DE202012012084U1 - Test kit (combi rapid test) for the synchronous detection of biomarkers in stool for the detection of pathological changes in the gastrointestinal tract, especially in the intestine - Google Patents

Abstract

Test kit for detecting a malignant event, in particular a tumor, in the gastrointestinal tract, consisting of a first sample tube containing a first stool sampling device, a first buffer solution containing buffer 1 = 10-70 mM phosphate buffer (pH 6.7-7.6) or buffer 2 = 10-70 mM Hepes buffer (pH 7.6-8.2) or Buffer 3 = 10-70 mM triethanolamine (pH 7.3-7.7) or mixtures thereof, a second sample tube containing a second stool sampling device, a second buffer solution, buffer 1 = 10-70 mM phosphate buffer (pH 6.7-7.6) or buffer 2 = 10-70 mM Hepes buffer (pH 7.6-8.2) or buffer 3 = 10-70 mM triethanolamine ( pH 7.3-7.7) or mixtures thereof, a test cassette containing a lateral-flow test system with a stationary phase nitrocellulose membrane, the nitrocellulose membrane in turn containing tumor M2-PK antibody selected from polyclonal antibody # 3198, polyclonal antibody 214618 or monoclonal niger antibody clone AT4M3, clone PAT4M3AT, clone 5D2-3B3 or mixtures thereof and hemoblobin antibody, preferably monoclonal hemoglobin antibody clone from 55081, clone from 119210, clone LS-B49414, clone LS-C129648, clone LS-C83309, clone LS- C8308 or clone B1232 M or mixtures thereof, 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 content of tumor M2-PK greater is positive as 4-6 units / ml of stool extract and the analysis result of the second stool sample is if the content of hemoglobin is between 15 and 40 μg hemoglobin per gram of stool and the four possible test results are indicated by four different colors, letters, numbers, characters and / or geometric shapes are presented.

Description

Field of the invention

The invention relates to a commercially applicable, cost-reducing new test kit, (combined rapid test cassette), in vitro medical device, lateral flow cassette, point of care test, combined rapid test, bioassay, rapid lateral flow test strips, platform) for carrying out a method for detecting biomarkers in the human and / or animal stool. The test kit is used for health analysis, early detection and "colorectal cancer screening" by the detection of pathological changes in particular a malignant event in the gastrointestinal tract (esophagus, stomach, small intestine, biliary tract, pancreas and colon), especially tumors and tumor precursors (polyps, adenomas) of the intestine. More particularly, the invention relates to a combined rapid test and combination quick test cassette and their components, as well as the reagents necessary for the synchronous performance of an immunochromatographic detection method. Synchronous in the linguistic meaning of simultaneous, but also synchronous in the technical meaning of the common, optimal coordination of effective pairings (the chromatographic conditions, membranes, reagents, dispensing rate, etc. see descriptions) for the detection of the two biomarkers tumor M2-PK and hemoglobin in a combo quick test cassette.

The invention also relates to a simpler method for evaluating the test and displaying the results ("risk light").

The combination rapid test is used for the "pre-filtering" of subjects in the context of a colorectal cancer screening program. Since performing the non-invasive combination rapid test is significantly less fearful than conducting an invasive colonoscopy in the subject, the test leads to an increased participation in the colonoscopy.

Background of the invention

Cancer, especially colorectal cancer is the most common cancer in Germany (65,000 new cases, 27,000 deaths). Europe-wide, 200,000 people die each year from this malignant disease. Due to the development of colorectal cancer, the consequences of colorectal cancers are due to successful screening, i. H. the detection of precursors (polyps / adenomas) and their removal highly minimized. Every 20 minutes, a human being dies of colon cancer in Germany. Colon cancer almost always develops from initially benign growths of the intestinal mucosa (mucosa). The cancer grows slowly and for a long time without the affected person noticing anything. Complaints usually appear only when the tumor is large or has already formed metastases. Left untreated, colon cancer often causes death within 12 months.

Due to its genesis, the consequences of colon cancer can be almost completely avoided by successful screening of asymptomatic subjects. Precursors (polyps / adenomas) can be detected early by screening. Colonoscopy is considered the best method (gold standard) in the context of colorectal cancer screening. Colonoscopy is an invasive procedure that can only be performed by specially trained physicians (eg gastroenterologists). This procedure involves risks (eg, performation, risks due to sedation) Cash service offered. The attendance rate is only 2-3% and is in spite of considerable education and marketing campaigns, "glittering world" now even declining! The reason for this lies among other things in the procedure of the investigation, which many people find unpleasant (psychological inhibition threshold).

