CA2358506A1

CA2358506A1 - Method and device for determining an analyte

Info

Publication number: CA2358506A1
Application number: CA002358506A
Authority: CA
Inventors: Bernd Pevec
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-01-09
Filing date: 2000-01-07
Publication date: 2000-07-20
Also published as: EP1141713A1; AU2288200A; WO2000042434A1

Abstract

The invention relates to a method and a device for the differential determination of a quantity of at least one analyte in a liquid sample. A test strip which has at least two areas is used, these areas being composed in such a way that they can provide information about the quantity of the at least one analyte. The test strip also ensures that the sample or the portion of the sample flows laterally through the strip and the at least two areas by having a predetermined capacity for binding the at least one analyte in the first area. When this capacity is exceeded by a too great quantity of the at least one analyte, the at least one analyte is bound in at least one second area, the capacity of the second area being essentially equal to the capacity of the first area and the binding of the at least one analyte in the at least one first area and the at least one second area being measured by a detectable property.

Description

UB
Translation A Process and a Device for the Differential Determination of the Amount of at Least One Analyte The subject matter of the present invention is a process for the differential determination of the amount of at least one analyte in a sample with the use of a solid phase and a device for carrying out said process.
In today's analytics, test strips have frequently been used. Test strips meeting the demands of a short-term test are basically based on the principle of fixing a reagent to a solid matrix, with said reagent reacting with the analyte to be detected. If required, an alteration of color, a change in color or another detectable property can be measured. Thus, for example, a coloration serves for detecting the presence of an agent to be identified.
Here, only WO-A-95/13542, WO-A-96/09546, EP-A-0 492 326 will be mentioned as examples of a very extensive state of the art.
WO-A-98/22824 pertains to a so-called inhibitor assay. This assay configuration is based on labeled compounds which correspond to the analyte or which are identical therewith. The labeled compound is provided on the test strip and competes with the analyte existing in a sample. The labeled compound leaves the initial zone and can be detected. In this process a linear way of proceeding is basically not possible.
US-A-5,527,509 pertains to an assay determining the analyte by an enzymatic reaction of a low-molecular analyte and a subsequent color reaction of the -2_ anaiyte. So-called capture assays being the subject matter of the present invention are not mentioned in said publication. A linear embodiment of said assay is not possible.
US-A-5,252,496 pertains to a color-forming reaction. Basically, it discloses only qualitative assays. If the assay is performed with two zones, one zone only serves as a control.
US-A-5,229,073 pertains to a non-linear competitive immunoassay.
WO-A-98/36278 pertains to a capture immunoassay having various detection zones comprising capture reagents having different capture concentrations each such that a linear realization of the assay described in said publication is not possible.
Similar circumstances exist in the processes described in WO-A-96/34287, WO-A-96/34271, US-A-4,059,407, US-A-4,042,329, and EP-A-0 833 159 which are either not capture assays or not configured in a linear manner.
Many test strips or similar systems have in common that often only one single kind of dyeing is performed which excludes any quantification or such a test.
Said test is merely used to enable a yes/no statement. Either the analyte exists in a detectable amount resulting in the formation of a color or the analyte does not exceed a previously defined limit, i.e. it falls below the limit of detection of the respective test strip or test system such that a coloration is not formed.
Moreover, an operational check has to be provided by introducing an additional reaction-independent parameter in order to exclude systematic errors. Although this test strip design is sufficient for many applications, questions such as the degree of severity of an exposure or a disease or the change of the amount of analyte during a rehabilitation process or a therapy trial cannot be answered satisfactorily.

