MXPA02005777A - A device for analytical determinations. - Google Patents

Abstract

There is described a device for carrying out analytical determinations such as, for example, immunoassays. The device comprises an assay cassette which includes a series of discrete reagent chambers which contain, successively, the reagents required to perform an analytical determination. A sample loaded into the assay cassette may be brought into alignment with successive reagent chambers by rotation of the reagent chambers relative to the sample.

Description

A DEVICE FOR ANALYTICAL DETERMINATIONS FIELD OF THE INVENTION The present invention relates to a device for performing analytical determinations and, in particular, to a device for performing a specific binding test, such as, for example, an in-assay.

BACKGROUND OF THE INVENTION In the field of analytical determinations there is a great interest in the development of simplified test systems, which allow an inexperienced user to perform complex test procedures without great error. There is also a lot of interest in the development of clean and efficient test systems, which require a minimum handling of liquid reagents and that can be automated to allow the test procedure to be carried out with minimal intervention on the part of the user. This has particular relevance in the field of health care, where there is a growing need for test systems, especially diagnostic ones, that can be operated efficiently and safely in the doctor's office, in the clinic, in the veterinary operating room or even in the patient's house or in the field.

P1529 t ?? dt *? A f t ..,. • -r 'af? Fir ?? -? «? IiM? Tr¡i- ** - J An approach to solving this problem is provided in EP-B-0, 320, 240, which describes a device for use in analytical determinations, which requires a minimum handling of 5 liquid reagents. This device consists of two components: an independent test cassette consisting of a series of separate reagent chambers, separated from each other by a separating means, which contain in successive order all the reagents and 10 wash solutions required to perform a test procedure, and a sample transport body that is adapted to place the sample in the test cassette. The sample to be tested is loaded into the sample transport body, which is inserted 15 then in the test cassette. To complete the test procedure, the sample transport body is inserted into the test cassette, breaking the separation medium between the reagent chambers as it moves through the cassette, to expose the sample to the samples. 20 test reagents in the successive reagent chambers. At the same time several tests can be carried out, for example, a sample and its corresponding control, thanks to a specially designed sample transport body. Although it is possible to carry out several tests of At the same time, reliable results are not obtained, since the analysis of the sample and the control is carried out separately with each one of the series of reactions, of which, each one uses its reagent in different dosage. While the device of EP-B-0, 320, 240 provides a minimum handling of liquid reagents, the design is not particularly suited for the automation of the test procedure, mainly because the overall dimensions of the device change during the operation, as the sample transport body is inserted into the test cassette. In this way, a device that combines the advantages of a minimum handling of liquid reagents with the capacity for the automation of the test procedure is still needed. From U.S. Patent 4,859,603 a portable diagnostic kit for performing analytical tests is known. This known device is designed to test a single sample per device and does not provide the possibility of including a control specimen. To perform an analysis, the test specimen is applied to the specimen support element on a disc, which is placed under a series of receptacles containing the different reagents that will be used in the test. The receptacles have, P1529. each, a sleeve that is cut during the rotation of a housing member, by means of a cutting means. When the cuff is cut, the contents of the receptacle abundantly flood the test specimen. A reagent is continuously present in the reaction zone and the introduction of the next reagent is carried out simply by flooding the test specimen with the next reagent without removing the reagent that was already present. It does not provide a well-defined concentration or the amount of reagent for the chemical reaction, in addition, the device is not suitable for tests such as immunoassays.

SUMMARY OF THE INVENTION The inventors have now developed an improved device for use in making analytical determinations, which is considerably more susceptible to automation, since the overall shape and dimensions of the device remain constant during the analytical procedure. This device provides a test system whose operation is easy, adequate, efficient and clean for the inexperienced user. In accordance with the present invention, a test cassette is provided for use in the performance of analytical determinations, the cassette of P1529 A te, test consists of: a sample receiving part and a reactive carrier part that can rotate with respect to each other; the reactive carrier part has therein formed a series of separate reagent chambers containing, in successive order, the reagents required to effect the analytical determination; the reagent chambers are placed in an eccentric position with respect to the axis of rotation and the sample receiving part defines an access port, also located in an eccentric position with respect to the same axis of rotation, so that it can be brought into alignment with the successive reagent chambers by means of the relative rotation of the reactive carrier part and the sample receiving part. A device for use in performing at least one analytical determination, consisting of a test cassette and a sample loading body, wherein the test cassette consists of a sample receiving part and a reagent carrying part that can rotate one with respect to the other; the reactive carrier part has formed therein a series of independent and successive reagent chambers, separated from each other by means of partitions or separations; the separate reagent chambers contain, in successive order, the reagents P1529. required to perform the analytical determination; the reagent chambers are placed in a practically circumferential location with respect to the axis of rotation and the sample receiving part consists of an access port placed in a practically circumferential location, with respect to the same axis of rotation, in such a way that the port access can be aligned with the reagent chambers by means of the relative rotation of the reactive carrier part and the sample receiving part, and a sample charge body consisting of at least one sample collection area, provided with less an absorbent body for the collection of the sample to be analyzed; the absorbent body projects down to the reagent chambers to contact a reagent disposed in the chamber; the absorbent body is compressed as the partition or separation passes and enters the successive reagent chambers in series, by means of the relative rotation of the reactive carrier part and the sample receiving part. In a preferred embodiment, the test cassette is adapted to be used with a body for sample loading in order to form a device for use in analytical determinations. The test cassette consists of a reactive carrier part, which can be formed from a P1529.