Object of the invention:

• 1. Lowering the psychological inhibition threshold.
• 2. Increase Participation Rate in Screening Colonoscopy Solution: To provide a simple, robust, cost-effective, routine-fit lateral flow test, in addition to the synchronous detection of two particular biomarkers (M2-PK and hemoglobin) for the detection of pathological gastrointestinal diseases in particular Colon cancer and its precursors is suitable. The combination rapid test, which is simple and easy to carry out, is suitable for lowering the psychological inhibition threshold compared to the screening colonoscopy. Even the positive findings of only one biomarker, M2-PK and / or hemoglobin, causes the measure colonoscopy for the purpose of further diagnosis by the Doctor. This leads to a higher detection rate of colorectal cancer and its precursors through the colonoscopy, since with the upstream combination rapid test, a preselection of the subjects takes place. Since the combination rapid test determines two important biomarkers for colorectal cancer synchronously, this test serves as a double filter before colonoscopy.

Colon cancer with its precursors (polyp-cancer-sequence) begins on the inside of the intestine (mucosa) and then expands into other tissue areas (submucosa, muscularis, serosa). If tumor growth exceeds the mucosal border one speaks of a colon cancer in early or advanced stage. Since the stool has direct contact with the mucosa of the intestine, biomarkers as indicators of pathological changes in the gastrointestinal tract are earlier and more specifically analytically detectable than in the blood. For this reason, the stool specimen material is preferable to the blood specimen. Only when the tumor has reached the submucosa, early biomarkers can be detected in the blood. If these markers are detectable in the blood, they are often "late" forms of colon cancer and its precursors. In general, the measurement of biomarkers in the stool is advantageous in comparison to the measurement of biomarkers in the blood. The earlier a colorectal cancer and its precursors are detected, the better the prognosis. The initiated therapy (eg curative colonoscopy, surgery) leads to an almost 100% cure in early colorectal cancer.

The "gold standard" for testing a malignant, especially tumorous, process in the intestine is colonoscopy.

Only specially qualified doctors are allowed to perform the colonoscopy. The colonoscopy has the further disadvantage that it is not standardized. Also, the "wrong" subjects are colonoscopically. Colonoscopy (colonoscopy), which is a central component of early detection, makes about 70% of the findings inconspicuous. In only about a quarter of cases are precursors diagnosed and in 1% of cases a carcinoma. Screening colonoscopy is therefore a very inefficient and expensive screening method, as too many healthy subjects are colonoscopically (lower health costs!).

Therefore, it is desirable to use a non-invasive, inexpensive and sensitive test that measures multiple biomarkers in sync to colonoscopy to target subjects with a positive test finding to colonoscopy (reduction of mortality). The noninvasive combination rapid test according to the invention makes it possible to greatly increase the participation rate in a screening program for the detection of colon cancer and its precursors and thus to achieve a significant increase in the survival rate of patients (in contrast to test subjects!) With colon cancer and its precursors. Colorectal cancer and its precursors, if recognized in time, provide almost 100% chance of recovery for the patient; Findings: Elevated levels of M2-PK and / or hemoglobin lead to the diagnosis of colorectal cancer with the help of anamnesis, etc. The diagnosis, in turn, leads to a handling instruction of the physician z. As surgical therapies and these in turn lead to the cure of colon cancer. For example, surgery through surgery can lower lethality.

As with all diagnostic procedures, so-called sensitivity and so-called specificity are the decisive quality features in in vitro tests. Sensitivity is the likelihood of identifying a sick person as ill. This quality measure is medically very important, since in an early detection measure, of course, as few affected people may be overlooked. Specificity is the likelihood of recognizing a healthy person as healthy. Again, this measure is important in the early detection because it should be avoided that healthy people who are falsely diagnosed as ill, receive a superfluous treatment.

State of the art

Guaiac test (gFOBT) (sample material: stool)

This chemical stool test works on the guaiac resin base. A chemical reaction detects hidden (occult) blood levels in the stool samples of the patients. Since this test due to disorders such. As food, reducing agents, vitamin C has a low sensitivity and specificity, he is considered obsolete and outdated. In addition, only strongly bleeding forms, but not weakly bleeding forms of colon cancer or its precursors can be detected. It is also known that there are bleeding, non-bleeding and intermittently bleeding forms of colon cancer and its precursors. Depending on the time of sampling, consequently, colon cancer and its precursors can be overlooked.

