligand structure and the component(s) to
be retarded;
[0031] see below under the title "Separation zone". After the
passage of SZ, the analyte will migrate with the transport flow to
the detection zone (DZ), in which a complex containing the Capturer
and the analyte and/or R* are formed.
[0032] In those cases where it is intended to retard one or more
disturbing components, the formation of signal complexes will take
place in the absence thereof. The detection of signal complexes in
DZ may be taken as a qualitative or quantitative measure of the
analyte.
[0033] In those cases where it is intended to retard the analyte,
the point of time for the formation of a signal complex will be
changed, or, if the analyte-ligand binding in SZ is irreversible,
the formation of a signal complex may be completely inhibited. The
formation of a signal complex in DZ will be a measure of the
capability of the analyte to bind to the ligand in SZ.
[0034] FIGS. 1-3 illustrate different variants of flow matrices
according to the invention.
[0035] FIG. 1 is a simple variant having an ASZ, an ARZ, a SZ and a
DZ. ARZ and ASZ are separated.
[0036] FIG. 2A differs from the variant in FIG. 1 primarily by
having five separation zones With the same ligand. ARZ and ASZ are
separated.
[0037] FIG. 2B is the same as the variant in FIG. 2A except that
ARZ and ASZ coincide.
[0038] FIG. 3 illustrates the variant of flow matrix of the
invention that is used in Example 1 with three separation zones,
two zones (SZ1) thereof exhibiting a certain ligand and one zone
(SZ2) exhibiting another ligand. ASZ and ARZ (=AR*Z) are
separated.
[0039] A more detailed description of FIG. 1 is given under the
title "Matrix and transport flow", and of FIGS. 2-3 in the
introduction to Example 1. The flow matrices represented by FIGS.
1-3 may in principle have any of the geometric embodiments
below.
Matrix and Transport Flow
[0040] The matrix is of the same type as those previously used in
so-called immunochromatographic determination methods (flow matrix)
and defines the room in which reactants and sample components are
transported. The matrix may thus be the internal surface of a
single flow channel (for example a capillary), the internal surface
of a porous matrix having a penetrating system of flow channels
(porous matrix) etc. The matrix may be in the form of monolith,
sheet, column, membrane, separate flow channel(s), for example of
capillary dimensions, or aggregated systems of such flow channels
etc. They may also be in the form of particles packed in column
cartridges or in cut grooves, compressed fibres etc. Another
alternative is so-called nanocolumns for liquid chromatography,
i.e. silicon or quartz plates having channels of about 2 .mu.m or
less prepared by microlithography (see e.g. He. B. et al., Anal.
Chem. 1998, 70, 3790-3797). The inner surface of the matrix, i.e.
the surface of the flow channels, should be sufficiently
hydrophilic to pen-nit aqueous media (primarily water) to be
transported through the matrix, either by means of capillary force
or by means of applied pressure or suction. The smallest inner
dimension of the flow channels (for round channels measured as a
diameter) should be sufficiently great to permit transport through
the matrix of analyte, added reactants, and components that
interfere in the detection zone and that are to be retarded in SZ.
The rule of thumb is that suitable matrices may be selected among
those with flow channels having a smallest inner dimension in the
range of 0.1-1000 .mu.m, with preference for 0.4-100 .mu.m if the
matrix has a system of communicating flow channels. Flow channels
having their smallest dimension in the upper part of the broad
range (up to 1000 .mu.m) are primarily of interest for flows driven
by externally applied pressure/suction.
[0041] Suitable matrices are often built up from a polymer, for
example nitrocellulose, polyester, polyethersulphone, nylon,
cellulose nitrate/acetate, cellulose, regenerated cellulose.
Advantageously, these membranes may be provided with a tight
backside of e.g. polyester.
[0042] The material of the matrix as well as the physical and
geometric design of the flow channels may vary along the~flow
depending on the intended use of a certain part of the matrix [WO
96/22532 (Pharmacia AB); WO 94/15215 (Medix)]. One and the same
matrix may comprise several transport flows that are parallel or
directed radially from a common centre, for example in the form of
separate channels. In some of the most important embodiments, at
least the detection zone and the most adjacent parts of the matrix
should be in such a form that the transport flow into, in and out
of DZ may take place laterally in the matrix, i.e. at least this
part of the matrix is in the form of a membrane strip or plate
having cut grooves or the like.
[0043] Various flow matrices that may be used in the type of tests
in question are described in prior patent publications. See e.g.
U.S. Pat. No. 4,861,711 (Behringwerke), WO 88/08534 (Unilever),
U.S. Pat. No. 5,120,643 and U.S. Pat. No. 4,740,468 (Abbott),
EP-A-284,232 and U.S. Pat. No. 4,855,240 (Becton Dickinson); WO
96/22532 (Pharmacia AB).
[0044] The most important embodiment of the invention at the
priority date is based on liquid transport in a flow matrix which
is in the form of e.g. a membrane strip (see FIG. 1). The strip is
made up of a matrix that defines a transport flow (1) and is
applied to a liquid-tight backing (2), suitably of plastic. On the
matrix there is an application zone for sample (3, ASZ) and a
detection zone (4, DZ) located downstream thereof. The transport
flow is in the direction from ASZ towards DZ. Between the sample
application zone (ASZ) and the detection zone there is a separation
zone (5, SZ). In the transport flow there may, if required by the
particular embodiment, also be application zones (6) for additional
reactants (R, for example R*, with application zone ARZ, for
example AR*Z). Between said zones there may be zones (7) the only
function of which is to transport reactants. The position of an
application zone ARZ (AR*Z) is determined by the test protocol to
be used, and may be upstream or downstream of or coincide with ASZ.
For the case that ARZ (for example AR*Z) is upstream of ASZ, it may
be advantageous if the addition of liquid in ASZ takes place
substantially simultaneously as the addition of liquid in the zone
ARZ (AR*Z) located upstream thereof. See our earlier filed
international patent application PCT/SE98/02463 (incorporated by
reference herein). For certain types of test protocols, ARZ (AR*Z)
may coincide with DZ.
[0045] In some embodiments it is advantageous if a reactant R. for
example R*, is pre-deposited. This is especially the case if ARZ is
located downstream of ASZ and the test protocol variant used is
simultaneous, i.e. the reactant R and the analyte are to migrate
into DZ substantially simultaneously.
[0046] In the cases where it is desired to use variants that are
sequential in the sense that the analyte is to be transported into
DZ before the reactant (R), R should be added after the sample has
passed ARZ if the application zone for reactant (ARZ) is downstream
of ASZ. Sequential methods may also be achieved if ARZ is upstream
of ASZ, in which case R optionally may be pre-deposited in ARZ.
[0047] In alternative embodiments, reactants (R), for example R*,
may migrate into DZ in separate transport flows from another
direction than that of the flow that transports the analyte into
DZ. See, for example, U.S. Pat. No. 4,855,240 (Becton &
Dickinson).