Another drawback of the common test strips or assay devices is the necessity to indicate limit exceeds in a safe manner. This means that often only a very small concentration interval between a base value, where the coloration already begins, and a maximum coloration exists. Actually, however, it is possible that large variations in concentration exist between the limit of detection for an analyte representing the sensitivity of the test system and the actual value to be measured. Thus, e.g., in the field of environmental analysis ratios of frequently exist, and even in the medical field analyte ratios of 1/103 are not unusual.
Therefore, the technical problem forming the basis of the invention is to avoid the above-mentioned drawbacks of the system existing in the art and to provide quantifying short-time tests.
The technical problem forming the basis of the invention is solved by a process for the differential determination of the amount of at least one analyte in a liquid sample with the use of a test strip. A differential determination has to be interpreted as an at least semiquantitative determination, that is an analysis exceeding a merely qualitative determination. Said test strip must have at least two zones and ensure a lateral flow of the liquid sample or a sample proportion through the test strip and said at least two zones. At least two zones are configured such that they are capable of enabling a statement on the amount of said at least one analyte due to the fact that a first zone has a previously defined capacity for binding said at least one analyte and a bonding of said at least one analyte takes place in at least one second zone if this capacity is exceeded by an amount of said at least one analyte being too large for this capacity, wherein the capacity of said at least two zones is essentially equal, respectively, and the bonding of said at least one analyte in said at least first and at least second zones is measured by a detectable property. Preferably, the amount of said at least one analyte is determined by an activity of said at least one analyte.

The analyte can be bonded to the test strip in a specific and/or unspecific manner. Preferably, the specific bonding of said at least one analyte takes place by an affinity to another ligand or an affinity to the material used for the test strip. In this case an immunoaffinity bonding by bonding partners bonded to the test strip surface in a specific or unspecific manner is possible. The precipitation of said at least one analyte on the used test strip illustrates the unspecific bonding of said at least one analyte to the test strip. The specific bonding between said at least one analyte and an affinity bonding partner is accomplished by, e.g., receptor/ligand interaction, enzyme/substrate interaction, anti-gen/antibody interaction and other affinity bonds such as avidin, streptavidin, protein A, IgG, and other systems known to the skilled person.
When carrying out the process of the invention, it is preferred to specify the defined capacity for the bonding of said at least one analyte on the test strip as a function of the amount of said at least one analyte expected in the sample to be analyzed in order to be able to determine said at least one analyte in the second zone if the capacity of said at least first zone is exceeded.
Preferably, the capacity difference of the capture component applied in the first and second zones of the inventive capture assay does not exceed f 10 %, in particular t 5 %. Generally, it has to be remarked that the linearity of the inventive assay is the more precise the more accurate the capacity of the capturer in said at least two zones has been adjusted. According to the circum-stances, when using more than two zones, it may be advantageous to provide the additional zone with a higher amount of capturer, f.e. to increase the capacity of this zone in order to improve the detection. When using several zones, preferably the last respective zone is provided with a higher capturer concentra-tion.
The advantage of the present invention is that essentially equal amounts of bonding agent (capturer) are applied. Thus, a linearity is achieved enabling the safe rating also of intermediate colors. Therefore, different from the other known multizone assays one does not depend on a titration (i.e. in fact a logarithmic representation) of an antibody, see, e.g., WO-A-98/36278. Also in displacement reactions, which basically are not linear due to the labeled analyte to be charged previously, linearity can be maintained by applying the labeled analyte outside the bonding zone. For example, this is not possible according to US-A-5,229,073.
Figure i exemplifies the operating principle of the inventive process.
Figure 2 illustrates a result of the inventive process if three zones having respective defined capacities are provided.
Figure 3 pertains to a device with the test strip according to Fig. 4.
Figure 4 pertains to the inventive test strip.
Figure 1 schematically illustrates the fixation of an antibody on a solid matrix, e.g., cellulose nitrate. Then, in ib) the so-coated phase is treated with the analyte. The analyte bonds to the respective antibody by immunoaffinity.
Subsequently, in ic) another antibody which bonds to said antibody and is also labeled, in particular with a gold colloid, is added to said antigen for detection.
This indicates the presence of the analyte. If the capacity of the first zone is not sufficient to bond all analytes in the sample, said analytes together with other sample components diffuse on the solid porous support further towards said at least second zone, where the analytes are bonded to the antibodies immobilized there.
Hence, according to the invention it can be differentiated whether the amount of analyte in a sample is below the limit of detection, exceeds the limit of detection or distinctly exceeds the limit of detection. Therefore, the first zone only bonds the maximally bondable amount of analyte, and with the present amount of analyte being exceeding a further bonding in said at least second zone or additional zones will occur.

