NZ725664B2 - Device and method for detecting blood group antigens by means of an incomplete antibody - Google Patents

Abstract

The present invention provides a device for determining a cell-bound analyte in a liquid sample, comprising a separating matrix having at least one indicator zone, wherein the indicator zone comprises a first antibody, or a fragment thereof, directed against the cell-bound analyte, and a binding element directed against the first antibody, the first antibody being an incomplete antibody; and wherein the separating matrix enables the liquid sample to flow through the indicator zone without spinning or centrifugation. ment directed against the first antibody, the first antibody being an incomplete antibody; and wherein the separating matrix enables the liquid sample to flow through the indicator zone without spinning or centrifugation.

Description

Device and method for detecting blood group antigens by means of an incomplete antibody Field of the invention The invention relates to devices and methods for ining blood group antigens by means of an incomplete antibody, in particular for simultaneously determining blood group antigens.

Prior art In blood group serological diagnostics, ters which are important especially in connection with transfusions or haemolytic e of the n are generally detected.

This includes inter alia the ion of antigens on the surface of erythrocytes which are characteristic for the blood groups. Further important antigen systems are also found on ocytes, granulocytes and lymphocytes, which likewise play a role in usion and/or transplantation.

It is known that, in order to determine the blood group antigens, the erythrocytes of the person to be tested (donor or recipient) are brought together with reagents which contain blood-group-specific antibodies. The tests are usually liquid tests, in which a test batch is produced by mixing an erythrocyte-containing sample with a sample containing antibodies directed against a specific blood group feature. The test batch is then incubated for a defined period of time and under defined conditions and, when the incubation is te or directly after a centrifugation step, the batch is checked either visually or by optical methods for possible agglutination or adsorption of the erythrocytes. The prevailing end-point measurement in blood group serology continues to be haemagglutination. Direct agglutinating antibodies are also referred to as complete antibodies in blood group serology.

Antibodies which cannot agglutinate erythrocytes directly are ously referred to in blood group serology as incomplete antibodies.

The simultaneous determination of blood group antigens using a l flow test format is known from . The examples of disclose the determination of blood group antigens by means of IgM antibodies, which are complete antibodies and lead directly to haemagglutination. The WO specification does not, however, disclose the determination of blood group antigens using an incomplete antibody with the aid of a lateral flow test.

Lateral flow tests are widely used nowadays as rapid tests, for example as pregnancy tests, for determining infection markers or as a drug . A lateral flow test arrangement consists of a solid carrier to which there is applied an work zone for the sample to be tested, a separating membrane on which binding elements, for example capture antibodies or antigens, are bound and on which g reactions can be detected, and an absorbent absorption region which allows the sample to be tested to flow through the separating membrane.

Test membranes of conventional lateral flow tests are generally described having chromatography-like separation. The e in the sample binds specifically to the binding ts fixed in a membrane, which are generally arranged as indicator zones in bands located one behind the other or one above the other. The binding complex is made visible by indicator particles, which are generally already present in the arrangement in dried form in a conjugate release pad. The conjugate e pad is typically disposed between the work zone and the membrane. The pre-coated coloured indicator les are coated, for example, with an antibody directed against the analyte being sought.

The most important blood group features which nowadays must routinely be clarified in pretransfusion tests on the patient and on donors are: A, B, D, C, E, c, e, Cw, K, k, Jka, Jkb, Fya, Fyb, M, N, S, s, P1, Lea, Leb, Kpa, Kpb, Lua, Lub. The ns or antigen epitopes to be tested are, by way of example, those of the ABO blood group system, of the Rh, Kell, Lewis-Hh, Duffy-Kidd, MNS, Lutheran and P system, of the blood group systems Diego, Yt, Scianna, ck, Colton, Chido/Rodgers, Gerbich, Cromer, Knops, Landsteiner-Wiener, Xg, Kx, Indian, Ok, Raph, John Milton Hagen, Langereis, Sid, FORS, JR and/or LAN, in particular A1, A2, AB, B, D, C, c, E, e, Cw, K, k, M, N, S, s, Jka, Jkb, Fya, Fyb, Kpa, Kpb, Jsa, Jsb, Lea, Leb, Lua, Lub, P1, I, H, Xga, U, Vw, Wra, Lan, Vel, Dia and/or Mia.

Because of their ve net surface charge and the zeta potential thereby exerted, erythrocytes have a natural statistical m distance of approximately 300 angstroms between the cells. That minimum distance can be bridged by antibodies of the IgM class in physiological medium because of the le size, but naturally not by antibodies of the IgG class. This means that, as a rule, only IgM-class antibodies are available in blood group serology for a direct end-point measurement by haemagglutination. Direct agglutinating antibodies are also referred to as complete antibodies in blood group serology (most IgM antibodies are complete antibodies).