It is moldable plastic material and it has formed in it a series of separate reagent chambers, and also a sample receiving part, which is adapted for the placement of a body for sample loading. The reagent chambers of the reagent carrier part contain, in successive order, the reagents for carrying out the analytical procedure. Advantageously, the series of reagent chambers, independent and separate, can be placed practically along the circumference of a circle centered on the relative axis of rotation of the reactive carrier part with respect to the sample receiving part. It will be understood that the precise arrangement, as well as the content of the reagent chambers, may vary according to the nature of the analytical procedure and are not subject of this invention. In the case of an immunoassay, the reagent chambers may consist of a washing chamber, a conjugate chamber, a series of back washing chambers and a final chamber for signal detection. One or more of the reagent chambers may be empty to facilitate the removal of excess liquid reagents from the sample collection area. The sample collection area consists of an absorbent body or pad of compressible material, P1529 these "liquid removal" chambers may, advantageously, have a smaller depth than the absorbent body or the pad so as to be able to compress it and, thus, squeeze out as much liquid as possible. The detection / measurement of the reading of an analytical procedure or test is normally performed in the reagent chamber that is at the end of the series, which in this procedure can be designated as the signal detection chamber. The signal detection chamber may be filled with a final reagent, for example, a colorimetric substrate for an enzyme or it may be left empty if a luminescence, fluorescence, fluorescence, or color intensity measurement of a dye or markers is to be carried out. particular. It can also be filled with a luminescence activating reagent, such as hydrogen peroxide, when a chemiluminescent label is used in a high sensitivity test. The signal detection chamber is preferably provided with at least one transparent wall or window, through which the qualitative or semiquantitative result of the analytical procedure can be determined by means of visual inspection, or through which it can be measured, by means of a measuring instrument, a signal indicating the result of the procedure, to provide a quantitative result. In one embodiment, the entire reactive carrier part can be formed of a transparent material. The inner surface of the reactive carrier part, which refers to the surface remaining inside, when the test cassette is assembled, may consist of a layer of breakable material, for example, a thin foil. This breakable layer has the function of sealing the contents of the reagent chambers before use. Accordingly, the layer can be applied to the entire interior surface or only in the area comprising the reagent chambers. An essential feature of the invention is that the reactive carrier part of the test cassette can be rotatable with respect to the sample receiving part, which can also be formed, advantageously, from a molded plastic material. The reagent chambers of the reactive carrier part are placed in an eccentric position with respect to the axis of rotation of the reactive carrier part, for example, in a circular band. The sample receiving part has an access port which is also placed in an eccentric position with respect to the axis of rotation of the reactive carrier part, so that it can be aligned with the successive reagent chambers, simply by giving it P1529. iadaaa .ia fa. .. return to the reactive carrier part. In one embodiment, the test cassette may be adapted for use in a body for sample loading, which has a sample collection area therein. The sample collection area can, advantageously, consist of an absorbent body or pad of polyether foam, PVC, polyurethane, nylon or cellulose or other compressible material. In the case of an immunoassay, a specific binding agent, such as an antibody or an antigen, can be immobilized in the sample collection area. The specific binding agents can be directly coupled to the surface of the plastic, using the methods already established, such as, for example, passive adsorption or covalent binding. In an alternate embodiment, they can be attached to the absorbent body or pad by passive adsorption or covalent attachment. In many cases, the sample that will be subjected to the analysis can be taken with a pipette or added without measuring to the sample collection area, where it will be absorbed by the absorbent material. Alternatively, the sample can be measured accurately using a pipette. It will be understood that the sample can be any liquid material, including body fluids, such as blood, serum, plasma, urine, milk, etc., as well as other liquids, such as samplesP15 9. of the environmental environment. Additionally it will be understood that the device of the invention can be used in a wide range of analytical determinations, in addition to immunoassays, as described in the foregoing. Control samples or calibrators may be added in other areas of the body for sample loading. These controls can be added immediately before starting a test, for example, at the same time the test sample is added or they can be added during manufacturing. In a preferred configuration, the sample loading body consists of two or more absorbent bodies or pads, where at least one functions as a sample collection area for the addition of the test sample and at least one for the addition of a sample. control sample. In the most preferred configuration, the body for sample loading consists of three absorbent bodies, a sample collection area, one for a positive control sample and one for a negative control sample. The access port, formed in the sample receiving part of the test cassette, is preferably adapted to receive at least a part of the body for sample loading, including the sample collection area, so that the latter is correctly positioned. with respect to the reagent chambers when the P1529 l ** *, f .. *. ** Device ** is in use. Preferably, the sample charge body is adapted to be placed in a cooperative niche on the external surface of the sample receiving part of the test cassette, where the sample collection area is located within the access port. Advantageously, the sample charging body may be sealed in the test cassette, for example, by means of a snap-fit mechanism. In this way, the device is sealed during and at the end of the analytical procedure, avoiding leakage of any of the liquid reagents. Before use, the access port may be covered by a removable cover, for example, a removable strip or patch, which is removed immediately prior to insertion of the body for sample loading. In an alternative embodiment, the body for sample loading may be fixed on the upper surface of the sample receiving part of the test cassette. Most preferably, the body for sample loading can be fixed in the test cassette, by means of a flexible joint or joint, which allows the body for sample loading to move between a first position, in which the body for loading sample is bent backward on the upper surface of the sample receiving part, where the sample collection area is P1529. . oriented upwards, and a second position, in which the sample collection area is placed in the access port. In this configuration, the sample can be deposited, by means of a pipette, on the sample collection area oriented upwards, when the body for sample loading is kept in the first position. The body for sample loading is then folded to the second position, where the sample collection area is placed in the access port. A press fit ensures that the device is sealed. The sample receiving part of the test cassette may also be provided with a penetration means, placed on the inner surface thereof (it refers to the surface remaining inside the assembled test cassette), adjacent to the access port . The penetration means is adapted to penetrate through the breakable layer that seals the reagent chambers of the reagent carrier portion of the test cassette. Preferably, the penetration means consists of a knife or blade capable of cutting the breakable layer. The penetrating means may be mounted on a spring means or equivalent, which allows it to move with respect to the inner surface of the sample receiving part.