In this context, the previous determination of the capacity to bond the analyte in the respective zone is important. The skilled person is aware that said capacity is determined by the respective problem to be solved. If, e.g., in the field of environmental analytics a pollutant has to be determined, it may be advisable to choose the capacity of said at least first zone such that the limit exceeding results in a weak coloration of said at least first zone. If the sample contains a distinctly greater amount of pollutant, also said at least second zone and optionally additional zones on the used test strip will be calored as a function of the corresponding pollutant concentration. In the medical field one may proceed in a similar manner by choosing the capacity of the respective zones such that information on a particular analyte titer becomes ascertainable. If a particular value (threshold value) of an analyte is indicative for a pathologic condition and said value has to be determined, it may be advantageous also in this case to choose the capacity of said first zone to be within the concentration range of this threshold value. If, however, for example an operational check by determining an analyte is desired, instead of exceeding a threshold value which could be detected in the first zone the presence of the analyte at all can be indicated such as, e.g., in the case of the determination of C-reactive protein.
In this case, preferably anti CRP antibody is immobilized in the first zone in an amount enabling the detection of a CRP normal value. An analytical objective can be attained in various ways by choosing the capacity of the inJividual zones appropriately.
A fine graduation of the measuring range to be detected can be attained by increasing the number of zones. However, for practical reasons the number of zones will regularly be between three and ten. In this case, the upper limit will regularly be determined by the readability or Interpretability of the results.
Figure 2 shows the example of a medicinal immunotest. Thus, e.g., in humans the amount of C-reactive plasma-bonded proteins, a common marker for bacterial inflammations, is about between 0.2 and 200 mg/I with values of up to 10 mg/I being regarded as normal. Concentrations exceeding this value indicate different forms of bacterial inflammations. Since, moreover, the plasma _ 7 _ concentration of C-reactive proteins very rapidly reacts to a positive therapy, in the practice actually all concentrations fall in the mentioned range.
The situation schematically depicted in figure 2 shows three different zones which may immunologically interact with C-reactive proteins. Regularly, the following three conditions can visually be read off: - no coloration; (+) a weak; and +
a strong coloration. If a test strip reveals a situation indicated as situation 1 in figure 2, namely a weak coloration of the first zone and no coloration of the second and third zones, the amount of C-reactive proteins is within the range of 0.2 to 5 mg/I. In this case, the capacity of the respective zones has been adapted to these amounts. Situation 2 shows an increased plasma concentration of C-reactive protein, since the coloration is stronger. This situation corresponds to a content of 5 to 10 mg/I. In situation 3 the first zone shows the maximally possible coloration and the coloration of the second zone is weaker, whereas zone 3 is not colored at all, which indicates an amount of 1l to 15 mg/I.
Situation 4 shaves a strong coloration of the first and second zones, whereas the third zone is not colored, which indicates a plasma level of 16 to 25 mg/I.
Situation 5 shows a maximum coloration of the first two zones and a weak coloration of zone 3, which corresponds to a plasma level of 25 to 50 mg/I.
Situation 6 shows a strong coloration of all three zones enabling one to conclude that >50 mg/I is present. Thus, this exemplifying three-zone model overall enables a six-step differentiation by a simple combination of the number of colored zones and the color intensities. In addition, an operational check is inherently present by providing the first zone with a capacity between 0.2 and mg/I since the first zone practically has to be weakly positive with any patient (basal level). In principle, with N zones it will be possible to distinguish a number of conditions corresponding to twice the number of N of in a safe manner with very varying graduations being possible by applying different reagent amounts to the respective zones, as has been exemplified above.
According to the invention, e.g., C-reactive protein (CRP), rheumatoid factors, troponin I, creatinine kinase (CK-MB), myoglobin; antigens of the organisms of Salmonella, Legionella, E. coli (EHEC), Aspergillus flavus and fumigatus;
glucose, dioxin, PCB, cocaine (ecgonine), heroin, amphetamine, and other pathologically indicative or relevant in environmental analytics can be measured as an analyte.
Advantageously, also two or more analytes can be determined in parallel by covering said at least two zones appropriately. For example, the following pairs of analytes being relevant in the clinical practice or in environmental analytics can be determined: CRP and antistreptolysin; troponin I and CK-MB; myoglobin and CK-MB; CK-MB, myoglobin, and troponln I; cocaine, heroin, and amphet-amine; Salmonella and E. coli.
In principle, any material having a certain porosity such as porous supports comprising cellulose nitrate, nylon, PTFE, polystyrene, latex, glass fibers, silica gel, aluminium oxide, cellulose, dextran, agarose, polyvinyl alcohol, also in the form of micro-beads or "magnetobeads", optionally preactivated by BrCN, NPCF, NHS chloroformate, tresyl chloride, squaric acid esters, and/or corresponding chlorosilanes may be employed for the test strip advantageously used in the inventive process.
For that reason the inventive process is particularly user-friendly since individual analytical problems can be solved by choosing the concentrations which are to be detected in the separate zones. Thus, e.g., the user himself can solve individual analytic problems by employing for the zones an agent interacting with the analyte.
The inventive test strip suitable for performing the inventive process has an essentially porous support 2 being penetrable for liquids and at least two zones 3, 4 having at least one capturer for at least one analyte, wherein said at least one analyte is indirectly or directly bonded to the capturer and the zones 3, each have previously defined bonding capacities for said at least one analyte which are essentially equal and the capturers for the respective analytes are identical. A typical test strip is illustrated in Figure 4.