[0048] In one and the same transport flow there may be several
detection zones intended for different analytes or different
concentration ranges of the same analyte. For the case that the
analytes are different, the Capturers in the respective DZ must, of
course, not exhibit any substantial cross-reactivity against any of
the analytes.
[0049] The transport flow from ASZ through the separation zone (SZ)
and further to the detection zone (DZ) may be a liquid flow driven
by capillary force. When necessary, the flow matrix may exhibit a
liquid reservoir (8) in the form of a porous matrix that is soaked
with transport liquid and applied upstream of ASZ and/or a sucking
porous matrix (9) placed downstream of DZ. The liquid reservoir and
the sucking matrix assist in maintaining the flow. Liquid flow may
also be achieved by means of pressure or suction through the
matrix. Thus, the pressure may be driven hydrostatically, for
example by a part of the matrix being designed as a minicolumn
placed vertically and with its outlet in direct liquid
communication with a horizontally located flow matrix. In the
latter form, the horizontally located part of the matrix may be in
the form of a strip/membrane. An alternative for transport of
analyte, reactants and disturbing components may be the application
of an electric field across the matrix.
[0050] Similar sequences of zones, like that in FIG. 1, may also be
constructed for other types of flow matrices, for example capillary
tubes and matrices in which the transport flow may be in depth.
[0051] One or more matrices/transport flows according to the above
may be placed together, for example on a common backing, optionally
with a liquid, barrier between them. Optionally, the flows may have
a common ASZ, a common ARZ (AR*Z) etc. As a rule, DZ is separate
for each transport flow.
[0052] In the above mentioned variants, matrices having a
separation zone may be used to determine one heteroform (analyte).
A matrix without separation zone may be used to determine all
heteroforms of the analyte that may be present in the sample in an
analogous manner to that for the analyte. By combining these two
types of zone sequences, relative as well as absolute quantities of
analyte in the sample may easily be measured.
[0053] Separation Zone (SZ)
[0054] The separation zone exhibits a ligand/structure having
binding capability for one or more sample components that would
have disturbed the detection in DZ. A characteristic feature is
that the separation is achieved by means of some type of
specific/selective binding reaction and not because the matrix in
SZ provides a mechanical obstacle for disturbing components
(filtration). Guiding principles for the choice of
separating/retarding ligand/structure, especially with regard to
specificity, binding strength (affinity), and kinetics are the same
as in affinity chromatography, including ion-exchange
chromatography, covalent chromatography, and biospecific analytical
methods in which solid-phase technology is used for capture. With
regard to binding strength (affinity, avidity) and kinetics, the
main object of the presently preferred variants of the invention is
to retard disturbing components in relation to the analyte so that
detection in DZ may take place without presence of these
components. Generally, this means that the disturbing components
should be retarded as effectively as possible or be bound as
strongly and quickly as possible in the separation zone.
[0055] The ligands that make separation in SZ possible may thus be
a) charged (anionic, cationic, amphoteric=ion-exchange ligands),
amphoteric/amphiphilic, bioaffine. chelating, sulphur-containing
(primarily thioether for so-called thiophilic affinity), those
permitting covalent chromatography (reactive disulphide such as
pyridyl disulphide) or .pi.-.pi. interaction, hydrophobic etc.
[0056] In those cases where disturbing components are to be
retarded, the rule of thumb is that the binding capability of the
ligand to one or more disturbing components should be stronger than
that to the analyte. This applies to the conditions used for the
separation in SZ. Factors that determine how the separation will
succeed are the length of the separation zone, ligand density,
ligand availability, temperature, flow velocity, buffer,
ion-strength, pH, etc.
[0057] Among biospecific affinity ligands, primarily so-called
immunoligands are noted, i.e. antibodies and antigen-binding
fragments thereof, and antigen and hapten. Other examples of
affinity ligands are lectin (for example, sialic acid-binding
lectins); Ig(Fc)-binding protein (such as Protein A and G); nucleic
acid, such as oligo- or polynucleotide in single or double-stranded
form, analogues of substrates for enzymes, enzyme inhibitors, etc.
For biospecific affinity ligands, the specificity may be directed
towards one or more binding sites on the component(s) to be
retarded. The corresponding binding sites should not be available
to the same degree on the analyte (by which is also intended the
case that they do not even exist in non-exposed form).
[0058] The ligands/structures in question may be anchored to the
separation zone, either by covalent binding to the matrix, via
physical or biospecific adsorption. Examples of the latter is the
interaction between biotin and streptavidin, between highly affine
antibody and hapten etc. The anchorage to the matrix may take place
via a polymer or other substituent which in turn carries
covalently, physically adsorptively, or biospecifically bound
ligands that are used in the separation. Another possibility is
deposition of polymeric particles which exhibit a desired type of
ligand. The particles may be of hydrophilic or hydrophobic
character and to which a compound exhibiting the ligand structure
has been adsorbed or covalently bound. The technique for binding a
separating ligand to the matrix SZ may basically be selected in the
same way as previously known for the Capturer in DZ. See, for
example, our earlier filed international patent applications
PCT/SE98/02462, PCT/SE98/02463 and PCT/SE98/02464 which are hereby
incorporated by reference with regard to the introduction of
Capturer into the detection zone. In this connection it may be
mentioned that there are commercially available membranes which
have covalently bound ligands, for example DEAE cellulose paper
(diethyl aminoethyl) (DE81, Whatman International Ltd,
England).
Detection Zone
[0059] The Capturer in the detection zone may be selected according
to the same rules as those applying to the ligand in the separation
zone, with the proviso that the binding capability of the Capturer
should be directed towards the analyte and/or towards an
analyte-related reactant. It is advantageous to choose highly
affine Capturers with rapid kinetics for capture of the ligand. It
is primarily of interest to use antibodies or antigen/hapten for
which it is often easy to find highly affine antibodies.
[0060] By analyte-related reactant is intended a reactant (R) that
is added and when migrating through DZ may bind to the Capturer in
an amount that is related to the presence of analyte in the sample.
Examples of analyte-related reactants are R* in the form of a)
labelled analyte analogue in competitive methods that use
competition for a limiting amount of solid-phase-bound anti-analyte
antibody, and b) labelled or non-labelled soluble anti-analyte
antibody in methods that use competition/inhibition between
solid-phase-bound analyte analogue and analyte for a limiting
amount of anti-analyte antibody in dissolved form.
[0061] The Capturer may be anchored to the detection zone by a
technique analogous to that used to bind the ligand to the
separation zone.
[0062] It may be suitable to combine a separation principle in the
separation zone with a different capturing principle in the
detection zone, e.g. ion-exchange chromatography for separation and
immunochemical adsorption for capture in DZ. In some situations it
may be practical to use the same principle for retardation and
capture in the two zones (e.g. two monoclonal antibodies having
different specificities, see the Examples).