P1529.

When a test cassette, which has a penetration means, is in use, the reactive carrier part and the sample receiving part rotate with respect to each other, so that the reagent chambers move in turn further of the penetration medium, allowing the penetration medium to penetrate through the layer of breakable material that seals the reagent chamber and then align it with the sample collection area of the body for sample loading, placed in the access port, putting the sample in contact with the contents of the reagent chamber. The test cassette may further comprise a layer of absorbent material placed between the inner surfaces of the reactive carrier part and the sample receiving part. The layer of absorbent material comprises an aperture aligned with the access port of the sample receiving part and may consist of an additional aperture aligned with the penetrating means forming part of the sample receiving part. The absorbent layer has the function of absorbing any excess liquid reagents released from the reagent chambers when the device is in use, thereby avoiding leakage and minimizing the possibility of entrainment or transport of reagents between adjacent chambers. The total capacity to absorb liquids from the absorbent layer must ideally exceed the total volume of liquid reagents incorporated in the device, so that when the test is complete, all liquid reagents remaining in the device will be absorbed. So that the device can be disposed of in dry form, such that it does not require facilities for the confining of liquids. The invention also encompasses a test cassette that can be operated without a separate body for loading the sample. In this embodiment, on the inner surface of the sample receiving part a sample collection area is located, at a small distance from the access port, so that the sample receiving part also functions as the body for sample loading. When the device is in use, the sample is added directly to one of the chambers of the reactive carrier part, via the access port of the sample receiving part. Preferably, a cover is placed over the access port to seal the device, a pressure adjusting cover can be advantageously used. By rotating the sample receiving part with respect to the reactive carrier part, the sample collection area is brought into contact with the sample. Then, the following are successively contacted: P1529.

Other reagent chambers with the sample collection area. In this embodiment, a penetration means may be located adjacent to the access port. The radial configuration of the test cassette provides several advantages over previously known devices. In particular, the radial configuration allows the inclusion of many more reagent chambers, while retaining a device that has a small overall size, by placing the reagent chambers on the circumference of a circle. In addition, because once the body for sample loading is in place, it does not move with respect to the test cassette, the entire device has a fixed size before and during the operation, and is thus more susceptible to the automation of the test procedure. Because the test cassette is completely independent and because all reagents were incorporated during manufacturing, there is no need for complicated reagent addition or washing steps. After the addition of the sample to the sample collection area, the body for sample loading is placed in its place, so that the sample collection area is placed in the access port in the sample receiving part of the sample. Test c. After the above, the body stops P1529.

JdmlAáJ- AtAA .. The sample charge remains in place during the entire test procedure. In the case of an automated test, the entire device is then placed inside a test instrument that can perform the test procedure, in accordance with a predetermined program. The test instrument must comprise a drive unit to perform the controlled rotation of the reagent carrier portion of the test cassette, with respect to the sample receiving part and may also function as a measuring instrument to measure the result or to take the reading of the test procedure. A portable and hand-held test instrument may be manufactured, which has a drive unit for rotating the test cassette and which is not a complicated measuring instrument for use in the patient's home or in the field. A more complex instrument that incorporates measurement instrumentation would be more suitable for use in the clinic or in the doctor's office. This last instrument can also be preprogrammed with the calibration data needed to provide a completely quantitative result. To facilitate the correct positioning of the test cassette with respect to the drive unit of the test instrument, the external surface of the test P1529. i I I - ÍSÍÍ? ¡: Í .. .., J .. &,,. *.