Immunological stool test (iFOBT) (specimen: stool)

Immunological in vitro tests are also looking for fecal occult blood that enters the stool through precursors (polyps) or early stages. Evidence is provided by the use of specific antibodies against hemoglobin, or against the hemoglobin / haptoglobin complex. More than 10 manufacturers / distributors offer these iFOB tests. However, the tests offered on the market show extreme differences in quality (in terms of sensitivity and specificity); these differences can only be recognized by specialists, but not the large number of physicians (users) established as required.

Furthermore, it should be noted that these so-called blood-in-stool tests only indirectly detect colon cancer and its precursors, as they are not specific for cancer and its precursors, but only for the detection of blood (unspecific for colon cancer). These tests, as well as the guaiac tests (gFOBT), can only detect highly bleeding forms, but not mildly bleeding forms of colon cancer or its precursors. It is also known that there are bleeding, non-bleeding and intermittently bleeding forms of colon cancer and its precursors. Depending on the time of measurement, colon cancer and its precursors can be overlooked.

With the gFOBT and the iFOBT, only bleeding tumors and their precursors can be detected. These tests "recognize" and "overlook" colon cancer and its precursors.
General: Occult and also pathophysiologisch has occult blood in the stool therefore only indirectly with colon cancer and its precursors to do!

Enzymatic stool test (M2-PK) (specimen: stool)

This is a test that can detect not faecal occult blood, but a typical for cancer enzyme in the stool. The enzyme always occurs in malignantly altered tissue of various cancers in larger quantities - including colon cancer or malignant colon polyps. In this test, which is also available on the market in the format of the lateral flow test and ELISA, the biomarker Tumor M2-PK has been demonstrated. Tumor M2-PK is a special isoenzyme of pyruvate kinase (PK). In normal tissue, pyruvate kinase (M1, M2-PK form) occurs as a tetrameric form (M2-PK tetramer). In cancer and its precursors, one increasingly finds the dimeric form of this PK (M2-PK-dimer = tumor M2-PK). Two different monoclonal antibodies have been generated against this particular tumor M2-PK molecule. These particular tumor M2-PK specific antibodies are used in the marketplace assays (used in ELISA and / or lateral flow format).
In general: causal and also pathophysiological, the biomarker M2-PK has to do directly with cancer and its precursors. The enzyme M2-PK (dimeric form of the isoenzyme M2-PK) is always detectable in all tumors investigated so far (eg 12 tumors of different tissues / organs), even in colon cancer and its precursors. Clinical studies have shown that tumor M2-PK tests (in the lateral flow and / or ELISA format) on the market can detect both bleeding and non-bleeding colorectal cancer and its precursors. These tests (test strips) can also "overlook" colon cancer and its precursors. Therefore, it is also an object of the present invention to provide better M2-PK tests (test strips).

Technical Solution: Test kit according to the invention comprising a combined rapid test consisting of a plastic cassette as well as two new, better prior art test strips for the determination of M2-PK and hemoglobin.

Blood test (sample material: blood)

Tumors release DNA (and / or methylated DNA) into the bloodstream. Colon cancer thus leaves a typical signature - a biomarker. This biomarker is detectable with a blood test. For this, the doctor takes a blood sample from the test person and sends it to a special laboratory for the relatively complex, expensive measurement and evaluation by PCR, which is not very robust for everyday life.

In general: A DNA methylation pattern, a signature, a typical trace (biomarker SEPT9, Septin9 blood test) is only indirectly and pathophysiologically linked to cancer and its precursors.

With the M2-PK tests, bleeding and non-bleeding tumors and their precursors can be detected. The M2-PK tests "overlook" but also colon cancer and its precursors. Therefore, a task of the present invention to achieve an improvement. This object is solved by the claims and the technical description of the present invention.

Disadvantage of the previously known tumor M2-PK tests (in lateral-flow PCT / EP00 / 09303 and / or ELISA format) as well as the gFOBT and iFOBT is that none of the three tests, taken alone, covers the entire diagnostically relevant period, in particular patient-related.