Analyte
[0063] By analyte is intended the compound or compounds that are
determined quantitatively or qualitatively. Quantitative
determination relates to the measurement of quantities in absolute
as well as relative terms. Qualitative determination of an analyte
refers to detecting the existence or non-existence of something
(yes/no test) or qualitative properties of a compound, such as
capability of affinity-binding to a certain ligand.
[0064] By relative measurement is intended that the measurement
value obtained is a ratio of the sum of one or more selected
heteroforms and the sum of another combination of heteroforms. An
example is the ratio of analyte amount and total amount of all
heteroforms with regard to a certain counterpart (total amount
includes the amount of analyte).
[0065] The invention is applicable to analytes that may function as
a binding reactant. This means that the analyte basically can be
any substance for which it is possible to provide a Capturer as
above. As specific examples may be mentioned antigen/hapten, enzyme
or antibody or nucleic acid which completely or partly are in
single-stranded form. The analyte may exhibit amino acid/peptide,
carbohydrate or lipid structure.
[0066] Particularly great advantages are obtained for analytes
existing together with heteroforms with regard to binding
capability to Capturer and/or an added reactant R, for example R*.
This applies particularly to the cases where the analyte is in
sample concentrations which are <10.sup.-7 M, especially
<10.sup.-9 M. As examples of this type of heteroforms may be
mentioned: a) Compounds which differ from each other in charge,
such as isotransferrins with, for example, CDT as analyte,
isohemoglobins with, for example, HbAlc as analyte; b) Compounds
which differ from each other in certain parts of the basic
structure, such as additionally inserted or cleaved (e.g. by
degradation) amino acids, or partial differences in peptide chains;
c) Compounds which differ from each other due to the fact that
different substances/structures have been added to a basic
structure, for example covalently bonded carbohydrate structures;
d) macromolecules consisting of two or more subunits which in the
macromolecule bind to each other via non-covalent bonds, such as
bioaffine bonds between receptor and ligand in receptor-ligand
complexes and between antigen and antibody in immunocomplexes, or
via cystine bridges, for example between the chains of an
antibody.
[0067] Examples of potential uses/analytes are:
[0068] a) The analyte is a heteroform which differs from other
heteroforms with regard to carbohydrate contents (glycosylation),
for example glycoproteins having the same or a similar protein
part. Variations in this type of heteroforms are known in a number
of disease conditions such as cancer, inflammation and liver
diseases. (Turner G A, "N-glycosylation of serum proteins in
disease and its investigation using lectins", Clin. Chim. Acta 208
(1992) 149-171; and Varki A, "Biological roles of oligosaccharides:
all of the theories correct", Glycobiology 3(2) (1993) 97-130).
Particularly may be mentioned the measurement of i) combinations of
asialo-, monosialo- and disialo-transferrin for which separation
may be performed by ion-exchange ligand and also by lectin ligand
in SZ, and ii) HbA1c which may be separated by means of
ion-exchange or boronate ligand. Variations in the carbohydrate
contents of proteins are also known in normal biological changes,
for example during the menstrual cycle and for differences in age
and sex.
[0069] b) The degree of glycosylation of recombinant proteins could
be determined by means of ion-exchange, lectin or boronate ligands
in SZ. The analyte will in this case be the fraction of a
recombinant protein that does not contain a carbohydrate structure
that binds to the ligand in SZ and therefore migrates most rapidly
through SZ.
[0070] c) Recombinant proteins into which a separation handle has
been inserted, for example a histidine sequence or an IgG-binding
sequence, and where total cleavage of the handle is important,
could be checked after separation in SZ by means of a metal chelate
ligand or and IgG(Fc)-ligand, respectively. The analyte will in
this case be the fraction of the recombinant protein from which the
histidine sequence or the Ig(Fc)-binding sequence, respectively has
been cleaved off.
[0071] d) Enzymes could be separated into an active and an inactive
form by means of a ligand in SZ which is a substrate analogue or an
inhibitor of the enzyme in question. The analyte will be the
inactive enzyme.
[0072] e) Proteins, peptides or other biomolecules which exert
their biological function by binding to a specific receptor could
be separated by means of a ligand in SZ which is a receptor for the
biomolecule The analyte will be the fraction of the molecules that
lack or have a reduced capability of binding to the receptor.
[0073] f) Proteins (e.g. IgE) may in vivo have autoantibodies (IgG,
IgA, IgM) bound thereto. These autoantibodies give rise to, on the
one hand, a differing response in immunochemical determination of
the protein, and, on the other hand, an altered turnover
rate/function. By using antibodies to the autoantibodies in
question as ligand in the separation zone, autoantibodies in free
and immunocomplex-bound form may be separated and the amount of the
free form of the protein (=analyte, e.g. of IgE) may be
calculated.
[0074] g) By means of a monoclonal antibody directed against a
certain binding site of a protein and immobilized to SZ, the
presence of heteroforms to the protein which do not exhibit the
binding site (=analyte) could detected by quantification in DZ.
[0075] h) The presence of different substances bound to transport
proteins, e.g. a drug bound to albumin, could be measured by using
suitable ligands in SZ. By the choice of a suitable ligand in SZ,
transport proteins with or without bound drug may be measured in
DZ.
[0076] i) IgG and IgA in serum may in certain rheumatic or
autoimmune diseases have an increased adsorption to different
surfaces. By anchoring ligands in the separation zone which are
capable of binding to IgG and IgA with changed properties, it will
be possible to measure the proportion of IgG and IgA with unchanged
adsorption properties (=analyte) in DZ. By having the corresponding
autoantigen/hapten as Capturer in DZ, specific autoantibodies of
IgG or IgA class could be measured with better sensitivity.
[0077] j) Many biologically active compounds (for example, peptides
or steroids) are transported in serum in the form of complexes with
binder proteins. By using antibodies against the binder protein as
ligand in SZ, the non-complex-bound (free) form of these compounds
(=analyte) could be determined inmmunochemically in the following
detection zone. Examples are triiodothyronine and thyroxine which
are transported bound to thyroxine-binding globulin (TBG) or
thyroxine-binding prealbumin (TBPA). Analogously, free forms of
estradiol and testosterone which are transported in bound form with
sexual hormone-binding globulin may be measured.
[0078] k) The binding capability of a first compound (=analyte) for
a second compound may be determined with the invention. In this
embodiment, one may have the second compound as ligand in SZ, and a
Capturer with a known binding capability to the analyte in DZ.
Capture/retardation in SZ will be a measure of the binding
capability of the analyte and may be measured in DZ.
[0079] This embodiment of the invention may be particularly
advantageous in the screening of different libraries of compounds
with the library members as ligands in SZ (chemical libraries, for
example).