According to the current prior art, blood groups generally cannot be detected by means of IgG antibodies by direct haemagglutination. Antibodies which cannot agglutinate erythrocytes directly are referred to analogously in blood group serology as incomplete antibodies (most IgG dies are incomplete antibodies).

This leads to the situation that it is necessary to work with so-called different phases and reaction times and temperatures according to whether (monoclonal) IgMs or, on the other hand, monoclonal IgGs or polyclonal antibodies are available for detection for a particular blood group property, which makes ised or homologised working procedures more difficult.

Provided that IgM antibodies are available, direct determination with haemagglutination as the end point is frequently possible without the admixture of further dies or intensifiers or proteolytic enzymes and t incubation iate spin). With the widely used gel technique, incubation is not necessary for the performance of such a test; the reaction mixture comprising the erythrocytes to be tested and the antibody reagent simply has to be pipetted into the work zones of the gel card and centrifuged for 9-10 s in a neutral physiological medium, that is to say a physiological medium which does not contain antibodies (for example DG Gel Neutral Card from Diagnostic Grifols). In another variant of the same technique, blood-group-specific antibodies of the IgM class have y been introduced into the gel matrix. The erythrocytes to be tested then simply have to be pipetted into the work zones of the gel card (for example DG Gel ABO RH (2D) from stic Grifols).

If the antibody available for determining a ular blood group feature does not belong to the IgM class, a technique/phase change is required in order to make haemagglutination possible as the end point. This is the case, for example, for the following of the abovementioned features, for which no commercially available IgM antibodies are available according to the t prior art: k, Fya, Kpa, Kpb and Lua. The following further features are likewise of interest, such as Dia, Jsa, Jsb, Coa, Cob, Wra, Xga. Commercial monoclonal IgM dies are not available for ing any of these antigens.

Since IgG antibodies are generally not capable of overcoming the distance that is present between two erythrocytes due to natural repulsion, on with an antigen-specific IgG antibody can achieve only isation (that is to say antibody binding, but not haemagglutination, therefore no diagnostic end point) of the cells that are positive for the particular antigen, without the visible end point of haemagglutination, which in turn is necessary for simple visual diagnostic detection: If, for example, an erythrocyte which carries the blood group feature Duffy a (Fya) is incubated with an anti-Fya antibody of the IgG class, the antibody-antigen reaction (sensitisation) , but this does not lead to the visible end point of haemagglutination. In order to achieve this, the sensitised cells must additionally be incubated with a class-specific antibody (in the present case anti-IgG), with the aid of which the cells sensitised with IgG antibodies can be bridged and the end point of glutination can be produced (indirect Coombs test). The gel technique, which is widely used for this purpose, es an tion time of 10-15 minutes at 37 °C for this test, with subsequent centrifugation for 9-10 minutes in an anti-human globulin or Coombs card (for example DG Gel Coombs Card from Diagnostic Grifols).

In the tube technique, which is likewise widely used, incubation prior to centrifugation for approximately 20 seconds is not necessary in the case of immediate spin. For the indirect Coombs test, incubation is first carried out for from 15 to 60 minutes at 37 °C with the bloodgroup-specific incomplete antibody, following which a plurality of g steps are ed before the anti-human globulin reagent is added and centrifugation is then carried out for seconds.

There is ore a need for a device and a method for determining cell-bound analytes, in particular blood group ns, for which no standardised IgM antibodies, in particular no commercially available IgM antibodies, are available. There is further a need for a device and a method for simultaneously determining at least two cell-bound analytes, wherein a standardised IgM antibody such as a commercially available antibody is available for only one of the two cell-bound analytes, so that the determination of both analytes in the prior art requires a phase or technique change. It is an object of the present invention to go someway s meeting these needs and/or to e the public with a useful choice.

Summary of the invention According to a first aspect, the present invention provides a device for determining a cellbound analyte in a liquid sample, comprising a separating matrix having at least one indicator zone, wherein the indicator zone comprises a first antibody directed t the cell-bound analyte, or a fragment thereof, and a binding element directed against the first dy, the first antibody being an incomplete antibody; and wherein the separating matrix enables the liquid sample to flow through the indicator zone without spinning or centrifugation.

According to a second aspect, the present ion provides a device for simultaneously determining a first and a second cell-bound e in a liquid sample, comprising a separating matrix, wherein said separating matrix is a membrane having a work zone for application of the liquid sample, at least two indicator zones which are able to interact with the cell-bound analytes, and at least one absorption region which absorbs the liquid after it has passed through the indicator zone, the indicator zones being situated between the work zone and the at least one absorption region, wherein (i) the first indicator zone ses a first antibody directed against the first cellbound e, or a fragment thereof, and a binding element directed against the first antibody, the first antibody being an incomplete antibody, and (ii) the second indicator zone (a) comprises a first dy directed against the second ound analyte, the first antibody being a complete antibody; or (b) comprises a first antibody directed against the second cell-bound analyte, the first antibody being incomplete, and a binding element directed against that first dy; wherein the membrane enables the liquid sample to flow h the indicator zone without spinning or centrifugation.