From the WO2007 / 071366A1 (Roche Diagnostics GmbH) a method is known in which both tumor M2-PK, and hemoglobin in the stool are determined. The quantitative determination of both parameters is carried out in two separate tests, the evaluation should be done via a mathematical algorithm. In the description of the cited document, a number of possible methods are mentioned, which may be the basis for the development of such an algorithm (see page 7, line 12 - page 9, line 21), without actually disclosing an evaluation algorithm. The technical teaching of the document therefore relates only to a method for searching an evaluation algorithm.

However, a solution for the complete diagnostic detection / diagnosis of all phases of the polyp-cancer-sequence is not described in the prior art. The task was therefore to develop a test for the detection of colorectal cancer and its precursors, which makes it possible to reliably and consistently detect all diagnostically detectable phases of the polyp cancer sequence as a single test. The test should allow the synchronous determination of hemoglobin and tumor M2-PK.

The object is achieved by the method according to the invention for the diagnosis / diagnosis of a pathological change of the intestine by means of a test kit (combi rapid test) by the specific detection of hemoglobin and tumor M2-PK. With the aid of the method according to the invention, it is possible to reliably detect pathological changes very early on the occurrence of one of the abovementioned analytes (hemoglobin, tumor M2-PK).

Surprisingly, it has been found that the technical problem of increasing the overall sensitivity in the present invention by the inventive use of the combined rapid test and two new, improved test strips for the determination of M2-PK and hemoglobin, and the necessary reagents for the synchronous implementation of the immunochromatographic detection method Compared to using two biomarkers dissolved on two separate or one cassette.

The invention therefore relates in particular to a combination rapid test and a combined rapid test cassette and components as well as the reagents necessary for the synchronous performance of an immunochromatographic detection method. Synchronous in the linguistic meaning of both simultaneously and synchronously in the technical meaning of the joint, optimal coordination of the chromatographic conditions (especially for the two new test strips) for the detection of the two biomarkers M2-PK and hemoglobin in a combination rapid test cassette.

However, this was only possible through the optimization of the cassette and all components (choice of membrane and specification (capillary flow rate, etc.)), the selection of so-called backing, the selection of detergents and solubilizers, Liquid Spreading vs. Protein spreading, specificity of the selected membrane (eg, nitrocellulose vs. nylon), selection of Sample Pads Selection and Specification, Conjucate Pads Selection and Specification, Absorbent Pad Selection and Specification, Ahesive Card Selection and Specification, Housing Selecting and Specification , Manufacturing Schemes, possible.

In vitro diagnostic test strips based on the immunochromatographic principle have long been state of the art. When considering a design stage immunochromatographic test strip through all product enhancements to the final manufacturing process, all principles of biology, chemistry, physics and engineering are used. The following patent numbers describe relevant technical teaching:
z. B. US 433734 . US 4376110 . US 4435504 . US 4703017 . US 4855240 . US 4954452 . US 5,028,535 . US 5075078 . US 95/16207 . US 5654162 . EP 0810436A1 , Test cassettes with more than one test strip (combination test cassette) in a test cassette belong to the state of the art (drug tests by means of a strip test). Also, these cassettes are known for the determination of hemo / haptoglobin complex and hemoglobin. For the synchronous determination of M2-PK and hemoglobin only single test cassettes are known. Both tests "overlooked" many pathological changes. The reason for this is, among other things, the "bad" set cut-off of the test strip and the poorly selected chromatographic conditions of the test system. A challenge for the manufacturer of a test system is the optimal choice the so-called "cut-offs". This cut-off can basically be taken from the histogram of the measured values of the test subject population. The technical problem is the distinction between "healthy" and "sick" subjects. This is technically solved by the test kit and the provision of the two new test strips with improved cut-off and chromatographic conditions. The object of the present invention is to improve the cut-off and poorly selected chromatographic conditions. This is technically solved by providing a test kit, combined rapid test cassette containing two new test strips with "well" adjusted cut-off, as well as the well selected chromatographic conditions for performing the combination rapid test.

From the literature WO 01/21826 A2 . WO 02/50546 A2 and WO 03/069343 A2 Various methods for the detection of tumor markers for tumors of the gastrointestinal tract are known in which the stool of a subject is examined for the tumor marker. These methods have proven themselves in practice, but still have a high number of false-negative and / or false-positive results. This is capable of improvement.