[0080] l) Degradation isoforms of proteins where amino acids have
been cleaved off, can be determined by the invention. For example,
degradation isoforms of creatine kinase (CK) are interesting
cardiac markers.
Detection in DZ and Labelled Reactant (R*)
[0081] Detection and quantification of signal complexes may be
performed by means of an analytically detectable reactant
(Reactant*=R*). For those cases where the analyte per se is
detectable and is part of a signal complex, detection and
quantification may take place without using R*.
[0082] R* is usually a biospecific affinity reactant which is
labelled with an analytically detectable group, such as an
enzymatically active group, radioactive group, fluorescent group,
chromogenic group, hapten, biotin, particles, etc. Analytically
detectable reactants (R*) also include reactants which per se have
binding sites or properties which may be detected analytically when
the reactant is part of the signal complex. Examples of such
binding sites are Ig-class- and Ig-subclass-specific determinants
when the reactant is an antibody and the antigen-binding part
thereof is used to form the complex in the detection zone.
[0083] Usual forms of analytically labelled reactant are labelled
antibody and labelled antigen/hapten. Labelled antibody has its
primary use in
[0084] A) non-competitive techniques, such as sandwich technique,
in which the capturer is
[0085] a) an antibody which is directed against the same antigen
(=analyte) as the labelled antibody, or
[0086] b) an antigen/hapten, or
[0087] B) competitive techniques in which competition takes place
between an analyte and a solid phase-bound analyte analogue for a
limiting amount of anti-analyte antibody and the detection of free
or occupied sites on the solid phase may be performed by means of
labelled anti-analyte antibody and anti-anti-analyte antibody,
respectively.
[0088] Labelled antigen/hapten has its primary use in
[0089] A) competitive techniques in which a labelled antigen/hapten
is allowed to compete with an unlabelled antigen/hapten for a
limiting amount of antibody (Capturer), or
[0090] B) sandwich-techniques in which antigen/hapten-specific
antibody is determined with anti-antibody as Capturer.
[0091] Examples of variants of the invention in which an
analytically detectable reactant (R*) is not utilized are those
where the analyte per se is detectable when it is part of the
complex in DZ. This is illustrated with enzyme as analyte in
combination with a substrate that gives an analytically detectable
product, for example a substrate that gives a coloured or
fluorescent product that should be insoluble.
[0092] R* may, but need not, exhibit binding capability to the
disturbing components that are separated in SZ. To the extent that
R* has binding capability, the application zone thereof should be
located downstream of the separation zone (SZ), unless it is
desired to measure the level of disturbing heteroforms by means of
the amount of R* binding to SZ.
[0093] A particularly useful labelling group is particles which
optionally contain one of the above mentioned detectable groups,
such as fluorophoric group or chromogenic group (fluorescent and
coloured particles, respectively). Useful particles often have a
size in the range of 0.001 to 5 .mu.m, with preference for the
range of 0.05 to 5 .mu.m. The particles may be of colloidal
dimensions, so-called sol (i.e. usually spherical and monodisperse
having a size in the range of 0.001 to 1 .mu.m). Especially may be
mentioned metal particles (for example, gold sol), non-metal
particles (for example, SiO.sub.2, carbon, latex and killed
erythrocytes and bacteria). Also particles of non-colloidal
dimensions have been used. These particles have been more or less
irregular and more or less polydisperse (for example, carbon
particles<1 .mu.m; Pharmacia AB, WO 96/22532).
[0094] When particles are the label group in the invention, the
complex in DZ may often be detected visually or by optical
measuring equipment (e.g. a CCD camera coupled to a computer with
special software for image analysis or a laser scanner).
[0095] For particles as the label group, it is referred to WO
88/08534 (Unilever); U.S. Pat. No. 5,120.643 (Abbott); EP-A-284,232
(Becton Dickinson) and others.
Samples
[0096] The invention is primarily intended for biological samples,
for example, blood (serum, plasma, whole blood), saliva, tear
fluid, urine, cerebrospinal fluid, sweat, etc. The invention is
also applicable to other samples, such as fermentation solutions,
reaction mixtures, solutions containing a certain protein for which
the binding capability to a ligand in SZ is to be investigated,
etc. See above under the title "Analytes". It may be particularly
interesting to use the invention for analysis of environmental
samples.
[0097] In addition to the method, the invention also relates to an
apparatus and a kit, respectively, containing the above defined
flow matrix.
[0098] The inventions disclosed in the above-mentioned
international applications PCT/SE98/02462, PCT/SE98/02463 and
PCT/SE98/02464 may in relevant parts constitute preferred
embodiments of the present invention. All three applications have
been incorporated by reference.
PATENT EXAMPLES
Example 1
Test Strip For Measurement of the Proportion of Free IgE, IgE Bound
to IgG and Antibodies to IgE
[0099] In FIGS. 2A, 2B and 3 the direction of the transport flow is
indicated by an arrow (10). In each variant there may at the
beginning of the transport flow be a zone ASZ (11) for sample,
downstream thereof a zone DZ (12), at the end of the transport flow
a sucking part (13), and between each type of zone, parts which
only serve as transport zones (14).
[0100] FIG. 2A: The variant according to this figure has five
separation zones (SZ) in which the ligand may be the same or
different or be present in different amounts (15-19) and an AR*Z
(20) for reagents.
[0101] FIG. 2B: This is the same sequence of zones as in FIG. 2A
except that ASZ (11) and AR*Z (20) coincide (21). This zone
sequence may also be used for the cases where the analyte per se is
detectable when it is part of a signal complex in DZ. An AR*Z is
then not necessary.
[0102] FIG. 3: The sequence of zones according to this figure
exhibits two types of separation zones SZ1 (22, 23) and SZ2 (24),
respectively, and separately AR*Z (25) downstream of SZ1 (23) and
SZ2 (24).
[0103] Background: Free IgE and IgE complex-bound to autoantibody
(IgA, IgG and IgM) may be of interest to measure. Above all,
however, free IgE should be quantified correctly. In the current
tests for measurement of I-E, the autoantibodies may bind to the
same epitopes on IgE as the reagent antibodies (anti-IgE antibody)
and this may then give rise to falsely too low total IgE levels
that vary depending on the design of the test. By separating IgG,
IgM and IgA before the measurement of IgE, free IgE may be
detected. The amount of autoantibodies should also be quantified
both as complexes and as free IgG antibodies directed against
IgE.