The reagent of the test cassette may be provided with a locating means, such as a tooth, placed substantially centered on the axis of rotation of the test cassette. It will be very evident that the relative rotation of the sample receiving part and the reactive carrier part can be achieved by holding the sample receiving part in a fixed position, while moving the reactive carrier part, keeping the reactive carrier part in a Fixed position while moving the sample receiving part. In automated systems, the preferred configuration is one in which the sample receiving part remains fixed and the reactive carrier part rotates with respect thereto. In this way, the sample collection area can be kept aligned with a measuring means, for example, an optical measuring instrument. The invention provides a clean and efficient device, which does not require the handling of any type of liquid and that can be operated by an inexperienced user, to quickly provide qualitative analysis results. This device is, ideally, adapted to the field of diagnosis, specifically, to immunoassay or DNA amplification or hybridization tests. It can be used in the doctor's office or in the clinic to help the doctor in the P1529. ÍA? ± A ?? .. * AI diagnosis or to monitor the progress of a disease. A useful example would be in the field of cancer diagnosis and prognosis. The detection / measurement of blood markers associated with cancer is becoming an increasingly powerful tool to help in all stages of cancer treatment, including the detection of early neoplasms, the diagnosis of the disease and the monitoring of progress of the disease or the response to treatment. By using the device of the invention, a doctor or nurse will be able to perform rapid and quantitative tests to detect the presence of markers associated with the cancer, either in the clinic, the doctor's office or next to the patient's bed. In addition, because the device is suitable for use by an inexperienced user and does not require that liquid reagents be dispensed or measured to obtain a quantitative result, it is also adapted for use in home testing. In a further embodiment of the invention, the test cassette may be adapted to perform a variety of analytical determinations in parallel. This can be achieved by having two or more series of separate reagent chambers in the reactive carrier part, each series of chambers containing in successive order, P1529. * & * > the reagents required for an analytical determination and the corresponding number of access ports in the sample receiving part. Conveniently, the two or more series of reagent chambers may be positioned substantially along the circumferences of a series of concentric circles centered on the axis of rotation of the reagent carrier portion with respect to the sample receiving portion. A body for sample loading, for use in this test cassette, may consist of two or more sample collection areas adapted to be placed within two or more access ports when the device is in use. Alternatively, two or more bodies can be used for sample loading, each adapted to be placed in a specific position in the sample receiving part. In the modalities of the test cassette device may also include the particularity of multiple access ports, adapted to operate without a body for sample charge, separated, in order to perform more than one test simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be described with further reference to the accompanying drawings, in which: P1529.

FIG. 1A is a plan view of the body for sample loading for use in the device of the invention. Figure IB is a bottom plan view of the body for sample loading. Figure 2 is a plan view of the test cassette of the invention. Figure 3 is a plan view of the device of the invention, which illustrates how the body is loaded for sample loading in the test cassette. Figure 4 is a bottom plan view of the test cassette. Figure 5 is an expanded view of the test cassette. Figures 6a and 6b are cross-sectional views of an analytical device of the invention in operation. Figure 7 is a plan view of a test cassette according to the invention, in which the sample loading body is attached to the sample receiving part of the test cassette. Figure 8 is an expanded view of a device according to the invention, which does not require a body for separate sample charging.