According to a third aspect, the present ion provides a method for producing a device according to the first or second aspect, comprising applying a first antibody directed against a cell-bound analyte, or a fragment f, and a binding element directed t the first antibody in the indicator zone, wherein the first dy is an incomplete antibody.

According to a fourth aspect, the t invention provides a method for determining at least one cell-bound analyte in a liquid sample, sing applying the sample to the separating matrix of the device according to the first or second aspect, wherein said sample is present in an amount sufficient to cause the sample liquid to flow through the indicator zones without spinning or centrifugation and to cause the analytes in the sample liquid to form a complex in the indicator zones.

According to a fifth aspect, the present invention provides use of the device according to the first or second aspect for analysing blood.

Also described is a device for determining a cell-bound analyte in a liquid sample, comprising a ting matrix having at least one indicator zone, characterised in that the indicator zone comprises a first antibody directed against the cell-bound e, or a fragment thereof, and a binding element directed against the first antibody, the first antibody being an incomplete antibody.

According to a preferred embodiment, the device comprises a membrane (2) having an work zone (5) for application of the liquid sample, at least one indicator zone which is able to interact with the cell-bound analyte, and at least one absorption region (3) which absorbs the liquid after it has passed through the tor zone, the indicator zone being situated between the work zone (5) and an absorption region (3), characterised in that the indicator zone comprises a first antibody ed against the cell-bound analyte, or a fragment thereof, and a binding element ed against the first antibody, the first antibody being an incomplete antibody.

According to a further preferred embodiment, the device contains tubes filled with gel material. The gel technique is used to determine the ination reaction of erythrocytes.

The gel column acts as a filter which slows or stops the migration of the agglutinated erythrocytes relative to non-agglutinated ocytes and thereby effects separation.

According to the invention, the tor zone of the gel contains an antibody directed against the cell-bound analyte, or a fragment thereof, and a g element directed t the first antibody, the first antibody being an incomplete antibody.

Also bed is a device for simultaneously determining a first and a second cell-bound analyte in a liquid sample, comprising a membrane (2) having an work zone (5) for application of the liquid sample, at least two indicator zones which are able to interact with the cell-bound analytes, and at least one tion region (3) which absorbs the liquid after it has passed through the indicator zone, the indicator zones being situated n the work zone (5) and the at least one absorption region (3), characterised in that (i) the first indicator zone comprises a first dy directed t the first cell-bound analyte, or a fragment thereof, and a binding element directed against the first antibody, the first antibody being an incomplete antibody, and (ii) the second indicator zone (a) ses a first antibody directed against the second cell-bound analyte, the first antibody being a complete antibody; or (b) ses a first antibody directed against the second cell-bound analyte, the first antibody being incomplete, and a binding element directed against that antibody.

Surprisingly, the inventors of the present application have found that, by applying a first incomplete antibody and a second antibody directed against the first antibody together in an indicator zone, it is possible to configure a device having a separating matrix, preferably in the form of the membrane of a lateral flow test device or as a gel matrix, in such a manner that it is le to ine a ound analyte by means of an incomplete dy as the first antibody. As a result, it is possible for the first time to determine a cell-bound analyte, for which no standardised, such as commercially available, antibodies of the IgM type are available, using a separating matrix such as a lateral flow test device. This results in a considerable shortening of the time ed for determining such analytes, which previously could generally be determined only by means of the indirect Coombs test, which requires an additional tion step. The procedure described herein is also surprising for the skilled person because he would have assumed that, for example when using anti-IgG molecules as the second antibody, these were neutralised by the non-analyte-specific IgG molecules present in a high concentration in whole blood.

It was therefore not possible in the prior art to determine two blood group antigens simultaneously (that is to say in a single lateral flow device or in a single gel card having a plurality of gel tubes for determining a plurality of parameters), the first being ined by an IgG antibody and the second by an IgM antibody, without a technique or phase change thereby being required. The present ion therefore offers the advantage of simultaneous determination using a single lateral flow set-up, which requires only a single homogeneous method step without different media and different incubations.

Also described is a method for ing the above devices, comprising applying a first antibody directed against a cell-bound analyte, or a fragment thereof, and a second antibody directed t the first antibody, or a fragment thereof, wherein the first antibody is an incomplete antibody.

Also bed is a method for determining at least one ound analyte, comprising applying a first antibody directed against a cell-bound analyte, or a fragment thereof, and a binding element directed against the first dy in the indicator zone, wherein the first antibody is an incomplete antibody.

Also described is the use of the devices according to the invention for analysing blood, in particular for ining blood group antigens or antigen epitopes.