[0104] The most common tests measure free antibodies by methods
which use IgE bound to a solid phase (corresponding to DZ) with
which a heavily diluted serum sample is allowed to interact. If the
serum sample contains anti-IgE antibody, the latter is bound to the
solid phase forming an immunocomplex. After unbound serum
components have been washed away, anti-IgG antibody that is
labelled (R*), e.g. with enzyme, is added. Excess of labelled
antibody (R*) is removed and the amount of enzyme-labelled anti-IgG
antibody (R*) bound to the immobilized immunocomplex is determined
by the addition of a suitable substrate. The sensitivity of these
tests is limited by the unspecific binding of IgG to the solid
phase. The IgE-specific part of the IgG population is generally
very small and may be difficult to distinguish from the amount of
unspecifically bound IgG. By capturing IgG to the solid phase and
measuring the binding of IgE, this limitation may be avoided.
[0105] When measuring IgG-complex bound IgE, IgG is captured to a
solid phase s (corresponding to DZ) which supports covalently bound
anti-IgG antibody (Capturer). By adding labelled anti-IgE antibody
(R*), the amount of complex-bound IgE may be measured.
[0106] The use of an immunoassay technique based on lateral liquid
transport in membranes as described above where the flow first
passes through one or more separation zones (SZ) and then a
detection zone (DZ), opens many possibilities for simple
measurement of IgE-IgG related parameters. If e.g. a sample that
contains a mixture of free IgE and IgE bound to a human anti-IgE
antibody of IgG class first is made to pass through a zone
containing solid phase-bound anti-human IgG antibody (Ligand in SZ)
and then a zone containing solid phase-bound anti-IgE antibody
(Capturer in DZ), the sample content of complex between IgE and
anti-IgE antibody of IgG class will be bound in the separation zone
while free IgE passes to the detection zone where it is determined
by adding labelled anti-IgE antibody (R*) upstream of the detection
zone (12) but downstream of the separation zones (15-19) for
passage only through the detection zone (addition in zone 20 in
FIG. 2A). By having anti-IgE antibody (R*) pass also the separation
zone, the amount of IgE-IgG complex captured in the separation zone
by binding to anti-human IgG (Ligand) may also be determined (ASZ
and AR*Z coincide) (addition in zone 21 in FIG. 2B, ASZ common with
AR*Z). In the separation zone there are, in addition to complex
between IgE and anti-IgE antibody of IgG class, also free
antibodies against IgE. The amount of the latter may be determined
by having labelled IgE (R*.sub.1) pass through the separation zone.
Labelled IgE (R*.sub.1) is then added in a separate test to the
membrane strip upstream of SZ. See FIG. 2B.
[0107] When the amount of IgG is very high in serum, several bands
with high concentrations of anti-IgG must be used as SZ. Both
complex-bound and free anti-IgE antibodies will then be distributed
over several bands due to the total amount of IgG, and the sum of
the signal intensities of these bands gives the amount of
antibodies against IgE.
[0108] In the example below, the test principle of artificially
prepared complexes of IgE and IgG is demonstrated. The complexes
have been prepared with monoclonal antibodies against IgE, and
antibodies against mouse-IgG have therefore been bound to the
separation membrane. In the detection system, antibodies to IgE
directed against other epitopes than the complex-forming antibody
have been used. This makes it possible to measure the complex
equally well as free IgE in the detection system.
[0109] Separation membrane 1 (SZ1): Sheep anti-mouse IgG(Fc)
(Ligand 1) was coupled to polystyrene aldehyde particles (0.29
.mu.m diameter, IDC, Portland, Oreg., U.S.A.) by mixing 1.0 mg/ml
of antibodies and 20 mg/ml of polystyrene aldehyde particles in 25
mM phosphate buffer, pH 6.6, at +4.degree. C. for 20 hours. The
particles were washed in 20 mM borate buffer, pH 8.6, and were
reacted with 15 mg of NaCNBH.sub.3 (Sigma-Aldrich Chemie,
Steinheim, Germany) per 50 mg of particles for 20 hours. The
particles were then washed in 20 mM borate buffer, pH 8.6, by
repeated suspension, centrifugation and decanting. The particle
suspension was diluted in 3% trehalose, 20 mM borate buffer, to 25
mg of particles/ml. The diluted suspension was sprayed on strips
(20 cm.times.3 cm) of membranes of nitrocellulose (nitrocellulose
on polyester, 5 .mu.m pore size, Whatman International Ltd,
England) in two 0.3 cm wide lines which were parallel to the long
sides of the strips. The spraying equipment (IVEK linear striper,
IVEK Corporation, Vermont, U.S.A.) delivered about 50 .mu.g of
polystyrene particles/cm for each line. The membranes were dried at
room temperature and then cut to smaller pieces (0.5 cm.times.3
cm).
[0110] Separation membrane 2 (SZ2): Mouse IgG (Ligand 2) was
diluted in 20 mM borate buffer to 3.4 mg of protein/ml. The diluted
antibody was sprayed on strips (20 cm.times.4 cm) of membranes of
nitrocellulose (the same type as above) in a 0.3 cm wide line
(spraying equipment as above) with about 6.8 .mu.g of
antibodies/cm. The membranes were dried at room temperature and
then cut to smaller pieces (0.5 cm.times.1 cm).
[0111] Detection membrane (DZ): Mouse anti-IgE monoclonal antibody
(directed against domain 4 on IgE, Capturer) was diluted in 20 mM
borate buffer to 1.0 mg of protein/ml.
[0112] The diluted antibody was sprayed on strips (20 cm.times.4
cm) of membranes of nitrocellulose (the same type as above) in a
0.15 wide line (spraying equipment as above) with about 1 .mu.g of
antibodies/cm. The membranes were dried at room temperature and
then cut to smaller pieces (0.5 cm.times.4 cm) so that the line
with antibody was parallel with a short side.
[0113] Combination membrane: See FIG. 3. A piece of separation
membrane 1 (0.5 cm.times.3 cm, SZ1, 22 and 23, respectively, in
FIG. 3) were mounted to apiece of separation membrane 2 (0.5
cm.times.1 cm. SZ2, 24 in FIG. 3) and the thus obtained combined
separation membrane was in turn joined to a strip of the detection
membrane (0.5 cm.times.4 cm, the line =DZ=12 in FIG. 3) (short side
to short side with a gap between them).
[0114] The pieces were kept together on the bottom side by adhesive
tape. On the top side were placed pieces of nitrocellulose (0.5
cm.times.0.3 cm) (A100, 12 .mu.m, Schleicher and Schull, Dassel,
Germany) which somewhat overlapped two adjacent short sides. The
latter pieces were kept in place by more adhesive tape. A cellulose
filter (13 in FIG. 3) (0.5 cm.times.2 cm; GB 004, Schleicher and
Schull, Dassel, Germany) overlapping the free short side of the
detection membrane was mounted as a sucking membrane. The sequence
of zones was ASZ, SZ1, SZ2, DZ.
Preparation of Carbon Particle Conjugate (R*):
[0115] Carbon suspension (stock solution): 2 g of carbon particles
(sp 100, Degussa, Germany) were suspended in 200 ml of 5 mM borate
buffer, pH 8.4, and sonicated (VibraCell 600 W, 1.5 cm probe,
Soniced Materials, Danebury, Conn., U.S.A.) in an ice-bath for
3.times.5 minutes at 100% amplitude and with 9.9+2 seconds
pulse.