P1529. AAA &DETAILED DESCRIPTION OF THE INVENTION With reference to the drawings, Figures 1A and IB illustrate the body for sample loading 1, which is a plastic element having a sample collection area. In this embodiment, the body for sample loading has three pads of absorbent material 2 attached thereto. At least one of these pads functions as the sample collection area for the addition of the test sample. To the rest of the pads, the positive or negative control samples can be added. The arrangement of the pads, shown in Figure IB, is only intended to be illustrative and alternative configurations may be used. For example, three or more pads can be arranged radially. Figures 2 and 3 schematically illustrate the test cassette of the invention. In the preferred configuration, the test cassette is disk-shaped and is formed of a moldable plastic material. Figure 2 is a plan view illustrating the sample receiving part of the test cassette 3. The sample receiving part is provided with an access port 4 and can further be provided with a shaped niche to cooperate with the body for loading shows. Figure 3 schematically illustrates the manner in which P1529. that the body 1 can be placed to load the sample in the niche formed in the sample receiving part of the test cassette, so that the sample collection area 2 is placed in the access port 4. Figure 4 is a view on the bottom of the test cassette illustrating the reactive carrier part 6. The reagent carrying part has a series of reagent chambers 7-18 formed therein. The modality illustrated in Figure 4 has a total of 12 reagent chambers, although it will be understood that the devices can be manufactured with a variable number of reagent chambers, as required. Chamber 18 is the signal detection chamber. On the outer surface of the reagent carrier part a location means 19 is provided to facilitate placement of the test cassette on a test instrument. In this embodiment, the location means consists of a tooth that is adapted to cooperate with a drive unit provided by the test instrument. Figure 5 is an expanded view schematically illustrating the construction of the test cassette, which consists of a sample receiving part 3 and a reactive carrier part 6. The reagent carrying part is coupled to the sample receiving part, so what P1529. can rotate, with respect to the sample receiving part, around a common axis of rotation. In the embodiment shown in Figure 5, this is achieved by extending the edge of the sample receiving portion to form a lip 20, causing the outer circumference of the reactive carrier portion to be slightly smaller than the inner circumference of the sample portion. lip of the sample receiving part, so that the reactive carrier part fits and can rotate within the sample receiving part. The test cassette is additionally provided with a layer of breakable material 21, such that a thin foil which, when the test cassette is assembled, is attached to the upper surface of the reagent carrier part to seal the reagent chambers . A layer of absorbent material 22 is provided between the frangible layer and the sample receiving part 22. The absorbent layer contains a first opening 23 which is aligned in register with the access port formed in the sample receiving part and a second opening 24 which it is aligned in register with a penetrating means attached to the underside of the sample receiving part (not shown), so that the penetrating means can be brought into contact with the layer of breakable material when the test cassette is assembled. Figures 6A and 6B illustrate the steps of the P1529. operation of an analytical device of the invention. The body 1 for loading the sample is placed within a cooperative niche in the sample receiving part, which places the absorbent material that forms the sample collection area 2 within the access port. Figure 6A shows a cross-section of the device in a first position with the access port aligned with one of the reagent chambers 13. The absorbent material projects through the sample carrying body and through the absorbent layer 22 to be placed in contact with the content of the reagent chamber. The breakable layer 21, which seals the reagent chamber 13, has previously been cut by the action of the penetrating means 25, which is positioned adjacent to the access port and attached to the inner surface of the sample receiving part. The penetration means is shown in the cutting process of the breakable layer sealing the reagent chamber 14, the reagent chamber 15 remains sealed. As shown in Figure 6A, the reagent chambers are completely separated from each other. The direction of movement of the reagent chambers with respect to the absorbent body 2 and the penetrating means 25 is indicated. Figure 6B shows a cross section of the P1529. device in a second position, the reactive carrier part has been rotated with respect to the sample receiving part in the indicated direction. The absorbent body 2 is now positioned between the reagent chambers 14 and 15, illustrating the manner in which the absorbent body can be compressed between the surface of the sample carrying part and the body for sample loading, as it moves between the chambers of reagents. This compression helps remove excess fluid from the absorbent body. The reagent chamber 15 is shallower than the reagent chambers 13, 14, 16 and 17 and contains no liquid reagents. The absorbent body will be compressed as it moves through the chamber 15, facilitating the removal of an additional excess of the liquid reagents. The penetration means is placed between the reagent chambers 15 and 16, which is still sealed by an intact breakable layer 21. Figures 6A and 6B illustrate the manner in which the penetrating means can move with respect to the inner surface of the loading part for the sample. In Figure 6A, the penetrating means is shown in an extended position within a reagent chamber, while in Figure 6B it is shown in the retracted position while moving between the reagent chambers. Figure 7 illustrates a test cassette, where the body for sample loading is attached to the part P1529 I-i * AXA? A ".: .. M! **. * Sample receiver or is integrally formed with it and can be moved around a flexible or articulated joint 26. Figure 8 illustrates an additional modality of the invention being a test cassette that does not require a body for separate sample loading. The sample collection area 2 is located on the internal surface of the sample receiving part, near the access port 4. Alternative arrangements of the sample collection area are possible, for example, it may be formed in a circumferential ring around the access port. The opening 23 in the absorbent layer is elongated to align with the access port and admit the sample collection area. During use, the sample to be tested is loaded into the first chamber of the reactive carrier part of the device through the access port 4. The access port is then sealed by means of the closure 27. The pads that form The sample collection area is projected down to the chambers of the sample carrier part and, in this way, comes in contact with the sample added to the first chamber and then, successively, with the other reagents required to complete a test . The construction and operation of the device of the invention will be further understood with reference to P1529. the following experimental examples, together with the accompanying figures, in which: Figure 9 shows the results of a test for human IgA, using the device of the invention, as described in example 4. The concentration (ng / ml) of the IgA solution, added to the foam pads in the body for sample loading, is shown on the X axis and the color density of the foam pads at the end of the test is shown on the Y axis. 10 illustrates the effect of the number of washings on the signal to noise ratio in an IgA test, using the device of the invention, as described in examples 4 and 5. The number of washings is shown on the X axis and the density Color of the foam pads on the Y axis. Figures 11 and 12 show the standard curves of the detection of c-erbB2, using the device of the invention, as described in example 7. The concentration (HNU / ml ) of the solution of ce rbB2, added to the body foam pads for sample loading, is shown on the X axis and the color density of the foam pads at the end of the test is shown on the Y axis.

Example 1 Preparation of body-bound foams for sample loading Open cell PVC foam sheets (supplied by Duflex Ltd., Derby, GB), with a thickness of 3 mm, were placed on the flat sector at the end of a body for sample loading and an ultrasonic welding horn (supplied by Renfrew Stylengineering Ltd., Leicester, GB), configured to weld the tip of the foam, but leaving inside cushions, was used to attach the foam to the body material for sample charge. To ensure the bond between the foam and the plastic surface, a period of 30 seconds at 100 watts was used. This procedure created reaction zones of the foam at the end of the body for sample loading, which were held firmly in place by the welding process.