On the other hand, various methods for the detection of tumor markers for tumors are known which are based on detection of the tumor markers in blood or serum (see, for example, US Pat. Anticancer Research 19: 2785-2820 (1999) ; WO 03/065003 A2 ; Leman ES et al., Cancer Res. 67 (12): 5600-5605 (2007) and Leman ES et al., Clinical Cancer Research 14: 1349-1354 (2008) ). On the one hand, while this is advantageous for diagnosing a tumor disease at all, on the other hand, the detection of a biomarker for a wide range of tumor diseases does not give any definite information about the type and localization of the affected organ or tissue, since tumor markers are rarely tissue-specific and therefore very rare a positive result of a blood analysis on the tumor marker is no reliable conclusion on the affected tissue is possible. In addition, complicated and complicated diagnostic methods are therefore necessary in order to determine the type of tumor actually present. Markers in the blood, plasma or serum are therefore used mainly for the therapy and follow-up of tumors.

In addition, this detection method requires that tumor markers formed in the tumor reach the bloodstream or the serum in sufficient quantities. In the early stage of carcinogenesis, especially in colon cancer, not enough amounts of these tumor markers enter the bloodstream, so that detection via blood or serum tests is not possible.

Synchronous detection of biomarkers

In addition, the synchronous, precise detection of the two biomarkers according to the invention (M2-PK and hemoglobin) additionally requires the technical solution for joint coordination of the determination (reliable detection = no exceeding of the statistical confidence interval) of both biomarkers in a test cassette. That is the common chromatographic conditions

Please refer 1 to 6

This is achieved by selecting the common, optimal coordination of the chromatographic conditions for the detection of the two biomarkers M2-PK and hemoglobin in a combination rapid test cassette in terms of an inventive parameter selection.

In particular, by optimizing the cassette and its components (membrane selection and specification (capillary flow rate, etc.), selection of so-called backing, selection of detergents and solubilizers, liquid spreading vs. protein spreading, specificity of the selected membrane (e.g. Nitrocellulose vs. Nylon), Selection of Sample Pads Selection and Specification, Conjugate Pads Selection and Specification, Absorbent Pad Selection and Specification, Ahesive Card Selection and Specification, Housing Selecting and Specification, Manufacturing Schemes.

• Technisches Problem: schlechte klinische Sensitivität und Spezifität durch angewendete Screeningverfahren (gFOBT, iFOBT, Tumor M2-PK, Screening-Koloskopie.
• Aufgabe der Erfindung: Verbesserung der klinischen Sensitivität und Spezifität, insbesondere der Sensitivität
• Technische Lösung: Verwendung der erfindungsgemäßen Kombi-Test-Kassette wodurch wesentliche Vorteile (Erhöhung der Gesamtsensitivität) gegenüber der Verwendung von zwei Testkassetten (eventuell von zwei unterschiedlichen Herstellern) erreicht werden. Nach dem Stand der Technik existiert im Markt ein Lateral-Flow-Test (ScheBo M2-PK Quick Test) zur gerichteten Hinführung der asymptomatischen Probanden zur Koloskopie.
• Technisches Problem: Verbesserung der Sensitivität und Spezifität.
• Technische Lösung: Verwendung der erfindungsgemäßen Kombi-Test-Kassette, welche die synchrone immunchemische Bestimmung der Biomarker okkultes Blut und Tumor M2-PK ermöglicht.

The preparation of this combination cassette according to the invention with two test fields (use of nitrocellulose membranes) is carried out according to the In Vitro Diagnostic IVD Directive 98/78 / EC and after ISO 13485 and quality management QM ISO 9001 ,

• Technical problem: poor clinical sensitivity and specificity due to applied screening methods (gFOBT, iFOBT, tumor M2-PK, screening colonoscopy.
• Object of the invention: Improvement of the clinical sensitivity and specificity, in particular the sensitivity
• Technical Solution: Use of the combination test cassette according to the invention, whereby substantial advantages (increase of the overall sensitivity) compared to the use of two test cassettes (possibly of two different manufacturers) are achieved. According to the state of the art, there is a lateral flow test (ScheBo M2-PK Quick Test) for directing asymptomatic subjects to colonoscopy in the market.
• Technical problem: improvement of sensitivity and specificity.
• Technical solution: Use of the combination test cassette according to the invention, which enables the synchronous immunochemical determination of the biomarker occult blood and tumor M2-PK.

The combination test cassette according to the invention contains two lateral flow test strips. Both test strips do not correspond to the prior art, but each represent better lateral flow test methods according to the invention. on the combination test cassette, in the preferred embodiment of the invention, there is an optimized M2-PK test M2 = PK PK + test and a better iFOB test = iFOB + test in the prior art.