[0116] Carbon particle conjugate (R*): 35 .mu.g/ml of Fab'2 of
anti-IgE monoclonal antibody (directed against domain 3 in IgE) and
a suspension of carbon particles (250 .mu.g /ml) were mixed for 3
hours. Bovine serum albumin (BSA) was added to 1% and the particles
were mixed for another 30 minutes and then washed by means of
centrifugation in 1% BSA (0.1 M borate buffer, pH 8.5, 0.05%
NaN.sub.3) and diluted to 0.8 mg carbon/ml in the wash buffer. The
ready carbon particle conjugate was stored at +4.degree. C. in the
wash buffer.
Sample Material
[0117] Preparation of complex between IgE and IgG: 1 mg of IgE
(ND)/ml and 5 mg/ml of mouse anti-IgE monoclonal antibody (of IgG
class and directed against domain 2) were reacted in 50 mM
phosphate buffer, pH 7.5, for 2.75 hours at room temperature. The
sample mixture (0.35 ml) was separated on Superdex.TM. 200 prep
grade, 16/60 (Amersham Pharmacia Biotech AB, Sweden). The
separation gave two discernible complex peaks, one peak
corresponded to IgE-IgG and one peak corresponded to
IgG-IgE-IgG.
Control with .sup.125I-Labelled Proteins (Labelled anti-IgE
Antibody and Labelled IgE
[0118] Separation membrane 1 (Ligand=anti-mouse IgG): Mouse anti
IgE antibody (against domain 2 of IgE) and IgE were labelled with
.sup.125I (Chloramine T) to a labelling degree of 0.03 for anti-IgE
antibody and 1.5 for IgE. The labelled proteins were diluted in 6%
BSA (50 mM phosphate buffer, pH 7.5): anti-IgE antibody to about
2.4 .mu.g/ml and IgE to 0.06 .mu.g/ml. .sup.125I anti-IgE antibody
(domain 2) was mixed with unlabelled anti-IgE antibody (against
domain 2) for measuring higher levels of anti-IgE antibody. A
sucking membrane (0.5 cm.times.2 cm, GB004, Schleicher and Schuell,
Dassel, Germany) was attached with tape to one end of a piece of
separation membrane 1 (0.5 cm.times.4 cm) with adsorbed sheep
anti-mouse IgG(Fc). 10 .mu.l of 0.1 M borate buffer, pH 8.5 (6%
BSA, 0.05% NaN.sub.3), followed by 10 .mu.l of a solution of
.sup.125I-protein were applied to the free end of the separation
membrane. The lateral flow was then initiated by the addition of
4.times.10 .mu.l of 0.1 M borate buffer, pH 8.5 (1% BSA, 0.05%
NaN.sub.3) to the free end. After all liquid had migrated into the
membrane, it was cut to pieces for measurement of the radioactivity
in the different zones of the sheet (separation and transport
zones). The measurement was made in a gamma counter, and the
proportion of .sup.125I-protein (labelled anti-IgE antibody and
labelled IgE, respectively) that had been captured in the different
zones was calculated after correction for the amount of free
radioactive iodine. IgE did not bind any more to the separation
zones in which anti-IgG antibody was the ligand than to the
intermediate transport zones. More than 85% IgE passed through the
membrane. On the other hand, all labelled anti-IgE antibody was
bound to the two separation zones when up to 120 ng of anti-IgE
antibody were added. When 1000 ng of anti-IgE antibody were added,
200 ng were bound in each anti-mouse IgG zone (separation zone) and
500 ng passed. For IgG in human serum this capacity may be
sufficient if the serum is diluted {fraction (1/100)} (about 1000
ng of IgG) and more anti-IgG antibody (against human IgG) is used
as firmly anchored ligand.
[0119] Separation membrane 2 (Ligand=mouse IgG): This membrane was
introduced to bind any anti-mouse IgG antibody that may have been
released from the separation membrane 1 and which otherwise would
be bound to the detection zone resulting in an increased background
signal (anti-mouse IgG antibody has two Fab parts and may therefore
simultaneously bind to R* and Capturer which both are mouse-IgG).
The amount of sheep anti-IgG that was released could advantageously
be bound with a separation zone containing mouse IgG before the
detection zone. By means of this capturing zone (SZ2) the
non-specific binding in the detection zone could be reduced by more
than 6 times.
Standard Protocol for Combined Separation and Immunochemical
Determination
[0120] 20 .mu.l of wash buffer (1% BSA, 0.9% NaCl, 1% Tween 20, 0.1
M borate buffer, pH 8.4, 0.05% NaN.sub.3) were applied to the edge
of the free end (ASZ=11 in FIG. 3) of the separation membrane 1 on
a combination strip according to the above (Sequence SZ1, SZ2, DZ).
Then 10 .mu.l of IgE standard (IgE, 4-500 kU/l, 0.01-1.2 .mu.g/ml)
and sample (IgE-IgG complex with about 1 .mu.g complex/ml and
IgC-IgE-IgG complex with about 1.3 .mu.g complex/ml), respectively,
were added. Both sample and standard were diluted in 50 mM
phosphate buffer, pH 7.5, containing 6% BSA and 0.05% NaN.sub.3. A
lateral flow was initiated by placing a 0.6 cm.times.0.6
cm.times.0.3 cm cellulose sponge containing wash buffer, 0.1 M
borate buffer, pH 8.4 (1% BSA, 0.9% NaCl, 1% Tween 20, 0.05%
NaN.sub.3) on the free end of the separation part of the strip. The
test solution migrated through the separation zones (22, 23, 24 in
FIG. 3) and the detection zone (12 in FIG. 3) and into the sucking
cellulose sponge (13 in FIG. 3). After 7 minutes flow, 10 .mu.l of
conjugate (R*) of carbon particles and anti-IgE antibody (0.8 mg
carbon/ml in 0.1 M borate buffer, pH 8.4 (1% BSA, 0.05% NaN.sub.3)
were added in the position between the detection zone and the
separation part (25) of the strip. After another 5 minutes flow,
the detection zone was coloured grey to black. The blackening was
read in a laser scanner (Ultroscan, Amersham Pharmacia Biotech AB,
Uppsala. Sweden), the peak intensity was calculated and the
concentration determined by reading against the IgE standard curve.
The higher the IgE concentration, the blacker the signal.
[0121] As a comparison, strips having the separation zone 1
replaced by nitrocellulose without ligand (both standard and
sample) were evaluated in the same way.
