Test kit (combined quick test) for the synchronous proof of biomarkers in faeces for detecting of pathological changes in the gastrointestinal tract, particularly in the intestine

Abstract

The invention relates to a test kit for better carrying out a method for detecting biomarkers in human or animal stool, which can serve as an indication of a pathological, particularly a malignant event in the gastrointestinal tract (esophagus, stomach, small bowel, biliary tract, pancreas, and bowel). The invention teaches a novel and more efficient methods, uses and embodiments of a combined rapid test. The combined rapid test cassette used for implementing the test kit and the optimally coordinated reagents thereof contains two lateral flow test strips for the synchronousin the technical meaningdetection of the biomarkers M2-PK and the biomarker hemoglobin. The test serves as a dual filter for diagnosing probands as part of a colon cancer screening program. The test is very cost-efficient and cuts costs in the health system by the examination at an early stage of colon cancer and the consequences thereof.

Claims

1. A test kit for detecting biomarkers in human stool, consisting of a first sample tube, having a first stool sampling device, a first buffer solution, having buffer 1=10-70 mM phosphate buffer with a pH 6.7 to 7.6 or buffer 2=10-70 mM HEPES buffer with a pH 7.6 to 8.2 or buffer 3=10-70 mM triethanolamine with a pH 7.3 to 7.7 or buffer 4=10-70 mM acetate buffer with a pH 5.7, or mixtures thereof, a second sample tube, having a second stool sampling device, a second buffer solution, buffer 1=10-70 mM phosphate buffer with a pH 6.7 to 7.6 or buffer 2=10-70 mM HEPES buffer with a pH 7.6 to 8.2 or buffer 3=10-70 mM triethanolamine with a pH 7.3 to 7.7 or buffer 4=10-70 mM acetate buffer with a pH 5.7, or mixtures thereof, a test cassette, having a lateral flow test system with a nitrocellulose membrane as the stationary phase, the nitrocellulose membrane, in turn, having monoclonal tumor M2-PK mouse antibody clone PAT4M3AT, IgG1, and gold-coupled monoclonal mouse antibody clone 1 E3, IgG1, and monoclonal hemoglobin mouse antibody clone M1202100, IgG1, and gold-coupled monoclonal mouse antibody clone HB11-2312, a first opening for applying a stool sample from the first sample tube, a second opening for applying a stool sample from the second sample tube, wherein the analysis result of the first stool sample is positive if the amount of tumor M2-PK is greater than 41 units/ml stool extract, and the analysis result of the second stool sample is positive, if the content of hemoglobin exceeds 24 g hemoglobin per gram of stool.

2. The test kit according to claim 1, wherein the detection of tumor M2-PK and/or hemoglobin is carried out by means of specific monoclonal or polyclonal antibodies, which do not cross-react with other components of the stool.

3. The kit according to claim 1, wherein both buffer solutions include an acetate buffer having a pH of 5-6.

4. The test kit according to claim 1, wherein the determination of hemoglobin of the second stool sample is carried out on the basis of a hemoglobin/haptoglobin complex.

5. The test kit according to claim 1, wherein an antibody is used for determining tumor M2-PK which is selected from the group consisting of monoclonal tumor M2-PK mouse antibody clone PAT4M3AT, IgG1, and gold-coupled monoclonal mouse antibody clone 1 E3, IgG1.

6. The test kit according to claim 1, wherein an antibody is used for determining hemoglobin, which is selected from the group consisting of monoclonal hemoglobin mouse antibody clone M1202100, IgG1, and gold-coupled monoclonal mouse antibody clone HB11-2312.

7. The test kit according to claim 1, wherein a capture antibody is bound to gold colloids.

8. The test kit according to claim 1, wherein a four possible test results are represented by four different colors, letters, numbers, characters, and/or geometric shapes.

9. The test kit according to claim 1, wherein the detection of tumor M2-PK and/or hemoglobin does not cross-react with other pyruvate kinase isoenzymes.

10. The test kit according to claim 1, wherein both buffer solutions include an acetate buffer having a pH of 5.7.

11. The test kit according to claim 9, wherein the isoenzymes are M1-PK, M2-PK in tetrameric form, L-PK, R-PK.

Description

EXAMPLES

(1) The invention is further explained by the following examples.

Example 1

Test Kit

(2) The test kit based on an immunoassay consists of two sampling and preparation devices and a test cassette.

(3) Both sampling devices contain a rod that is capable of receiving the required amount of stool (4-30 mg, preferably 25 mg). The sampling devices further include one tube each for receiving samples, which are filled with buffer solution.

(4) The aqueous buffer solution for the tumor M2-PK test has the following components:

(5) Buffer 1=10-70 mM phosphate buffer (pH 6.7 to 7.6) or buffer 2=10-70 mM HEPES buffer (pH 7.6 to 8.2) or buffer 3=10-70 mM triethanolamine (pH 7.3 to 7.7), preferred mixtures thereof volume 1:1:1 each.