Example 2 Method for coating foams with antibody In this example, the foams were coated with an anti-human IgA antibody. The foam segments in the body for sample loading were washed once in 0.01 M Tris / Cl buffer, pH 7.5. The foam pieces were coated with IgA antibody P1529. * ^ ia «M '* > "*** Aa." - mouse monoclonal antihuman (supplied by Zymed Laboratories, Inc., USA) at 10 ug / ml in 0.01 M Tris / Cl solution, pH 7.5, by adding 20 ml to each sector of foam The coating was continued for 12-16 hours at 4 ° C in a sealed wet chamber The control foam segments in the body for sample loading were washed once with 0.01 M Tris / Cl buffer solution, pH 7.5, and three times with Tris / Cl 0.01 M buffer, pH 7.5, containing 0.05% (v / v) of Tween 20. Both the coated foams and the controls were varnished by washing them three times with Tris buffer. / Cl 0.01 M, pH 7.5, containing 0.1% (w / v) of BSA and 1% (w / v) of lactose. The body for sample loading could be used immediately in a test. The suppliers of reagents were the following: Trizma-base from Sigma Chemical Co. / Sigma-Aldrich Chemie Gmbh; NaCl of J.T. Baker 0278 or Sigma S7653; Tween 20 from Merck / KEBO Lab, Denmark; bovine serum albumin (BSA) Fraction V, from Sigma A4503; a-Lactose of Sigma L3625; MgCl2, J. T. Baker; distilled water from Bie & Berntsen, Denmark. To test the coating of the antibody in the foam sectors, the whole body for sample loading was placed in SuperBlock (supplied by Pierce Chemical Co., USA) for 2 minutes at room temperature P1529. (22 ° C). The foams were then pressed to remove the excess liquid, using absorbent paper. Then, 25 μl of human IgA, in the concentrations shown in Table 1, were added in a solution of 0.05 M Tris / Cl, pH 7.5, containing 0.1 M NaCl, 1 M MgCl 2, 1% (w / w). v) of BSA and 0.1% (v / v) of Tween 20 to the three foam sectors of the body for sample loading. The foams were then incubated at room temperature (22 ° C) for 5 minutes and transferred dry. Then, 25 μl of rabbit antihuman IgA conjugated with alkaline phosphatase (supplied by DAKO A / S, Denmark), diluted 1:25 in 0.05 M Tris / Cl buffer, pH 7.5, was added to each foam sector. contained 0.1 M NaCl, 1 mM MgCl 2, 1% (w / v) BSA and 0.1% (v / v) Tween 20, for an additional incubation period of 5 minutes. The foams were washed three times with 0.01 M Tris / Cl, pH 7.5, containing 0.05% (v / v) of Tween 20 and then, after transfer, 25 μl of the BCIP / NBT substrate was added to each foam sector. (supplied by Zymed Laboratories, Inc., USA). After incubation for 5 minutes at room temperature (22 ° C), the foams were washed with distilled water to stop the color development reaction and in the foams the scan was then carried out to determine the color density, using a DUOSCAN P1529.

T1200 (supplied by AGFA, Germany) connected to a PC, making use of the software program "Cream for Windows", version 1.0 (supplied by Kem-En-Tech A / S, Denmark). The software "Cream" provides a quantitative measure of the density of color in foams. The results in Table 1 show that the foams were coated with the specific antibody, a standard curve was obtained with the increase in IgA concentrations and the bottoms that are on the uncoated foams are low.

P1529. l? AAÁ, L.Í A? ..

Table 1: AGFA browser / software Cream color density signal.

Example 3 Preparation of the base of the test cassette The reagent carrying part was supplied by Renfrew Stylengineering Ltd. Leicester, GB The component was made from PVC sheet (1.5 mm thick), using a vacuum forming process . The reagent carrying part was placed in a carrier or support and the appropriate chambers were filled with the liquid reagents, in accordance with the specific test procedure. An aluminum foil sheet (which P1529.

? SLtk Á-ít% I, a * A *? * T, ijaH.a.aJ? * * ^ * ÜA? * I * J ^ ?? ^ *? ^ A ?? * ^. *? *? ^ *, ílí. A * A had a side coated with lacquer) was cut to fit the diameter of the reactive carrier part and placed on the component, with the lacquered surface juxtaposed to the surface of the reactive carrier part. Next, a domestic iron was used, with the heat placed in "silk", to seal the lamella on the surface of the part for handling the reagent. The component was then examined for leaks and signs of overheating in the reagent chambers. To finish the reagent handling part, the lamella was cut around the edge and the surface of the lamella covering the first chamber was removed to allow placement of the body for sample loading.

Example 4 A test for human IgA in the test cassette This example describes a test for human IgA that was performed on the test cassette. A manually operated mechanism was constructed, on which the reactive carrier part was seated, as prepared in Example 3. On the upper surface of the reagent carrier part a sheet of absorbent material was placed (supplied by Renfrew Stylengineering Ltd ) and then, in place, the sample receiving part of the test cassette was cut out. The sample receiving part of the cassette P1529. of test has a blade placed on the inner surface, the blade is adapted to pierce the lamellae of the agent chambers, thereby allowing the liquid to reach the sample collection area in the body for sample loading. To perform the test, the sample is added to the body foam sample collection area for sample loading and, after a brief incubation to allow the IgA to bind to the antibody on the surface of the foam, in place cut the body for sample loading on the sample receiving part of the test cassette and the sample receiving part was manually rotated to bring the foam sectors successively through the broken compartments of the reagents. To perform or run the test on the test cassette, a body for sample loading was prepared in accordance with the procedure of Example 2. After coating with antibodies, using 25 μl of mouse monoclonal anti-human IgA, the foam sections were washed with Tris / Cl 0.01M, pH 7.5, and whole body for sample loading was placed in SuperBlock (supplied by Pierce Chemical Co.) for 2 minutes at room temperature (22 ° C). After the transfer, 25 μl of human IgA was added to 400, 200, 100, 50, 25 or 0 to the three foam sectors of the body for sample loading.