Detailed description of the invention Definitions In connection with the present invention, the following expressions are to have the meanings given below: The expression "complete antibody" means an antibody which leads to the agglutination of erythrocytes in the physiological saline . te dies include IgM antibodies or fragments thereof, provided the fragments are still capable of agglutination. The IgM antibodies can be monoclonal or polyclonal.

The expression "incomplete antibody" means an antibody which, when incubated with erythrocytes, does not lead to the agglutination thereof. Incomplete antibodies include IgG antibodies, IgA antibodies, IgD antibodies and IgE antibodies including their subclasses or dy fragments, provided the fragments are still capable of binding a second dy directed against the entire antibody. These antibodies can be monoclonal or polyclonal.

Methods of producing antibodies of the various classes are known to the skilled person.

The expression "cell-bound e" means any molecule that is naturally bound to the surface of a cell, preferably of a human cell, in particular of an erythrocyte. They include, for example, receptors or blood group antigens, blood group antigens being preferred.

The expression "blood group antigen" es antigens of the ABO blood group system, of the Rh, Kell, Lewis-Hh, Duffy-Kidd, MNS, an and P system, of the blood group systems Diego, Yt, Scianna, Dombrock, Colton, Chido/Rogers, Gerbich, Cromer, Knops, Landsteiner-Wiener, Xg, Kx, Indian, Ok, Raph, John Milton Hagen, Langereis, Sid, FORS, JR and/or LAN, in particular A1, A2, AB, B, D, C, c, E, e, Cw, K, k, M, N, S, s, Jka, Jkb, Fya, Fyb, Kpa, Kpb, Jsa, Jsb, Lea, Leb, Lua, Lub, P1, I, H, Xga, U, Vw, Wra, Lan, Vel, Dia and/or Mia.

Production of the lateral flow device A method that is le in principle for producing a lateral flow device is described in DE 10330982 A1 and , but it is altered as indicated hereinbelow. The disclosure of DE 10330982 A1 and is incorporated herein by reference.

The method for producing a device described herein comprises applying a first antibody directed t a cell-bound analyte, or a fragment thereof, and a binding element directed against the first antibody in the indicator zone, wherein the first antibody is an incomplete antibody.

The first antibody directed against a cell-bound analyte and the binding element directed against the first antibody can be applied to the membrane in the region of the indicator zone either together or separately from one r. Where they are applied separately from one r, it is preferred that a drying step takes place after the application of the first antibody, before the binding element is applied. The concentration of the first antibody is determined empirically and depends on the affinity for the cell-bound analyte. The tration of the g element can be optimised by test series on the basis of the concentration of the first antibody and the properties thereof.

The analyte to be determined is preferably a blood group antigen. The first dy is particularly preferably directed against a cell-bound analyte selected from the blood group antigens A, B, AB, D, C, E, c, e, Cw, K, k, Jka, Jkb, Fya, Fyb, M, N, S, s, P1, Kpa, Kpb, Lua, Lub, Lea, Leb, Mia, Dia, Jsa, Jsb, Coa, Cob, Wra and Xga, particularly preferably against k, S, Fya, Kpa, Kpb, Lua, Lea, Leb, Mia, Lua, Lub, Dia, Jsa, Jsb, Coa, Cob, Wra and Xga. The first antibody is an incomplete dy in this case, preferably an IgG or IgA dy, particularly preferably an IgG antibody. For example, the following antibodies can be used: anti-Fya: clone P3TIM (Merck Millipore, VL); anti-S: clone P3S13JS123 (Diagast, ref. 78007); anti-k: clone P3A118OL67 (Merck Millipore, FA); and anti-D: clone ESD-1 (Alba Bioscience).

The g element is preferably selected from an antibody directed against the first antibody, or a fragment thereof, and a lectin or a fragment thereof. The antibody directed against the first antibody is particularly preferably an anti-IgG antibody. Anti-IgG antibodies are commercially available, particularly red are the clone MS-278 (Merck Millipore) and as a polyclonal antibody, for example, mono-type or anti-IgG uman in (Medion Grifols Diagnostics). Where the first antibody is an IgA dy, the second antibody is an anti-IgA antibody. gA antibodies are cially available. The anti-IgG or anti-IgA antibodies can be of the IgM or IgG type, a monoclonal gG of the IgM class being preferred. Preferred lectins are protein A and protein G.

Where a device for simultaneously determining a first and a second cell-bound analyte as described herein is to be produced, (i) the first indicator zone comprises a first antibody directed against the first cell-bound analyte, or a fragment thereof, and a binding element directed against the first antibody, wherein the first antibody is an incomplete antibody, and (ii) the second indicator zone comprises (a) a first antibody directed against the second cellbound e, wherein the first antibody is a complete antibody; or (b) a first antibody directed against the second cell-bound analyte, n the first dy is incomplete, and a binding element directed against that antibody.