(6) Detergent 1=CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate, 10 mM-50 mM (Sigma), detergent 2=sodium dodecyl sulfate (SDS) 0.01%-0.1%), for example from Biochrom, detergent 3=lauryldimethylamine oxide (10 mM-50 mM), for example from Biochrom, or mixtures thereof.

(7) The aqueous buffer solution for the hemoglobin test contains the following components:

(8) Buffer 1=10-70 mM phosphate buffer (pH 6.7 to 7.6) or buffer 2=10-70 mM HEPES buffer (pH 7.6 to 8.2) or buffer 3=10-70 mM triethanolamine (pH 7.3 to 7.7), or mixtures thereof.

ANTIBODIES USED

(9) The four antibodies required for the measurement (one each in the liquid phase and one antibody in the solid phase) being antibodies capable of specific binding may be polyclonal antibodies, preferable however monoclonal.

(10) The polyclonal and monoclonal antibodies are obtainable according to prior art by the classic methods of immunizing animals with the respective antigen or, preferably, by using the hybridoma method of Koehler and Mielstein.

(11) Polyclonal and monoclonal antibodies that bind the human pyruvate kinase and also the isoenzymes of the pyruvate kinase, belong to prior art. Preferred are polyclonal antibodies and monoclonal antibodies that bind the dimeric form, i.e. the tumor form of the pyruvate kinase.

(12) The quaternary structure of a protein relates to the spatial arrangement of the subunits of the protein.

(13) Normal M2-PK has a tetrameric structure (quaternary form). The M2-PK of healthy persons is different from the tumor M2-PK. Tumor M2-PK has a dimeric structure.

(14) Polyclonal antibodies and monoclonal antibodies against these special forms (tumor M2-PK dimeric or M2-PK tetrameric) are obtainable by conventional immunization methods, as well as by the hybridoma method (Kohler-Mielstein technology).

(15) Thus, the tumor form of M2-PK can be cleaned and used as an antigen. Another option is to buy genetically expressed M2-PK also in the tumor form and to use it for immunization. Another way is to synthesize specific fragments, i.e., amino acid sequences and to use them for the immunization.

TUMOR M2-PK ANTIBODIES

(16) Preferred for spraying onto the nitrocellulose membrane is a monoclonal mouse antibody (clone PAT4M3AT, IgG1). This clone has been generated by hybridization of myeloma cells with B lymphocytes of the mouse. Recombinant human tumor M2-PK from E. coli with amino acids 1-531 was used as the immunogen. The antibody is available, amongst others, from the company ProSpec (East Brunswick, USA). Optionally, a monoclonal mouse antibody (clone AT1 E3, IgG1) can be used. Recombinant human tumor M2-PK was used as the immunogen. The antibody is available, amongst others, from the company Novus Biologicals (Littleton, USA). Particularly preferred for the coupling to the gold is a monoclonal mouse antibody (clone 1 E3, IgG1). Recombinant human tumor M2-PK with the amino acid seq 47-574 was used as the immunogen. The antibody is available, amongst others, from the company Novus Biologicals (USA). Optionally, a polyclonal antibody generated in sheep (Ig fraction) from the company Randox (United Kingdom) can be used. Recombinant human tumor M2-PK from E. coli served as the immunogen.

HEMOGLOBIN ANTIBODIES

(17) Preferred for spraying onto the nitrocellulose membrane is a monoclonal mouse antibody (clone M1202100, IgG1). The antibody was produced by immunizing with human hemoglobin. The antibody is available, among others, from the company Thermo Scientific (Rockford, USA). Alternatively, a monoclonal antibody against human hemoglobin can be used. This antibody from the clone 7202 SPR-5 has an affinity constant of 110.sup.10 l/mol and an isoelectric point of 5.8. It can be obtained from the company Medix (Finland). For the hemoglobin antibody coupled to gold, the monoclonal mouse antibody (clone HB11-2312) was used. Purified human hemoglobin was used as the immunogen. The antibody can be obtained from the company Thermo Scientific (Rockford, USA).

(18) One of the most important conditions for the selection of the antibody is that it does not cross-react with other components of the stool, especially not with other pyruvate kinase isoenzymes (e.g. M1-PK, M2-PK (tetrameric form) L-PK, R-PK).

(19) The above hemoglobin antibodies are particularly suitable for binding to a membrane (solid phase), in a preferred form nitrocellulose. The antibodies mentioned above do not cross-react with hemoglobin from swine, horse, sheep, and cattle. The above hemoglobin antibodies are preferably suitable for binding to latex or nanoparticlespreferably gold colloids(liquid phase). They do not cross-react with hemoglobin from sheep, horse, cattle, or swine. The antibodies bind hemoglobin A0 and A1 equally well, A2 partially with a lower binding constant, and AS partially with a very poor binding constant. Solid phase antibodies are preferably mixed 1:1 w/w. Liquid phase antibodies are preferably mixed 1:1 w/w.