Results
[0122] The standards (IgE) gave the same intensity on the
blackening curve in both measuring systems. The complexes (IgE-IgG
and IgG-IgE-IgG) were detected by a strong black signal in DZ if SZ
1 was replaced by nitrocellulose without ligand. If SZ1 contained
anti-mouse IgG as ligand, no signal could be detected in DZ for the
complexes.
1TABLE 1 Sample Separation zone (SZ1) Immune complex Without ligand
Ligand = anti-mouse IgG IgE-IgG complex 131 kU/l <4 kU/l
IgG-IgE-IgG complex 141 kU/l <4 kU/l The separation zone with
anti-mouse IgG thus captured up to more than 97% of the
complexes.
Example 2
Determination Method for CD-Transferrin in Patient Samples
[0123] Separation membrane having anion-exchanging properties: A
sheet of nitrocellulose membrane (5 .mu.m, nitrocellulose on
polyester, Whatman International Ltd, England) was placed in a
solution of 0.1% polyethylene imine (PEI, Sigma, St Louis, Mo.,
U.S.A.) in ultrapure water (Milli Q, Millipore Corp., Bedford.
Mass., U.S.A.). The solution was shaken for 3 hours and then placed
in 0.1% in Tween 20 for 30 minutes, air-dried and then stored in a
plastic bag at +4.degree. C. The modification degree of the
membrane was checked with bromophenol blue (pK=4.1).
[0124] The function of modified membranes to interact with charged
proteins was confirmed by transporting .sup.125I-labelled proteins
(bovine serum albumin, tetrasialo- and asialo-transferrin which had
been labelled by the Chloramine T method) in a lateral liquid flow
in strips of the sheet. The protein having the highest pI had the
strongest tendency to migrate with the liquid flow. If the liquid
in different tests contained an increasing concentration of NaCl
(0-1000 mM), the migration rate was affected most for the proteins
having the lowest pI. Both these function controls support the fact
that positively charged groups had been introduced in the treatment
with polyethylene imine, and that these groups can function as
ion-exchanging groups towards protein and NaCl.
[0125] Detection membrane: Anti-transferrin monoclonal antibody was
coupled to polystyrene-aldehyde particles (0.29 .mu.m diameter,
IDC, Portland, Oreg., U.S.A.) by mixing 1.3 mg/ml antibody and 22
mg/ml polystyrene-aldehyde particles in 25 mM phosphate buffer, pH
6.6, at +4.degree. C. for 18 hours. The particles were washed in 20
mM borate buffer, pH 8.4, and were reacted with 5 mg of
NaCNBH.sub.3 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) per 40
mg of particles per ml for 18 hours. The particles were washed in
20 mM borate buffer, pH 8.6, and diluted in 20 MM borate buffer
containing 6% trehalose to 14 mg particles/ml. The diluted
suspension was sprayed on strips (20 cm.times.4 cm) of membranes of
nitrocellulose (5 .mu.m, nitrocellulose on polyester backing,
Whatman International Ltd, England) in a 1.4 mm wide line in the
middle of the strip and in parallel with the long side of the
strip. The spraying equipment was the same as in Example I and now
delivered 14 .mu.g of polystyrene particles/cm. The membranes were
dried at room temperature and stored in a plastic bag at +4.degree.
C.
[0126] Combination membrane: See FIG. 1. The end of a strip of the
separation membrane (0.5 cm.times.3 cm) (=SZ=5 in FIG. 1) was
mounted by means of tape on the underside to the end of a strip of
the detection membrane that had been shortened by 0.5 cm (0.5
cm.times.3.5 cm the line with antibody=DZ=4 in FIG. 1). The gap
between the ends was bridged with an overlap by a piece of
nitrocellulose membrane (0.3 cm.times.0.5 cm, A100, 12 .mu.m,
Schleicher and Schuell, Dassel, Germany) which was kept down by
tape. As sucking membrane (9 in FIG. 1), a cellulose filter (0.5
cm.times.2 cm, GB 004, Schleicher and Schuell, Dassel, Germany) was
mounted by tape so that it overlapped the free end of the strip
derived from the detection membrane.
Carbon Particle Conjugate (R*)
[0127] Carbon suspension (stock solution): 2 g of carbon particles
(sp 4, Degussa, Germany) were suspended in 100 ml of 5 mM borate
buffer, pH 8.4, and sonicated with the same apparatus as in Example
1 in an ice-bath for 5 minutes at 100% amplitude and 5+5 seconds
pulse.
[0128] Carbon-particle conjugate: 100 .mu.g/ml of anti-transferrin
monoclonal antibody and carbon suspension (250 .mu.g/ml) were mixed
for 2 hours. BSA was added to 1% and the particles were mixed for
another 30 minutes and then washed by means of centrifugation in
0.1 M borate buffer, pH 8.5 (containing 1% BSA and 0.05% NaN.sub.3)
and diluted to 1.9 mg carbon/ml with wash buffer. The ready carbon
particle conjugate was stored at +4.degree. C. in wash buffer.
Sample Materials
[0129] Tetrasialo-transferrin: Tetrasialo-transferrin was isolated
from an iron-saturated preparation of human transferrin (mainly
tetrasialo-transferrin) by ion-exchange chromatography on Mono Q
(Amersham Pharmacia Biotech AB, Uppsala, Sweden).
[0130] Asialo-transferrin: An iron-saturated preparation of
transferrin (Sigma, St Louis, Mo., USA) was treated with
neuramidase (Behringwerke, Marburg, Germany), whereupon
asialo-transferrin was isolated by ion-exchange chromatography on
Mono Q (Amersham Pharmacia Biotech AB, Uppsala, Sweden).
[0131] Isoelectric points (pI): These values were determined for
the respective isoform preparation and for BSA by isoelectric
focusing in Phast System (Amersham Pharmacia Biotech AB, Uppsala,
Sweden). Asialo-form pI=5.7, tetrasialo-form pI=5.3 and bovine
serum albumin pI=4.7.
[0132] Transferrin standard: Asialo-transferrin prepared as above
was diluted in 20 mM BIS-TRIS pH 6.3 containing 0.2% BSA, 0.1%
Tween 20, 0.1 mM Fe.sup.3+-citrate, 1 mM NaHCO.sub.3 and 0.05%
NaN.sub.3 to the concentrations 0.07-16.6 .mu.g transferrin/ml and
was used as standard.
[0133] Serum samples: 11 serum samples and 6 serum controls were
diluted {fraction (1/50)} in 20 mM BIS-TRIS pH 6.3 containing 0.1%
bovine gammaglobulin (Sigma, St Louis, U.S.A.), 0.1% Tween 20, 0.1
mM Fe.sup.3+-citrate, 1 mM NaHCO.sub.3, and 0.05% NaN.sub.3. The
serum samples were previously analysed with regard to CDT by means
of CDTect (Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden).
CDTect measures CD-transferrin.