The first cell-bound analyte is preferably selected from the blood group antigens k, Fya, Kpa, Kpb, Lua, Lub, Mia, Dia, Jsa, Jsb, Coa, Cob, Wra, Xga and S and the second cell-bound analyte is preferably selected from A, B, AB, C, D, E, c, e, Cw, K, Lea, Leb, Jka, Jkb, Fyb, P1 and s.

The first antibody directed against the second cell-bound analyte according to alternative (a) is a te antibody, in particular an IgM antibody, which leads directly to haemagglutination. The first antibody directed against the second cell-bound analyte according to alternative (b) is an lete antibody, preferably the first antibody according to alternative (b) is an IgG or IgA antibody. The binding t directed against the first antibody according to alternative (b) permits determination by haemagglutination. The binding element is preferably an IgG or IgM antibody. atively, a lectin such as protein A or protein G may also be used.

The membrane of the device used according to the t disclosure is a porous membrane. Preferred membrane materials are, for example, nitrocellulose (for example UniSart from Sartorius, HiFlow from Millipore, Whatman, AE99 or FF85/100 from Whatman Schleicher & Schuell), hylene (Lateral Flo from Porex ation) or nylon (Novylon from CUNO). The membrane preferably has as large a pore size as possible, since a high porosity of the membrane facilitates the penetration in ular of cell components of the sample to be determined, for example of ocytes, into the porous structure. The use of absorbent membranes is particularly advantageous. However, the device bed herein is not limited to those properties. Preference is given to any membranes having a high capillary flow rate, where the capillary flow rate is the time [s] required for a dye solution to cover a distance of 40 mm on a given membrane. Membranes whose capillary flow rate is < 100 are particularly red.

In a preferred embodiment described herein, a sealing element is arranged on the porous membrane downstream, in relation to the direction of flow, of the work zone of the device bed herein. Two- or three-dimensional sealing elements are used, which are placed on the porous membrane and with which a sample work zone separated from the remainder of the surface of the porous membrane is created. According to the present sure, the sealing element acts primarily as a liquid barrier and permits the directional distribution of sample liquid and test reagents into the porous ne. According to the present disclosure, the sealing element further seals off the sample work zone in order to prevent liquid from undesirably entering the other regions of the lateral flow device. red embodiments of the sealing element are the web or trough or funnel shape. The sealing element is cut the material used to produce the sealing element. In the case of the funnel or trough shape, the sealing element is provided with an inner opening, preferred variants of which are round, square or rectangular shapes which, in the case of the funnel shape, taper s the underside (membrane contact side) of the sealing element.

Preferred materials for the sealing element are materials which do not absorb water (hydrophobic). In a particular embodiment, the materials are coated on one side with an ve film, for example a pressure-sensitive or self-adhesive acrylate adhesive. The sealing element can thus be bonded directly to the surface of the porous membrane. atively, the sealing element can be connected, for example adhesively bonded, to the lateral flow casing, the lateral flow casing in this embodiment pressing the sealing element on the surface of the porous membrane and the ons of the sealing element thereby being achieved.

Preferred materials for g two-dimensional sealing ts are any form of adhesive tape or adhesive foil (for example Tesa 4124 from Beiersdorf AG, ARcare 7815 from Adhesives Research). Preferred materials for forming three-dimensional sealing elements are flexible, -pore elastomer materials or flexible silicone materials with different material thicknesses, preferably 3-5 mm (for example EPDM140 cellular rubber from Pitzner, silicone rubber or solid , hardness 40 deg. or less, from Castan).

In a further preferred embodiment, multiple sealing ts consisting of one piece with, for example, 20 individual cavities (trough shape) are arranged on one membrane.

As a result of this design, the device described herien is capable of ing liquid samples which contain cells, such as whole blood, without thereby filtering off the cells. Furthermore, the sealing element allows large sample volumes to be applied to the porous membrane (work zone) without flooding it. The sealing element thus supports the use of the absorbing properties of the porous membrane. Furthermore, the g element s a directional sample flow. The device described herein can, however, function well with or without a sealing element.

For the tion region (absorption pad) of the device described herein, preference is given to mechanically stable materials, preferably having water absorption capacities of -30 g/100 cm2 (for example Millipore). The contact between the absorption pad and the lateral flow membrane of the device described herein is established by pressure and overlapping with the porous membrane. Precise positioning of the absorption pad on the membrane is achieved by vely bonding the absorption pad to the backing sheet ng the lateral flow membrane.

In a further embodiment, the components of the device described herein are applied to a ate or backing sheet for the purposes of mechanical strengthening. The device described herein can, however, on with or without a backing sheet. Preference is given to mechanically stable materials which do not absorb water, preferably with material thicknesses of 100 μm or more, which are coated on one side or on both sides with an adhesive film, for example a pressure-sensitive or self-adhesive acrylate ve (for example 0.005" polyester W/GL-187, G&L). The porous membrane and the absorption pad are fixed to the backing sheet. In the case of a backing sheet that is ve on both sides, the adhesive second side is used for fixing the stack to further surfaces, for example inside the lateral flow casing.