(20) Detergent 1 (CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM (Sigma)), detergent 2=sodium dodecyl sulfate (SDS) 0.01%-0.1%), e.g. from, detergent 3=lauryldimethylamine oxide (10 mM-50 mM), e.g., from Biochrom, or mixtures thereof.

(21) The sampling device for carrying out the hemoglobin test contains one of the buffers described above (1.5-2.5 ml). The person being tested moves the dosing tip into the stool and transfers the stool sample into the empty tube filled with the corresponding buffer.

(22) The sampling device for the tumor M2-PK test that the physician hands over to the person to be tested does not include any buffer. The patient moves the dosing tip into the stool and transfers the stool sample into the empty tube.

(23) The test kit according to the invention further includes a test cassette, in which the two immunoassays being improved over prior art are carried out by lateral flow technology. For this purpose, the test cassette contains two preferably circular recesses for the application of the stool samples and two preferably elongate recesses for reading the test results.

(24) The test cassette contains in its interior nitrocellulose with a preferred capillary flow rate of 135 sec/4 cm as the stationary phase.

(25) During the investigations for the development of the test kit according to the invention, it has been found out that nitrocellulose has a strong influence on the spread of the results. This spread is particularly large in freshly prepared nitrocellulose, but becomes smaller after three to six months storage (maturation). The nitrocellulose used in the present invention has been stored for three to six months before being subjected to further testing. The use of such matured nitrocellulose enables a significant reduction in the spread of the results. In particular, it could be achieved that significantly lower variations occur between different batches of the produced test kits than for fresh nitrocellulose.

(26) The antibodies are coupled to colloids, preferably gold colloids or latex colloids, preferably gold colloids with 20 nanometers in diameter.

(27) The stationary phase=nitrocellulose (preferably with a capillary flow rate of 90 sec/4 cm) contains hemoglobin antibody 1, hemoglobin antibody 2, preferably in a mixing ratio as specified above.

(28) The stationary phase =nitrocellulose (preferably with a capillary flow rate of 135 sec/4 cm) further includes tumor M2-PK antibody (from clone P1F3), tumor M2-PK antibody (from clone P5A1), preferably a mixture of antibodies of clones P1F3 and P5A1 in the following mixing ratio volume 1:3.5 w/w.

(29) Antibodies from clone P1F3 and clone P5A1 are particularly useful as capture antibodies (stationary phase) for binding to a membrane, preferably nitrocellulose. Tumor M2-PK antibodies from clone P1A6 are preferred for binding to gold colloids or latex colloids, preferably gold colloids with 80 nanometers in diameter.

(30) Devices and protocols for the test kit production, especially for the production of the two test strips: Sealing devicefilm sealing device (company Kopp), packaging systems company Reichenbach. Sealing temperature 60, Type MSC 440 watts. Biodot device. 2. Biodot device. Cutter of the company Zeta Corporation, www.zetacorporation.com, device type GC1800-081101. Device is CE marked. Protocol for laminating and cutting the nitrocellulose membrane number of laminated M2-PK cards. Device setting and operation laminator Matrix 2210 of the company Kinematic. Protocol for laminating and cutting the nitrocellulose membrane devices setting, operation laminator Matrix 2210. Implementation device settings and operation. Device settings and operation cutter GSI-800 of the company Zeta Corporation. Production of cassettes and installing the ScheBo M2-PK Quick Kits. Production of test cassettes. Storage of partially finished products in refrigerators. Quality control. The temperature and humidity of the premises, where the production is made, is recorded. Air conditioner FUJITSU DG Inverts for air conditioning and keeping the humidity and temperature constant. Compliance with the air temperature and humidity is of particular importance for the quality assurance of the test membranes. Selecting the operating mode/setting the thermostat 1, setting the fan speed.

(31) Critical success factors for the optimum coordination of the two test strips in the combined cassette are in particular:

(32) a) Heidelberger curves for Hb determination,

(33) b) Heidelberger curves for M2-PK determination.

(34) Surprisingly, it has been found that when 6.7% ox gall (natural wetting agent, cleaned, from the company Schmincke, Part No. 50031, Schmincke, Erkrath, Germany) is admixed as a final concentration to the extraction buffer for M2-PK, particularly good reproducible immunochromatographic results are obtained.

(35) Surprisingly, it has been found that when 2.8% ox gall is admixed as a final concentration to the extraction buffer for hemoglobin, particularly good reproducible immunochromatographic results are obtained.

(36) Technical Synchronous Determination of the Two Analytes in the Combined Cassette.

(37) For optimization of the time shift of the chromatography start time, the user starts the chromatography by dripping M2-PK stool sample extract into the left sample window of the cassette (then sets the timer to 5 minutes). Dripping of the Hb stool sample extract occurs after 1 minute after dripping the M2-PK stool extract into the right sample window of the cassette. I.e. the two chromatographies are started at shifted times. The result of the two tests is, however, read after expiration of the 5-minute stop time.