[0134] Standard protocol for combined separation and immunochemical
determination: 2 .mu.l of sample (dilution series of transferrin
and diluted serum samples, respectively) were applied at 1 cm from
the edge (ASZ=3 in FIG. 1) of the free end of the membrane part
with separation zone on a combination strip according to the above.
A lateral liquid flow was initiated by placing a 0.6 cm.times.0.6
cm.times.0.3 cm cellulose sponge (8 in FIG. 1) soaked with 20 mM
BIS-TRIS buffer, pH 6.5, containing 15 mM NaCl and 0.1% Tween 20 on
the free end of the separation zone. In the separation zone (5 in
FIG. 1) the analyte (CD-transferrin) and its heteroforms (other
transferrins) are attracted by positive charges firmly anchored in
the zone (Ligand introduced in the PEI treatment) so that a
heteroform having a greater negative charge (other transferrins) is
attracted more than a heteroform having a smaller negative charge
(CD-transferrin), i.e. CD-transferrins migrate easier with the
liquid flow than trisialo-, tetrasialo-, pentasialo- etc
transferrin. During its migration through the combination
strip/matrix, a certain proportion of the total amount of
transferrin will therefore be able to bind to the anti-transferrin
antibody (Capturer) in the detection zone (DZ=4 in FIG. 1). After 4
minutes flow, 5 .mu.l of conjugate (R*) between carbon particles
and anti-transferrin antibody (1.8 mg carbon/ml in 0.1 M borate
buffer, pH 8.4, containing 30% trehalose, 1% Tween 20, 1% BSA,
0.05% NaN.sub.3) were added between the separation zone and the
detection zone (in zone (6) in FIG. 1 (=AR*Z)). After another 5
minutes, the flow was stopped and the blackening in the detection
zone was read with a laser scanner (Ultroscan, Amersham Pharmacia
Biotech AB, Uppsala, Sweden) and the concentration was calculated
by reading against measurement values for the dilution series of
asialo-transferrin. The higher the level of CD-transferrin is in
the sample, the stronger is the blackening signal.
2TABLE 2 Results Invention CDTect arbitrary Sample U/L units/L 1 5
0.09 2 11 0.24 3 13 0.22 4 17 0.30 5 18 0.49 6 22 0.44 7 22 0.57 8
26 0.55 9 26 0.64 10 38 0.87 11 40 1.24 12 40 1.51 13 58 1.71 14 78
1.60 15 86 2.18 16 90 2.85 17 110 3.36
[0135] The measurement values obtained with the method of the
invention showed very good conformity with those obtained with
CDTect (correlation coefficient 0.971). The invention is
considerably faster and simpler to perform than CDTect.
Example 3
Test Strip with Sambucos Nigra Lectin in the Separation Zone
[0136] Separation membrane: A sheet (4 cm.times.12 cm) of cellulose
(cellulose filter 54, Whatman International Ltd, England) was
activated with cyano-diethyl-aminopyridine (CDAP) (Kohn and
Wilchek, Appl. Biochem. Biotechnol. 9 (1984) 285-304). The
activated sheet was placed in a solution of 0.1 mg/ml of Sambucus
Nigra lectin (binds sialic acid which is in the terminal position
of a carbon chain; Vector Laboratories Inc., Burlingame. Calif.,
U.S.A.) in 0.1 M NaHCO.sub.3, pH 8.4. The solution was shaken for 2
hours, and the sheet was then placed in a) 0.1 M NaHCO.sub.3, b)
0.5 M NaCl, c) distilled water, d) 0.1 M acetate buffer, pH 4.5, e)
0.1 M NaHCO.sub.3, pH 8.4, f) 0.5 M NaCl, g) distilled water, h)
0.1 M acetate buffer, pH 4.5, i) 5 mM BIS-TRIS, pH 6.4, containing
0.1% Tween 20. Between the different baths, excess liquid was
sucked offby means of kitchen roll paper. After the wash procedure,
the sheet was air-dried and stored in a plastic bag at +4.degree.
C.
[0137] Before the sheet was used, the sheet was mounted to
self-adhering plastic (75 .mu.m self-adhering polyester film;
Gelman Science Inc, Ann Arbor, Mich., U.S.A.).
[0138] Membranes with detection zone and combination strip: These
membranes can be produced in analogy with Example 2. See also FIG.
1. The ligand in SZ is now lectin.
[0139] Carbon-particle conjugate (R*) and .sup.125I-labelled
proteins. See Example 2.
[0140] Control of separation membrane by means of
.sup.125I-labelled proteins: Tetrasialo- and asialo-transferrin and
bovine albumin were labelled with .sup.125I (Chloramine T,
labelling degree 0.08-0.13). The labelled proteins were diluted in
10 mM BIS-TRIS pH 6.4 containing 0.1% Tween 20, 0.04 mM
Fe.sup.3+-citrate and 0.05% NaN.sub.3 to about 0.3 .mu.g/ml.
Additionally, 0.4 mg BSA/ml was added.
[0141] A (0.5 cm.times.4 cm) strip of the separation membrane and a
piece of a sucking membrane of cellulose (0.5 cm.times.2 cm, GB004,
Schleicher and Schuell, Dassel, Germany) were joined by tape on the
underside so that their ends overlapped somewhat. 1 .mu.l of the
solutions of the .sup.125I-labelled proteins were applied at 1 cm
from the free end of a respective separation membrane. The lateral
flow was initiated by placing a cellulose sponge (0.6 cm.times.0.6
cm.times.0.3 cm) on the free end of the separation membrane. The
sponge was soaked with 20 mM TRIS-HCL buffer, pH 7.5, containing
0.5 M NaCl, 1 mM CaCl.sub.2 with 0.1% Tween 20. The flow was
interrupted by removing the cellulose sponge after 2, 4, 6 and 10
minutes, respectively, and the membranes were cut 2 and 3 cm from
the free end of the separation membrane. The radioactive membrane
pieces were measured in a gamma counter and the proportion of added
.sup.125I-protein that had passed 2 and 3 cm was calculated. The
values for migration of 1 cm or more is shown in Table 4.
3TABLE 4 % of totally added .sup.125I-protein that had migrated
more than 1 cm in the separation membrane: Asialo- Tetrasialo-
transferrin transferrin BSA PI 5.7 5.3 4.7 % of total % of total %
of total Migration time min 2 min 54 10 86 4 min 74 10 87 6 min 78
11 91 10 min 91 11 92
[0142] Conclusion: It appears from the results that
tetrasialo-transferrin is heavily retarded in the separation
membrane by the Sambucus Nigra lectin, while asialo-transferrin and
BSA are not retarded to the same extent. The results indicate that
a separation membrane with Sambucus Nigra lectin may be combined
with a detection membrane in analogy with Example 2 and be used for
quantifying CD-transferrin in samples containing transferrin with a
greater content of sialic acid than CD-transferrin.
* * * * *