_g_ The inventive device for performing the inventive process accommodates the inventive test strip. The device has a housing wherein the test strip is arranged.
The housing consists of a top side having at least one opening at the sites of said at least two zones of the test strip, said top side extending over the area of said at least two zones of the test strip, and at least one additional opening for applying at least one sample to be tested and/or auxiliary agents for pertorming an assay, said additional opening enabling the liquid to contact the test strip.
Preferably, the device has an additional opening for taking up auxiliary reagents.
In another embodiment of the inventive device the device has a number of openings corresponding to the number of zones of the test strip instead of said one opening 12 extending over the complete area of zones 3, 4 of the test strip.
To be able to read off the result in the test strip zones, the openings in the housing have essentially to be congruent with the test strip zones.
Figure 3 illustrates a preferred embodiment of the inventive device. Here, the top side 11 of the housing 10 has openings at several sites. At the sites of said at least two zones 3, 4 of the test strip 1 an opening 12 is provided in the top side il of the housing 10 extending over the area of at least two zones 3, 4 of the test strip 1. Furthermore, there is provided an opening 14 through which the application of the sample to be investigated is effected. Optionally, also auxiliary agents for pertorming the inventive process can be added through opening 14. The opening 14 has a wall and is connected to the test strip 1.
After being applied, the sample soaks in the test strip due to the porosity thereof and migrates in lateral direction through the test strip and consequently through zones 3, 4 due to capillary forces. The size of the opening 14 determines the volume the opening can take up. The sample volume is precisely defined by the exact diameter and the height of the opening. If the volume provided by opening 14 is too small for a sample application, the sample uptake volume is increased by a tube which can be inserted into the opening. In particular, by inserting a tube having a defined volume (defined wall thickness and length) it is possible to provide a defined volume which is matched to the quantitative or semiquantitative assay which can be performed using the inventive device. The additional opening 15 is suitable for taking up auxiliary agents.