(38) Chromatography, particularly immunochromatographic test methods dependas the name impliessubstantially on the time as well as the binding affinities (the binding of the antibodies to the antigens in the liquid and solid phase, as well as the various kinetics within the nitrocellulose membrane), i.e. they are subjected to various kinetics.

(39) Test results, which are read later, are invalid! (All this is explained in the description of the combined test). The formation of the pink-red test line in the test region of the nitrocellulose test strip of the test membrane is biophysically the result of a complex agglutination reaction (various kinetics and affinities play a special role here, see also the Heidelberger curve to be described in more detail above).

(40) Parallel, i.e. synchronous, in the technical meaning, processing or determining of the analytes according to the invention.

(41) 1. Start of the chromatography M2-PK followed by a second start of the chromatography Hb.

(42) The synchronous determination, in the technical meaning, is a challenge, since the immunochromato-graphic determinations are very complex.

(43) Many parameters are of particular importance for achieving the multiple objects according to the invention:

(44) (All objects have been achieved at the ScheBo Biotech AG).

(45) These objects have been achieved, according to the invention, by, e.g., the identification of specific/essential active pairings:

(46) 1. Binding constant/affinity of the individual antibodies to the respective substances (proteins), the analytes according to the invention.

(47) Another critical factor is also the composition of the dried gold and the release of the gold from the PAD release, i.e. the resolution of the dried gold particles (in a glass-fiber matrix/dried sugar matrix) by the dropwise addition of the extraction buffer, which contains the corresponding analyte (stool sample extract).

(48) Preparation of the test kit according to the invention containing a dual cassette for immunochromatographic determination of two analytes.

(49) In order to accomplish this, we first tested the immunochromatographic determination of the two analytes in a so-called dipstick format. This dipstick format is much less complicated, since it does not contain the conjugate release PAD.

(50) The following tasks and challenges had to be solved according to the invention:

(51) Nearly none of the technical solutions according to the invention were obvious.

(52) General Remarks:

(53) For the preparation of the test strips, it is of particular importance to keep the humidity between 40 and 60% at 18 C. to 25 C.

(54) By series of tests performed in the R&D department at the ScheBo-Biotech AG, an optimum humidity of 35% at 20 C. was determined in an inventive manner.

(55) Tasks/Challenges:

(56) 1a. Slowly flowing liquid in the membrane or even stop/interruption of the chromatographic liquid flow.

(57) 1b. The sample in the liquid stops in the middle (in the flow direction of the membrane) of the membrane strip.

(58) Is the selected membrane too slow in flow speed for the application?

(59) Was the membrane turning hydrophobic?

(60) Has the correct wick been used?

(61) The nitrocellulose membrane contains surfactants and wetting reagents.

(62) The test membrane has a high background staining (this background staining was solved by non-obvious solutions according to the invention).

(63) It is necessary to clarify whether specific blocking reagents are to be used.

(64) Set the release of gold onto the nitrocellulose membrane in an optimum and complete manner (solved by non-obvious solution according to the invention).

(65) The liquid front is not straight-lined (solved by non-obvious solution according to the invention).

(66) The test line is too thick or fuzzy, blurred (solved by non-obvious solution according to the invention).

(67) A much too weak signal at the test line is reproducibly observed/found (solved by non-obvious solution according to the invention).

(68) The test line shows false positive/false negative results (solved by non-obvious solution according to the invention).

(69) The test kit according to the invention contains metal colloids, preferably gold colloid. Specific embodiments are, e.g., latex particles, and also nanoparticles. Latex particles may be stained and also provided with fluorescent dyes and have different sizes (i.e. size in the micrometer range but also in the nanometer range).

(70) Magnetic particles (initiate quantification using special reading devices (readers)) are also possible.

(71) The major technical challenges were specifically related to quality assurance. Creation of good-manufacturing protocols (solved by non-obvious solution according to the invention).

(72) Object/task: achieving an optimum, small inter- and inter-assay variance. Particularly important: very good lot-to-lot variance (i.e., a smallest possible lot-to-lot variance).

(73) This small lot-to-lot variance is to be ensured in the context of quality assurance in the production process according to the invention (solved by non-obvious solutions according to the invention).

(74) Production of a large number, more than 100,000 test kits with the same lot number, the same date of expiry (solved by non-obvious solutions according to the invention).

(75) Challenge: ensuring extremely good qualitybenefit for the customer (solved by non-obvious solutions according to the invention).

AGGLUTINATIONDEFINITION

(76) In the medical field, agglutination designates the adhesion or clumping of cell pathogens, etc.

(77) Agglutination reactions are used in laboratory diagnostics to make quantitative statements. The agglutination is measured by nephelometric determination methods.

(78) The red test line is formed in a complicated kinetics by an agglutination reaction. In the three-dimensional structure of the nitrocellulose of the test strip, there occurs an agglutination. This agglutination is visible to the naked eye as a pink line.

(79) This pink-red line is the result of a substance detection by an antigen/antibody reaction. This antigen/antibody reaction is subjected to a variety of effects. One of these effects is the so-called hook effect (also referred to as high dose hook effect), which takes place in falsely low determinations of analytes that occur in very high concentrations of sample solutions (i.e., high concentrations of the analyte in the particular case may feign false negative measurement signals).