P1529. ÍAAAlAé *. * ¡¡¡¡¡¡Ai a ^ lÍ ^. ^ J..a-. ^. ^. Altádi éég = íJ¡. ^^ ng / ml, in Tris / Cl 0.05 M solution, pH 7.5, which it contained 0.1 M NaCl, 1 mM MgCl 2, 1% (w / v) BSA and 0.1% (v / v) Tween 20. The foams were then incubated at room temperature, 22 ° C, for 3 minutes. The sample charge body was then cut in place on the sample receiving part of the test cassette and the foam sectors were transported through the first chamber of the reagent carrier part, which was filled with 250 pi SuperBlock , and then to the second chamber containing 250 pi of rabbit antihuman IgA conjugated with alkaline phosphatase (supplied by DAKO A / S, Denmark) diluted 1:25 in 0.05 M Tris / Cl buffer, pH 7.5, containing 0.1 M NaCl, 1 mM MgCl 2, 1% (w / v) BSA and 0.1% (v / v) Tween 20. The incubation time in the second chamber was 6 minutes. The foams were then transported through the following seven chambers, which were filled with 250 pi of Tris / Cl 0.05 M washing buffer, pH 7.5, containing 0.05% (v / v) of Tween 20. The chamber final (la 10a) contained 250 μl of the BCIP / NBT substrate (supplied by Zymed Laboratories, Inc., USA). The foams were incubated for 6 minutes and the color development was carried out in the foam structure. Afterwards, the body for sample loading was removed from the P1529. sample receiving part of the test cassette, the foams were washed with distilled water to stop the color development reaction and in the foams the color density detection scan was then carried out, using a DUOSCAN T1200 (supplied by AGFA, Germany ) connected to a PC using the "Cream for Windows" software program, version 1.0 (supplied by Kem-En-Tech A / S, Denmark). The software "Cream" provides a quantitative measure of the density of color in foams. The results of a typical test are presented in Figure 9.

Example 5 Investigation of the effect of the number of washes on the test cassette A test for human IgA was performed, practically as described in Example 4. A motorized version of the mechanism was used, where the rotation of the test cassette was controlled by a gradual speed motor controlled by a timer device. The incubation period in the mouse antihuman alkaline phosphatase conjugate was 4 minutes 25 seconds. After incubation in the conjugate, the motor transported the body for sample loading through eight washing chambers, which either contained 275 pi P1529. the washing buffer solution or they were empty. The final chamber (la 10a) contained the BCIP / NBT substrate (supplied by Zymed Laboratories, Inc. USA) and the incubation period was 4 minutes 25 seconds. The results in Figure 10 indicate that the optimal signal-to-noise ratio in the motorized version of the test cassette was obtained at seven washes.

Example 6 Coating of the foams with the SKBR-3 extract In this example, the PVC foam used for the sample collection area of the body for sample loading was coated with an aluminum sulphate extract of SKBR-3 cells (supplied by the University of Southern Denmark, Odense). The extract contained the human protein c-erb at 320,000 HNU / ml (measured by means of an ELISA kit supplied by Oncogene Sciences Inc.), which was produced thanks to the cells in culture. The cell extract was diluted to provide varying concentrations of c-erb in buffer solution of 0.01 M Tris / HCl, pH 7.5, and 25 μl were added to each piece of foam. The foams were incubated at 4 ° C overnight. They were then washed with the Tris / Cl wash buffer and then incubated with SuperBlock P1529. for ten minutes. After the transfer, 25 μl of anti-c-erb monoclonal antibodies (Mab 15) were added to the foam pieces., supplied by Neomarkers Inc., USA) at a dilution of 1/100 in Tris / Cl buffer and incubated for 5 minutes at 22 ° C. After washing three times and transfer, 25 μl of the anti-mouse IgA alkaline phosphatase conjugate (supplied by Amdex A / S, Denmark) was added to the foams and incubated for a further five minutes. After washing three more times with the Tris / Cl solution and making the transfer, 25 μl of the BCIP / NBT substrate (supplied by Zymed Laboratories, Inc. USA) was added to the foam pieces and, after 10 minutes , the foams were washed with distilled water and the color density was read with the AGFA / Cream system. The results in Table 2 show that the c-erbs in the cell extract were bound to the foam pieces and can be detected by means of specific mouse monoclonal antibody in a quantitative test.

P1529.

Table 2: Example 7 A test for c-erb in the test cassette In this example, the sample collection areas of a body for sample loading were coated with different concentrations of c-erb, as described in Example 6, and incubated with the mouse monoclonal anti-c-erb antibody at a dilution of 1/100 in Tris / Cl buffer for 15 minutes. After 3 washes with the Tris / Cl wash buffer, the sample charge body was cut out in the sample receiving part of the test cassette and the hand mechanism was used to continue the test. The first chamber contained conjugate of alkaline phosphatase-IgG P1529.