In a r embodiment, the device described , either with or without a backing sheet to which the components of the device described herein are applied, is ated in a casing, the membrane components thereby being pressed against one another and the casing supporting the function of the sealing element. The device described herein can, however, function equally as well with or without a casing in this case.

Determination methods The method is carried out by applying a liquid sample. The liquid sample preferably consists of blood or tuents of blood, particularly preferably of whole blood, ocyte concentrate, coagulated blood or test liquid, such as l blood. The sample may be diluted with a buffer in this case before it is applied.

The invention will be explained in r detail below by means of figures and examples, without being limited thereto.

Fig. 1 is, by way of example, a perspective view of a device according to the invention for lateral flow tests for simultaneously determining blood group antigens. In the present example, the device consists of a backing sheet 1, the porous membrane 2, the absorption pad 3 and the sealing element 4, which is two-dimensional in web form or three-dimensional in trough form. The porous membrane 2 is y fixed to the backing sheet 1 provided with a pressure-sensitive or self-adhesive acrylate adhesive. The absorption pad 3 is likewise fixed to the backing sheet 1, some of the absorption pad 3 overlapping the porous membrane 2. The sealing element 4 fixed to the upper side of the porous membrane 2 tes the work zone 5 from the remainder of the ne surface and permits the directional distribution of sample liquid and test reagents into the porous membrane 2. The indicator zone region 6 is arranged between the work zone 5 and the region of the porous membrane 2 that is in contact with the absorption pad 3.

Fig. 2 shows a successful simultaneous determination of the blood group ns Jka, Jkb, Fya, Fyb, S, s, k and P1. The donor is Jka-Jkb+Fya-Fyb+S-s+k+P1+. The sample was hereby applied to the work zone situated in the middle. The sample flows through both the indicator zones situated to the left of the work zone and the indicator zones situated to the right of the work zone.

Fig. 3 shows a ison of, on the one hand, the method according to the invention with a first IgG antibody directed against the blood group ns D, Fya and k and a second anti- IgG antibody directed against the first dy, and, on the other hand, a comparative method without the second antibody. On the right-hand side, gG is applied three times as a further negative control. Fig. 3a shows the dispensing plan used. Fig. 3b to 3e show the experimental results which were obtained using s from different donors.

Examples Example 1: Blood group determination Production of the test strips: The test strips consist of a work zone located in the middle of the membrane, as well as two indicator zone regions and two absorption regions at equal distances on both sides of the central work zone. Membranes of the Millipore HiFlow Plus 065 type are trimmed in strips to a size of 19 x 48 mm (width/length; x/y) for an 8- to 10-band design and vely bonded to a backing sheet (for example from G&L). Two absorption pads (Millipore) measuring 19 x 17 mm and overlapping the membrane by 7 mm are vely bonded to the ends of the membrane distal to the work zone. 6 mm-long bands (each 0.6 μl) of solutions of different blood-group-specific antibodies are applied to the indicator zone regions, so as to be offset in two linear rows, using a dispenser, for example AD3200 (Biodot): anti-Jka: clone P3HT7 (Diagast, ref. 78003); anti-Jkb: clone P3 143 (Diagast, ref. 78004); ya: clone P3TIM + anti-IgG clone MS278 (Merck Millipore, VL+JZ); anti-Fyb: clone SpA264LBg1 (Merck Millipore, FF); anti-S: clone P3S13JS123 + anti-IgG clone MS278 (Diagast, ref. 78007 + Merck Millipore, JZ); : clone P3BER (Merck Millipore, FE); anti- P1: clone P3MON2 (Merck Millipore, VN); anti-k: clone P3A118OL67 + anti-IgG clone MS278 (Merck Millipore, FA+JZ). All the antibodies are concentrated about 10 times before formulation.

The anti-Jka antibody is positioned to the left of the work zone in position x = 3 mm/y = 9 mm to y = 15. Three other antibodies (anti-Jkb, anti-Fya and anti-Fyb) are dispensed iteratively at intervals of x = 2.5 mm in parallel with the position of the anti-Jka antibody. The anti-S antibody is positioned to the right of the work zone in position x = 3 mm/y = 34 mm to y = 40.

Three other antibodies (anti-s, anti-k and anti-P1) are sed iteratively at intervals of x = 2.5 mm to the position of the anti-S antibody.