(80) Once the analyte concentration is too high, the antibody binding sites may be occupied by the analyte and the additional analyte molecules are no longer identified in the binding curve. There will be falsely low readings.

(81) A technical object was to avoid the hook effect.

(82) By parallel measurements of various dilutions of a sample, the presence of a high-dose effect can be noticed, and the measurement can be corrected accordingly (the object of the technical problem has been solved by non-obvious solutions according to the invention).

(83) The test kit according to the invention described in the claims has, in contrast to the embodiments of prior art, no high dose hook effect. This could be shown in a convincing manner by tests with samples having tumor M2-PK concentrations exceeding the cut-off by more than 200 times (up to 160,000 ng/ml).

(84) In general, the immunobioanalytic detection methods, especially the immunochromatographic methods, are subject to the following failures: 1. Failures caused by cross-reactivity and nonspecific binding. 2. Failures by matrix effects. 3. Failures by anti-animal antibodies. 4. Failures by endogenous components of the sample. 5. Failures by heterophiles and other cross-linking interferers.

(85) (These failures 1-5 could be solved by non-obvious solutions according to the invention).

(86) Of particular interest was the use of a special LowCross Buffer (produced by ScheBo Biotech AG). It includes various detergents, proteins, polyclonal antibodies, surface-active substances as well as substances, which change the surface tension.

(87) All this together with the above-described reagents, substances, etc. of the test kit according to the invention results in an optimum coordination of active pairings in the technical meaning. All this allows the synchronous determination of the two analytes in a combined test cassette.

(88) Surprisingly, it was found that for the solution of the common, synchronous (in the technical meaning) determination of the two analytes on the test strips, the humidity control and the degasification of the liquids when precisely dispensing antibody liquids and colloidal gold was of crucial importance.

(89) Creative, but also systematic working was necessary in order to provide the test kit according to the invention.

(90) Extensive test series were necessary for detecting active pairings and their common, optimum coordination.

(91) This is a basic requirement in order to develop a robust, reproducible and reliable test in the form of a rapid test (platform and/or point-of-care test).

(92) With the extremely large variability of the samples of the available material (stool probands), reagents and antibodies, two pairs each have to be coordinated with each other to obtain step by step reproducible results.

(93) When designing and developing such a complex platform, one comes across numerous physical and chemical phenomena that can affect the test result in an unexpected manner.

(94) Another embodiment of the test kit is the use, after testing all optimum, essential active pairings, of the qualitative, semi-quantitative, and quantitative determination of the analytes preferred according to the invention (biomarkers by means of other immunochemical test principles such as ELISA), agglutination, preferably the use of nanoparticles for initiating the agglutination in a liquid phase.

(95) Turbometric, nephelometric measurement technology according to the state of the art allows the generation of measurement data. These measurement data can be processed using appropriate computer software. By means of an internal calibration curve (also depending on the batch), for example, a determination of the two analytes according to the invention is possible. Various graphical representations, such as log-log, log-decadic representation, are also possible.

(96) A particular embodiment allows statistical evaluations, e.g., algorithms from the fields of fuzzy logic analyses, classification and pattern and image recognition, explorative data analysis, data visualization, robust and computer-supported statistics, initial data analysis (IDA), and evolving systems (ES), data mining and exploratory data analysis, fuzzy systems, neuronal networks, evolutionary algorithms. Preferred are the algorithms of Prof. Dr. Frank Klawonn. Prof. Klawonn has developed good evaluation strategies that improve data reliability. Prof. Klawonn is the director of the Institute for Applied Computer Science (Institut fr angewandte Informatik).

(97) Another embodiment provides an interface to WLAN.

(98) Temporal progresses of patients are also possible.

(99) An individual patient data series is possible for evaluation.

(100) Other apparatus, appliances, etc. for the measurement of the two analytes are conceivable.

(101) Other embodiments platforms ELISA are conceivable.

(102) Various arrays: liquid (Luminex) or solid phases (arrays) are possible. All immunochemical detection methods are conceivable.

AGGLUTINATION

(103) Surprisingly, it was found that the droplet size (volume in the nanoliter range) and the distance of these droplets is crucial.

(104) The droplet size and the distance between these droplets is of particular importance. There is thus not dispersed a line, but individual droplets. The capillary forces of the nitrocellulose membrane absorb droplets in fractions of seconds and cause a line to be formed. The antibodies present in the liquid are distributed in the three-dimensional structure of the nitrocellulose.

(105) Surprisingly, it was found that for achieving a very precise line, the drying level is crucial.

(106) Surprisingly, it was also found that the hydrophobicity is very, very important. To ensure that the nitrocellulose membrane to be used is dry, it was previously sealed in aluminum films that are coated on one side and that do not let pass any humidity, opened, provided with antibodies and immediately resealed in the aluminum films plus drying agent. Surprisingly, these production steps were important and were carried out in 6-minutes cycles.