«I-ílMHifctiThi. ** ..-. - - *** *. «, -,., .......,. ^ ~ ****** ^. * Í? & , .. ^. ± t .. ¿ií / l & anti-mouse in Tris / Cl conjugate buffer and incubation was five minutes. Then the body for sample loading was rotated through seven washes, using 275 μl of Tris / Cl wash buffer solution, reaching the BCIP / NBT substrate with a final incubation period of 10 minutes, before the removal of the Test cassette and wash with distilled water to stop the reaction. The results of the Agfa / Cream system are presented in Figures 11 and 12 and show that the test cassette has the ability to generate a standard curve for c-erb.

P1529.

Claims (19)

1. CLAIMS I 1. A device for use in the performance of at least one analytical determination, consisting of a test cassette and a body for sample loading (1), where: the test cassette consists of a sample receiving part ( 3) and a reactive carrier part (6), which can rotate with respect to each other; the reactive carrier part (6) has, formed therein, a series of independent and successive reagent chambers (7-18), separated from one another by means of partitions (7a-18a); the reagent chambers (7-18) contain, in successive order, the reagents required to perform the analytical determination and are placed in an almost circumferential location with respect to the axis of rotation, and the sample receiving part (3) consists of: an access port (4) placed in an almost circumferential location with respect to the same axis of rotation, so that the access port (4) can be aligned with the reagent chambers (7-18) by the relative rotation of the part reagent carrier and sample receiving part (3), and a sample loading body (1) consisting of at least one sample collection area (2), P1529. provided with at least one absorbent body (2) for the collection of the sample to be analyzed; the absorbent body (2) projects down to the reagent chambers (7-18) to come in contact with a reagent provided in the chamber, the absorbent body (2) will be compressed as it passes through the partition (7a-18a) on entering the successive reagent chambers (7-18) in a series, by the relative rotation of the reactive carrier part (6) and the sample receiving part (3). A device according to claim 1, wherein the inner surface of the reactive carrier part (6) consists of a breakable layer of material that can be operated to seal the reagent chambers (7-18) before use. 3. A device according to claim 2, wherein the breakable material is a thin foil. A device according to claim 1 or 2, wherein the sample receiving part (3) further consists of a penetration means (25) positioned adjacent to the access port, the penetration means (25) is adapted to penetrate the breakable layer sealing the reagent chambers (7-18) of the reactive carrier part (6). 5. A device according to claim 3, wherein the penetrating means (25) is a blade. P1529. ? ii Ajj-iA.AÁA «á, * - tfÜ ^. - «¿•.» * ÁAkm *,. * «. , ^ ^ te ^ t &«^ ¿*« - sa »jh ^ a ^^^ & isj« Ma. »» < ^ < TO. 6. A device according to claims 3 or 4, wherein the penetration means (25) is mounted on a spring means. A device according to any of the preceding claims, which further comprises a layer of absorbent material (22) located between the reactive carrier part and the sample receiving part (3), the layer of absorbent material (22) consisting of an opening (23) aligned with the access port (4). A device according to claim 7, wherein the layer of absorbent material (22) further comprises a second opening (24) aligned in register with the penetrating means (25). A device according to any one of the preceding claims, wherein one of the reagent carrying chambers (7-18) is a signal detection chamber provided with at least one transparent wall or window. 10. A device according to any of the preceding claims, wherein the reactive carrier part (6) has a series of separate and separate reagent chambers, placed practically along the circumference of a circle centered on the axis of rotation. 11. A device according to any of the P1529. preceding claims, wherein the series of reagent chambers (7-18) contain the reagents required to perform a particular analytical determination. 12. A device according to any of the preceding claims, wherein the absorbent body (2) is attached to the body for sample loading (1). A device according to any of the preceding claims 1 to 9, wherein the absorbent body is fixed to the inner surface of the sample receiving part (3), close to the access port (4). A device according to any one of the preceding claims, wherein the sample loading body (1) is adapted to be placed in a niche (5) formed on the external surface of the sample receiving part of the test cassette, so that the sample collection area (2) is placed in the access port (4), thus allowing the sample collection area (2) to be placed sequentially in alignment with the successive reagent chambers (7-18) , when the device is in use, by means of the relative rotation of the reactive carrier part (6) and the sample receiving part (3). 15. A device according to any of the preceding claims, for performing a variety of analytical determinations, where: P1529. the reactive carrier part (6) has formed therein two or more different series of separate and separate reagent chambers (7-18), which are placed practically along the circumferences of a series of concentric circles centered on the axis of rotation; the sample receiving part consists of two or more access ports (4) aligned in register with the respective series of reagent chambers (7-18), and - the access ports are aligned with the successive reagent chambers (7-) 18) by the relative rotation of the reactive carrier part (6). 16. A device according to any of the preceding claims, wherein at least one of the absorbent bodies (2) is used for a positive or negative sample or calibrator. A device according to any of the preceding claims, wherein the final reagent chamber (18) of the series is the signal detection chamber (18). 18. A device according to any of the preceding claims, wherein the reactive carrier part (6) further comprises a location means (19) for cooperating with the drive unit of a test instrument. P1529 • * i a-, »? Í ie & iHi &.k * s? & A > 19. A device according to any of the preceding claims, wherein the location means (19) is a tooth (19). P1529