- i l) -The opening 14 can be adapted to the respective requirements by additional supplements. If, e.g., a commercial membrane being capable of separating blood cells from plasma due to the defined pure size thereof is positioned directly beneath the opening 14 and in contact therewith, an analysis of plasma parame-ters can directly be performed in the test strip. This membrane may be, e.g., a glass fiber filter having a suitable density or a plastic membrane having narrow channels, preferably asymmetric channels, enabling the blood to soak in. Thus, the demands on a short-time assay in on-site analytics are met.
In this manner impairing parameters can selectively be filtered off. If the opening 14 is filled with, e.g., aluminium oxide, a sample can directly be degreased, which is very important in food analytics. In a similar manner, particles can be removed by the use of a filter membrane or soil samples can directly be investigated by the use of a filter together with an absorbing medium (for example activated carbon).
The inventive test strip can be prepared by procedures well-known in the art (WO-A-95/13542). Also WO-A-96/09546 and EP-A-0 291 194, which are incorporated herein by reference, pertain to the preparation of test strips which may also be employed according to the present invention. The system provided by the invention is advantageous in that it permits the preparation ~ of test strips which may be coated by the customer himself. For this, the zones on the test strip serving for the detection of the analyte are merely preactivated, e.g., by a chemical activation or the application of immunoreactive substances, e.g., protein A, protein G, streptavidin, etc. Then, the user has only to modify hls analyte by complementary structures or to bond antibodies for the analyte in the zones in order to use the test strips for his purposes.
The invention will be described more detailed by the following examples:

Example 1 The quantitative determination of C-reactive protein is performed as follows:
the reactive zones 3, 4 and a respective third zone are charged with 2.3 ug each of an anti-human CRP rabbit antibody on a cellulose nitrate strip 4 cm in width.
Subsequently, the surplus reactive groups of the cellulose nitrate are saturated with skimmed milk. Subsequently, a test strip consisting of said cellulose nitrate, thereon a porous glass fiber strip for taking up the anti-CRP gold conjugate and a bfood/plasma-separating glass fiber membrane are mounted on a self-adhesive plastic sheet. After the application of thick paper strips for taking up the required washing liquids, strips 5 mm in width are placed in a housing according to figure 3.
In order to perform the analysis, the opening 14 is filled with 20 ul of blood (this reproducibly corresponds to an plasma amount reaching the test strip suited for carrying out the test). After 2 min opening 15 is filled with buffer.
In this embodiment, the plasma is passed through the three reactive zones. The buffer first passes the glass fiber filter comprising the lyophilized gold-antibody conjugate and from there through the zones to which CRP is already bonded.
The gold conjugate bonds to the sites where CRP is present and tl-us generates a coloration.
Surplus reagent and other substances are washed out by the buffer which is added subsequently. The result can be read off after 5 min.
Example 2 Also troponin I and CK-MB can be measured in parallel in a manner analogous to that of example 1. Although both parameters are important indicators of a cardiac infarction, the amounts thereof increase differently in time according to the reason of the myocardial injury such that the parallel determination thereof provides important diagnostic information. For the determination thereof, two zones each containing a monoclonal murine anti-human troponin I and a CK-MB
antibody are applied. The difference with respect to the test according to example 1 is that lyophilized antibody enzyme conjugates are used instead of the antibody gold conjugate. Said antibodies are directed against a second domain of the analyte. In this case anti-troponin I is conjugated with peroxidase, anti CK-MB with alkaline phosphatase.
Subsequent to the reaction BCIP/INT (a phosphatase reagent leaving an orange precipitate at the phosphatase site) and TMB (a peroxidase reagent, a blue colorant) are introduced in opening 15. Subsequently, the test may be analyzed according to the description of example i.

Claims

1. A linear capture assay process for the differential determination of the amount of at least one analyte in a liquid sample with the use of a test strip having at least two zones configured such that they are capable of enabling a statement on the amount of said at least one analyte and the test strip ensures a lateral flow of the sample or a sample portion through the strip and said at least two zones due to the fact that a first zone has a previously defined capacity for binding said at least one analyte and a bonding of said at least one analyte takes place in at least one second zone if this capacity is exceeded by an amount of said at least one analyte being too large for this capacity, wherein the capacity of said at least second zone is essentially equal to the capacity of the first zone and the bonding of said at least one analyte in said at least first and at least second zones is measured by a detectable property.