(107) Surprisingly, it was remarkable that in order to ensure a constant flow over the membrane, the construction of the test strip was made in the proper order.

(108) In the reaction zone of the test strip (test resuit line region), the corresponding lines (pink-red lines) are formed.

(109) The colored test result line (a positive result) is the result of an antigen/antibody reaction, and the determined Heidelberger curve shows the measurement signal as a function of antibody excess and antigen excess. It is the aim to use the equivalence range according to the Heidelberger curve.

(110) The optimum use of the antigen/antibody reaction, i.e., the coordination of the stationary antibodies with the antibody-coupled gold particles present in the liquid phase in the equivalent area of the M2-PK test strip provides an optimum signal for M2-PK on the test strip.

(111) The optimum use of the antigen/antibody reaction, i.e., the coordination of the stationary antibodies with the antibody-coupled gold particles present in the liquid phase in the equivalent area of the Hb test strip provides an optimum signal for Hb on the test strip.

(112) The optimum use of the antigen/antibody reaction, in the equivalent area of the one M2-PK test strip and in the equivalent area of the Hb test strip was the object of the present invention.

(113) The object of the present invention is achieved by the optimum common coordination of active pairings, in particular by the proper choice of the antibodies, the proper choice of detergents, etc., but in particular by the reproducible, high-precision dispensing of the antibody solutions on the two nitrocellulose test membranes.

(114) This allowed the synchronous detection of the two analytes in stool samples from probands. All these active pairings and the technical solutions are described in the application. For this purpose, often non-obvious solutions were needed, lucky coincidences also led to positive results.

(115) The hemoglobin test tube and the M2-PK-tube must be handed over within 48 hours in the physician's office. The dosing tip with the stool sample is transferred into a tube filled with the above-described buffer. Both the tube for the M2-PK test and the tube for the hemoglobin test are vigorously shaken.

(116) The prepared stool sample extracts are applied immediately after each other on the two recesses. After 5-10 minutes, it is read with the aid of the marked line, whether one or both of the measured biomarkers are positive.

(117) Crucial for the reliable determination of a malignant event in the gastrointestinal tract is the choice of so-called cut-off. The cut-off marks the minimum concentration of the analyte in the sample, indicating a positive result. The cut-off of the hemoglobin test in the test kit according to the invention is between 18 and 38 g of hemoglobin per gram of stool, preferably 24 g of hemoglobin per gram of stool. The cut-off for tumor M2-PK is 3-6 units/ml of stool extract, preferably 41 units/ml.

(118) The detection limit is 150 ng hemoglobin/ml stool extract.

(119) The evaluation of the test result and the assignment to the risk levels, as shown for example in

(120) FIG. 10, can be made manually, semi-automatically or fully automatically. For example, a test cassette according to the invention can be manually loaded to a read-out device, which carries out an evaluation by means of a photocell or camera and then displays the result on a monitor. Preferably, the representation takes place in colors (risk impact scheme). Particular preferred is, for safety reasons, a parallel representation in another form (e.g., risk levels 1-4, or similar).

(121) Of course, the test device may also be configured such that a fully automatic evaluation is possible, e.g., by detection of the gold particles by electro-chemical means or by an optical recording (scanning) of the test kit.

Example 2

(122) The combined rapid test cassette according to the invention does not contain the lateral flow test strip existing on the market to determine the M2-PK, but a novel, improved lateral flow test strip according to the invention for determining the M2-PK (tumor M2-PK plus), e.g. Sartorius.

(123) Furthermore, the combined rapid test cassette does not contain a lateral flow test strip existing on the market for determining hemoglobin, but a novel, improved, lateral flow test strip according to the invention for determining hemoglobin (iFOB plus), e.g. Sartorius.

(124) The invention relates in particular to the combined rapid test and components, as well as all the necessary reagents for synchronously carrying out the immunochromatographic detection method. Synchronously in the linguistic meaning of at the same time, but synchronous also in the technical meaning of the common, optimum coordination of the chromatographic conditions for detecting the two biomarkers M2-PK and hemoglobin in a combined rapid test cassette.

(125) Detection Reagents:

(126) Various detection reagents may be used: latex beads, colloidal gold particles, colloidal silver particles, etc. One of the most important properties of these particles is that the population must be monodisperse with a constant spherical size.

(127) Preferred, according to the invention, are gold particles.

(128) The production of colloidal gold particles is in principle well known. Typically, a solution containing Au.sup.3+ is chemically reduced under rapid stirring, so that atomic gold particles precipitate, which aggregate in the course of time. Aggregation can be prevented by stabilizing agents. By choosing the right additive, the size of the colloids formed can be set. As an Au.sup.3+ source, H[AuCl.sub.4] is often used. Sodium citrate solution, sodium borohydride, or hydroquinone can be used as reducing agents. For stabilization, often sulfur compounds (such as alkanethiols) may be used. Solutions containing gold particles are available from various sources.