The anti-erythrocyte-specific validation antibody (Val = process control; rabbit IgG fraction of anti-human RBC, Rockland, 209-4139) is applied as a dot in x = 2.5 mm/y = 3 mm offset to the last band of the series of the blood-group-specific antibodies. The l dot (Ctl = negative control; contains all the constituents of the various dy formulations with the exception of the antibody) is applied in y = 3 mm offset to the Val dot. All the antibody solutions n 1 % BSA and 9.4 % APP3 solution [32.4 % (w/v) D(+)-trehalose dihydrate, 0.055 % (v/v) Genapol PF10, 21.8 % (v/v) methanol, PPB buffer: 15 mM potassium phosphate buffer/10 mM NaCl/0.05 % (w/v) NaN3]. The antibodies are d in 0.07M Tris/HCl buffer, having a pH of 7, with the exception of anti-P1, which is d in 0.01 M citrate buffer, having a pH of 4, as follows: anti-Jka 1:5, anti-Jkb 1:5, ya 1:5 + anti-IgG 1:25, anti-S 1:5 + 1:100, anti-s (small) 1:16.7, anti-k 1:10 + gG 1:100, anti-P1 1:10 and anti-RBC 1:10. After the antibodies have been dispensed, the membranes are dried for 1 hour at 45 °C and welded er with a sealing element in a polycarbonate casing n Grifols Diagnostics AG).

Test set-up: The blood samples can be taken in tubes ning conventional anticoagulants (for example EDTA, CPDA-1, ACD, citrate) or in native form.

In a test tube, 1 drop (50 μl) of anticoagulated whole blood is mixed with 4 drops (200 μl) of Diluent F (Medion Grifols Diagnostics) or 1 drop of erythrocyte nt is mixed with 8 drops (400 μl) of Diluent F, or 2 drops (100 μl) of the cells of coagulated blood are mixed with 2 drops of Diluent F.

Two drops (100 μl) of the resulting suspension are applied to the work zone of the described test arrangement. After 30 seconds, 6 drops (300 μl) of Diluent F are applied to the work zone. After 5 minutes, the results are read off and recorded.

Result: The test is valid if the anti-RBC validation dot (val) shows a clearly positive signal (red dot) and the control dot (ctl) tes a negative result. The presence of a red band indicates that the tested blood sample is positive for the particular blood group feature. The absence of a band in the corresponding position in the work zone means that the tested blood sample is negative for the corresponding blood group feature.

Fig. 2 shows the successful simultaneous determination of the blood group ns Jka, Jkb, Fya, Fyb, S, s, k and P1. The donor is Jka-Jkb+Fya-Fyb+S-s+k+P1+. e 2: Blood group determination by means of the method according to the invention and comparative e The test strip was produced analogously to Example 1. There were used as antibodies: anti- D, clone ESD-1 , human IgG; anti-k (cellano), clone P3A118OL67 (Millipore), human IgG; anti-Fya, clone P3TIM (Millipore), human IgG, and as the second dy: anti-IgG, clone MS278 (Millipore), mouse IgM.

Fig. 3a shows the dispensing plan used. Fig. 3b to 3e show the experimental results obtained with samples from different donors. It can clearly be seen that only the determination by means of a first antibody of the IgG class directed against the blood group n and a second antibody directed against that first antibody leads to a clearly detectable band, s the determination using the first antibody of the IgG class does not lead to a clearly recognisable band. On the right-hand side, anti-IgG is applied three times as a further negative control. Fig. 3b to 3e show that no band was obtained in the case of this negative control.

The term "comprising" as used in this specification and claims means "consisting at least in part of". When interpreting statements in this ication, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. d terms such as "comprise" and "comprised" are to be interpreted in similar manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the es of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an ion that such documents, or such sources of ation, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

Claims (28)

Claims

1. A device for determining a cell-bound analyte in a liquid sample, comprising a separating matrix having at least one tor zone, wherein the indicator zone comprises a first antibody, or a fragment thereof, directed against the cell-bound analyte, and a binding element directed against the first dy, the first antibody being an incomplete antibody; and wherein the separating matrix enables the liquid sample to flow through the indicator zone without spinning or centrifugation.

2. The device according to claim 1, comprising a membrane having a work zone for application of the liquid sample, at least one indicator zone which is able to interact with the cell-bound analyte, and at least one absorption region which absorbs the liquid after it has passed through the indicator zone, the indicator zone being situated between the work zone and an tion region.

3. The device according to claim 1 or 2, wherein the cell-bound analyte is a blood group antigen.

4. The device according to claim 3, n the cell-bound analyte is selected from the blood group antigens A, B, AB, D, C, E, c, e, Cw, K, k, Jka, Jkb, Fya, Fyb, M, N, S, s, P1, Kpa, Kpb, Lua, Lub, Lea, Leb, Mia, Dia, Jsa, Jsb, Coa, Cob, Wra and Xga.

5. The device according to claim 4, wherein the cell-bound analyte is selected from the blood group antigens k, S, Fya, Kpa, Kpb, Mia, Lua, Lub, Dia, Jsa, Jsb, Coa, Cob, Wra and Xga.

6. The device according to any one of claims 1 to 5, wherein the first antibody is an antibody of the IgG or IgA type, preferably of the IgG type.