2. The process according to claim 1, wherein the amount of said at least one analyte is also detectable by an activity of said at least one analyte.

3. The process according to claim 1 and/or 2, wherein the bonding of said at least one analyte at the solid phase is specific.

4. The process according to claim 3, wherein the specific bonding of said at least one analyte is effected by affinity, in particular immunoaffinity.

5. The process according to any one of claims 1 to 4, wherein the defined capacity for bonding said at least one analyte to the solid phase is deter-mined as a function of the amount of analyte to be expected in a sample to be analyzed.

6. The process according to claim 5, wherein the difference between the capacity of the respective capture components in the first and second zones does not exceed ~10 %, in particular ~5 %.

7. The process according to any one of claims 1 to 6, wherein said at least one analyte is C-reactive protein (CRP), rheumatoid factor, troponin I, creatinine kinase (CK-MB), myoglobin; antigens of the organisms of Salmonella, legionella, E. coli (EHEC etc.), Aspergillus flavus and fumigatus; glucose, dioxin, PCB, cocaine (ecgonine), heroin, amphet-amine.

8. The process according to any one of claims 1 to 7, wherein two or more analytes are determined.

9. The process according to claim 8, wherein CRP and antistreptolysin;
troponin I and CK-MB; myoglobin and CK-MB; CK-MB, myoglobin, and troponin I; cocaine, heroin, and amphetamin; Salmonella and E.coli are determined as analytes.

10. The process according to any one of claims 1 to 9, wherein the solid phase is selected from the group comprising cellulose nitrate, nylon, PTFE, polystyrene, latex, glass fibers, silica gel, aluminium oxide, cellulose, dextran, agarose, polyvinyl alcohol, also in the form of micro-beads or "magnetobeads", optionally preactivated by BrCN, NPCF. NHS
chloroformate, tresyl chloride, squaric acid esters, and/or corresponding chlorosilanes.

11. A test strip (1) for performing the process according to at least one of claims 1 to 10 on the basis of an essentially porous support (2) being penetrable for liquids and at least two zones (3, 4) having at least one capturer for at least one analyte, wherein said at least one analyte is indirectly or directly bonded to the capturer and the zones (3, 4) each have previously defined bonding capacities for said at least one analyte which are essentially equal and the capturers for the respective analytes are identical.

12. The test strip (1) according to claim 11 comprising as said porous support (2) a layer from cellulose nitrate, nylon, PTFE, polystyrene, latex, glass fibers, silica gel, aluminium oxide, cellulose, dextran, agarose, polyvinyl alcohol, and/or zones from micro-beads or "magnetobeads" being option-ally preactivated by BrCN, NPCF, NHS chloroformate, tresyl chloride, squaric acid esters, and/or corresponding chlorosilanes.

13. A device having at least one test strip (1) according to any one of claims 11 and/or 12, wherein the test strip (1) is arranged in a housing (10), the housing (10) has a top side (11) which has at least one opening (12) at the sites of said at least two zones (3, 4) of the test strip (1), wherein the opening (12) extends over the area of said at least two zones (3, 4) of the test strip (1) and at least one additional opening (14) for the applica-tion of at least one sample to be tested and/or auxiliary agents for performing an assay, in particular with a wall extending to the test strip and enabling a liquid to contact the test strip (1).

14. The device according to claim 13 having an additional opening (15) for the uptake of auxiliary reagents, wherein the opening (15) is positioned either between the openings (12) and (14) or before the openings (12) and (14).

15. The device according to any one of claims 13 or 14, wherein the opening (12) is replaced by at least two openings (16, 17) being essentially congruent with the position of said at least two zones (3, 4) of the test strip (1).