POLYMER COMPOSITION AND PROTEIN BINDING

(129) For the production of lateral flow tests, nitrocellulose, polyvinylidene fluoride, charge-modified nylon, polyethersulfone are used.

(130) The polymer surface size is dependent on pore size, porosity, thickness, and other structural characteristics.

(131) The surface increases non-linearly with the pore size, but increases linearly with thickness, and non-linearly with porosity. The protein binding to a given surface area depends on the density of the protein, its structure, and its Stokes radius (effective diameter). Furthermore, the binding of the protein to the polymer is dependent on the pH and the immobilization solution.

(132) In the combined rapid test according to the invention, preferably cellulose was used for the so-called sample pad, nitrocellulose for the test membrane, and cellulose for the absorbent pad.
Conjugate/sample pad=22 mm (+/0.2 mm)5 mm (+/0.2 mm).
Nitrocellulose membrane=25 mm (+/0.2 mm)5 mm (+/0.2 mm).
Absorbent pad=16.5 mm (+/0.2 mm)5 mm (+/0.2 mm).
The width of the test membrane=4 mm (+/0.2 mm).

(133) Capillary flow rate: usually 1-6 cm/min; according to the invention 3.74 cm/min.

DETECTION LIMIT

(134) The particularly preferred cut-off of 4 units M2-PK/ml was found in various studies that investigated the M2-PK stool test ELISA. The chromatographic conditions according to the invention, such as the cutoff value of the combined rapid test according to the invention were determined on the basis of the previously determined cut-off value of the M2-PK stool tests ELISA.

CAPILLARY FLOW RATES FOR NITROCELLULOSE MEMBRANES

(135) The analytical sensitivity decreases with the capillary flow, i.e. the nitrocellulose with the slowest capillary flow time results in the highest analytical sensitivity. Capillary flow times are between 240-75 sec/4 cm. Capillary flow rate according to the invention is 120 sec/4 cm.

List of the Components According to the Invention and of the Active Ingredients According to the Invention

(136) TABLE-US-00003 Component Reagents Amount Capture line Monoclonal anti- 0.20-130 g 1. Antibodies mouse anti-M2-PK protein, 0.5-3 2. Antibodies antibodies g/cm protein mouse anti-hemo- globin antibod- ies Capture line Colloidal gold 0.01-0.07 g conjugate particles to protein which anti-M2 antibody and anti-hemoglobin antibodies ac- cording to the invention were coupled. Control lines Special anti- 0.20-1.30 g antibodies mouse IgG anti- protein bodies e.g., 0.05-1 g/cm such as from the protein company Jackson Immunochemical (USA).

EXTRACTION/RUNNING BUFFER

(137) TABLE-US-00004 Reagents Amount Triton X-100 p.A. <0.50% Sodium azide <0.05%

(138) In the tests during the development of the test kit according to the invention, it has surprisingly been found that both the clinical sensitivity and specificity, but also the chemical-analytical sensitivity are considerably increased when the running liquid in the immunochromatographic test principle has a pH 5-6, preferably a pH of 5.7. This finding is particularly surprising since, according to the state of the art with respect to properties of proteins, these should be present in a partially denatured condition at a pH of 5.7. Therefore, it is surprising that in this area an improvement of sensitivity, specificity, but also of responsiveness is achieved. All immunochromatographic test kits so far available on the market for determining hemoglobin involve a pH from 7 to 7.5 in the running buffer. A final scientific explanation for this cannot yet be given. Possibly, without wishing to be bound to this explanation, there is a partial denaturation in such a way that the epitopes of the analytes are more accessible for the various antibodies. For carrying out this embodiment of the invention with a running liquid having a pH of 5-6, in particular 5.7, a corresponding buffer is needed, namely, an acetate buffer (pH5.7), which advantageously further contains 0.1% albumin as a stabilizer. Such buffer systems are familiar to the person skilled in the art, and do not require any further depression at this point.

DESCRIPTION OF THE FIGURES

(139) FIG. 1. Schematic view of a test strip in the plastic cassette of a lateral flow rapid test.

(140) TABLE-US-00005 1 Sample Port 2 Test Line 3 Control Line 4 Housing 5 Sample Pad 6 Conjugate Pad 7 Membrane 8 Absorbent Pad

(141) FIG. 2. Relationship between bubble point and pore size.

(142) The bubble point of the membrane is the pressure required to force the air through a wet membrane.

(143) FIG. 3. Effect of capillary flow rate on the analytical sensitivity of a lateral flow rapid test.

(144) Examples:
Flow rate=1.00 cm/min.fwdarw.effective analyte concentration=1.00.
Flow rate=1.25 cm/min.fwdarw.effective analyte concentration=0.65.

(145) FIG. 4. Effect of a detergent or wetting agent concentration on different performance characteristics of a membrane.