7. The device according to any one of claims 1 to 6, wherein the binding t directed against the first antibody is selected from an antibody directed against the first dy, or a nt thereof, and a lectin or a fragment thereof.

8. The device according to claim 7, wherein the element directed against the first antibody is an anti-IgG or anti-IgA antibody, preferably a monoclonal gG antibody of the IgM class, or the lectin is protein A or protein G.

9. A device for simultaneously determining a first and a second cell-bound analyte in a liquid sample, comprising a ting matrix, wherein said separating matrix is a membrane having a work zone for application of the liquid sample, at least two indicator zones which are able to interact with the cell-bound analytes, and at least one absorption region which absorbs the liquid after it has passed through the indicator zone, the indicator zones being situated between the work zone and the at least one absorption region, wherein (i) the first indicator zone comprises a first dy directed against the first cellbound e, or a fragment thereof, and a binding t directed against the first antibody, the first antibody being an incomplete dy, and (ii) the second indicator zone (a) comprises a first antibody directed against the second cell-bound analyte, the first antibody being a complete antibody; or (b) comprises a first antibody directed against the second cell-bound analyte, the first antibody being incomplete, and a binding element directed against that first antibody; wherein the membrane enables the liquid sample to flow through the tor zone t spinning or centrifugation.

10. The device according to claim 9, wherein the first and the second cell-bound analytes are blood group antigens.

11. The device according to claim 10, wherein the first cell-bound e is selected from the blood group antigens k, Fya, Kpa, Kpb, Lua, Lub, Mia, Dia, Jsa, Jsb, Coa, Cob, Wra, Xga and S and the second cell-bound e is selected from A, B, AB, C, D, E, c, e, Cw, K, Lea, Leb, Jka, Jkb, Fyb, P1 and s.

12. The device ing to any one of claims 9 to 11, wherein the first antibody in the first indicator zone (i) and/or the first antibody in the second indicator zone (ii) is an antibody of the IgG or IgA type, preferably of the IgG type.

13. The device according to any one of claims 9 to 12, wherein the binding element directed against the first antibody in the first indicator zone (i) and/or in the second indicator zone (ii) is selected from an antibody directed against the first antibody, or a fragment thereof, and a lectin or a fragment thereof.

14. The device according to claim 13, wherein the binding element is an anti-IgG or anti-IgA antibody or the lectin is protein A or protein G.

15. The device according to claim 13, wherein the binding element in the first indicator zone (i) and/or the second dy in the second indicator zone (ii) is an IgM or IgG antibody.

16. The device according to any one of claims 9 to 15, wherein the first antibody directed against the first cell-bound analyte and the binding element directed against the first antibody in the first tor zone (i) and/or the first antibody directed against the second cell-bound analyte and the second binding element directed against the first antibody in the second indicator zone (ii) are IgG antibodies.

17. The device according to any one of claims 9 to 16, wherein the second tor zone (ii) comprises an IgM antibody directed against the second cell-bound analyte.

18. A method for producing a device according to any one of claims 1 to 17, comprising applying a first dy directed against a cell-bound analyte, or a fragment thereof, and a binding element directed against the first antibody in the indicator zone, wherein the first antibody is an incomplete antibody.

19. A method for producing a device according to claim 18, wherein the first dy and the binding element are applied separately from one another or as a mixture.

20. A method for determining at least one ound analyte in a liquid sample, sing ng the sample to the separating matrix of the device ing to any one of the preceding claims 1 to 17, wherein said sample is present in an amount sufficient to cause the sample liquid to flow through the indicator zones without ng or centrifugation and to cause the analytes in the sample liquid to form a complex in the indicator zones.

21. The method according to claim 20, wherein the method does not se ting the cell-bound analyte with an antibody before the cell-bound analyte is applied to the separating matrix.

22. The method ing to either claim 20 or claim 21, wherein the liquid sample consists of blood or constituents of blood, preferably of whole blood, erythrocyte concentrate, coagulated blood or test liquid, such as control blood.

23. Use of the device according to any of claims 1 to 17 for analysing blood.

24. The use of claim 23 for determining blood group ns or antigen epitopes.

25. The use according to claim 24 for simultaneously determining a plurality of the following A, B, AB, D, C, E, c, e, Cw, K, k, Jka, Jkb, Fya, Fyb, M, N, S, s, P1, Kpa, Kpb, Lua, Lub, Lea, Leb, Mia, Dia, Jsa, Jsb, Coa, Cob, Wra and/or Xga blood group ns or antigen epitopes.

26. A device as claimed in any one of claims 1-17 ntially as herein described and with reference to any example thereof.

27. A method as claimed in any one of claims 18-22 substantially as herein described and with reference to any example thereof.

28. A use as claimed in any one of claims 23-25 substantially as herein described and with reference to any example thereof.