(146) TABLE-US-00006 1 Protein Binding 2 Capillary Flow Rate 3 Strip Consistency 4 Strip Width

(147) FIG. 5. Calculation of the bandwidth as a function of the distribution rate.
Membrane bed volume=10 L/cm.sup.2.
Reagent dispensing rate=1 /cm.
Bandwidth=reagent dispensing rate/membrane bed volume=1 /cm/10 L/cm.sup.2=0.1 cm.

(148) FIG. 6. Typical relationships between flow rate of a membrane and function of an immunochromatographic test.

(149) TABLE-US-00007 1 Surface Quality 2 Specificity 3 Sensitivity 4 Total Assay Time 5 Reagent Costs
Sensitivity=analytical sensitivity=detection in g=mass.

(150) FIGS. 7-9. show in an exemplary way a test kit according to the invention. In FIG. 7 can be seen the sampling devices. FIG. 9 shows the actual test cassette.

(151) FIG. 10. shows an example of the assignment of the various measurement results to risk levels. For ease of reading, the result advantageously is shown in a colored manner. To avoid false readings by, e.g., color-blind persons, additional distinguishing features can be employed (e.g., different shapes, numbers, and/or letters.

OBJECTS OF THE INVENTION

(152) Objects of the invention are therefore: 1. Tool=classifier for diagnosis and assignment in the truth matrix of findings: true and false classifications. Since it is a yes/no question, one can also say the test is positive (means abnormal) or negative (means normal). If the test with only one biomarker is positive, further evaluation of the positive diagnosis is indicated by a colonoscopy. 2. Tool for classifying, based on certain features (determination of M2-PK/hemoglobin). 3. Tool for deciding: should a colonoscopy be performed for further diagnosis: yes/no. 4. Tool for facilitating medical instructions by improved starting diagnosis (significant additional benefits). 5. Tool for facilitating medical decisions on measures, e.g., the application of other diagnostic procedures, e.g., colonoscopy, etc., or therapeutic measures, e.g., surgery, chemotherapy, etc. 6. Tool for improving the prediction accuracy, reporting on the basis of stool samples of probands. 7. Test kit with high overall sensitivity and overall specificity for hemoglobin and M2-PK for detecting colon cancer. The test kit is used as a dual pre-filter as part of the colon cancer screening by means of colonoscopy. 8. Test kit with lower lot-to-lot deviation that allows for the best possible comparability of test results not only within a production batch but also between batches. 9. Tool for predicting therapeutic success. 10. Tool for improving therapy monitoring. 11. Tool for visualizing the test result (risk impact scheme). 12. The test kit according to the invention is used for predictive problem prevention: 1. The prevention (Latin: praevenire, avert, prevent) in the sense of keeping undesirable developments from happening. a. Prevention of diseases, of pathogenesis, and disease seriousness (severity). b. Prevention of certain medical procedures, e.g., unnecessary, dangerous, expensive measures (surgery, chemotherapy, side effects). The test kit according to the invention is to be viewed in the context of an optimum damage/benefit/risk consideration, compared to the diagnostic colonoscopy. But also very important is the preservation of quality of life and the prevention of premature death. 13. Object of the test kit according to the invention: The test kit serves for the solution of a serious health problem worldwide. The test kit serves for the selection of abnormal probands (increase of efficiency).

(153) This tool is achieved, according to the invention, by providing a combined rapid test.

(154) Combined rapid test for synchronous analytical determination of the enzyme biomarker tumor M2-PK and the biomarker blood (hemoglobin).

(155) Combined rapid test including the test strip (tumor M2-PK plus)+the test strip (iFOB plus) on a test cassette.

(156) Combined rapid test for synchronous analytical determination of the enzyme biomarker tumor M2-PK and the biomarker hemoglobin/haptoglobin complex (Hb/Hp complex).

(157) Combined rapid test including the test strip (tumor M2-PK plus)+the test strip (Hb/Hp complex plus) on a test cassette.

(158) The preferred detection method is the immunochromatographic method. In a particular embodiment, immunochemical methods in array, mini-array format, also turbidimetric methods are further possible. Subject matter of the invention are further monoclonal antibodies.

(159) The use of specific antibodies in the combined rapid test that are specific both in their binding properties for tumor M2-PK or hemoglobin and can be used in immunochromatographic methods (e.g., are membrane-suitable, detergent-suitable).

(160) The use of a specific antibody (clone P1F3 AK specifically detects the spatial, dimeric conformation of the M2-PK) for providing the combined rapid test according to the invention. This tumor M2-PK-specific antibody preferably binds to one of the following epitopes or fragments thereof, or combinations (epitopes or fragments thereof) from these which have a minimum length of four amino acids:

(161) TABLE-US-00008 LAPITSDP (SeqID01) EAEAAIYH (SeqID07) VEASFKCC (SeqID02) SGAIIVLT (SeqID08) CSGAIIVLT (SeqID03) LQLFEE (SeqID09) TEATAVGA (SeqID04) QLFEELRR (SeqID10) LRRLAPITSDPTEATA (SeqID05) VEASFKC (SeqID11) KCCSGAIIV (SeqID06) KSGRSAHG (SeqID12)