The synchronous (simultaneous) detection of the biomarkers tumor M2-PK and hemoglobin with matched cut-offs in a combination cassette (a test kit with all the reagents and components needed for the test procedure) has advantages over the time-delayed processing (determination) of the two biomarkers (tumor M2-PK and hemoglobin) by means of two lateral flow cassettes (two test kits with two different assortments of reagents and components for assay performance) since the specific solid phase / liquid phase antibodies as well as all reagents and components of the combination are needed. Test kits are matched. For example, if a user purchased the ScheBo Lateral Flow Test to determine the Tumor M2-PK and purchased another Lateral Flow Test to determine hemoglobin from another manufacturer and run it with a time delay, he would use these two different ones and not Coordinated tests achieve inferior clinical sensitivity and specificity.

Tasks and technical solutions of the invention:

• 1. Saving of health expenditures through a targeted colonoscopy.
• 2. Provision of a targeted, simple, manageable "entry-level diagnostic" for the early detection of colorectal cancer.
• 3. Improvement of the prognosis of an individual colorectal cancer disease by early detection by providing the combi rapid test cassette according to the invention for the synchronous determination of the tumor M2-PK and iFOB determination for the optimized selection of such individuals (subjects), which require a targeted entry examination by colonoscopy. The technical problem of preselection for colonoscopy can be solved with the combi rapid test cassette according to the invention. Only those subjects who were found positive in the test result in the measurement of one of the analytes (tumor M2-PK and / or iFOB test) are specifically sent to the colonoscopy The combination rapid-test cassette according to the invention functions technically as a double "filter" selection process.
• 4. Provision of a lateral flow double test cassette according to the invention and all associated reagents, special antibodies (anti-tumor M2-PK (special epitope), anti-hemoglobin (special epitope), the special physico-chemical properties, eg special binding constants and Furthermore, the selected solid-phase and liquid-phase antibodies which are directed against the two different analytes possess, for example, in the lateral flow measuring method special properties (eg compatibility with glass fiber, nitrocellulose membrane, good coupling properties to eg gold colloid).
• 5. Use of special antibodies in the combination rapid test, which are both specific in their binding property for tumor M2-PK or hemoglobin and are useful in immunochromatographic methods (eg "membrane-friendly", "detergent-suitable").
• 6. Reduction of the lethality of colorectal cancer by the provision according to the invention of a combined rapid test for targeted introduction to the invasive method - colonoscopy - and thus targeted preparation (according to diagnostic decision tree) to a therapeutic method (surgery, chemotherapy).
• 7. Cost / benefit optimization of the screening measures for the detection of colorectal cancer and its precursors.

From the literature WO 01/21826 It is known that one can detect the tumor M2-PK on a test strip by means of a chromatographic method. For example, in this test, a test strip (nitrocellulose) can be used on which in different zones of the test strip the required antibodies are arranged either in soluble form or fixed in solid phase. The sample (eg stool extract, a mixture of many substances, but also the analyte to be detected in detergent solution) or the liquid portion of the sample or an extract can travel through the test strip and deliver a signal at the detection point, if e.g. B. Tumor M2-PK and / or hemoglobin is present in the sample. The use of highly specific capture antibodies according to the invention ensures the binding of the target proteins (eg tumor M2-PK) from the faeces sample extract. By the use of gold colloids on which a second highly specific antibody is formed in the presence of z. For example, tumor M2-PK is an immune complex. This analyte-detection complex is visible to the naked eye on the test strip as a line. The exact arrangement of the individual components on a test strip is known depending on the applied immunological method and the expert. A particular challenge is the use of optimal antibodies. In the combination rapid test according to the invention, special antibodies which specifically bind the tumor M2-PK were used.

The tumor M2-PK-specific antibody preferably binds (also stereospecifically the dimeric form of the M2-PK) to one of the following epitopes or fragments thereof, which are at least 4 amino acids in length:
LAPITSDP
VEASFKCC
CSGAIIVLT
TEATAVGA
LRRLAPITSDPTEATA
KCCSGAIIV
EAEAAIYH
SGAIIVLT
LQLFEE
QLFEELRR
VEASFKC
KSGRSAHG

The appearance of one of these epitopes or fragments thereof, which are at least 4 amino acids in length, or combinations of these in a stool sample extract, is indicative of pathological changes (colon cancer or its precursors) in the gastrointestinal tract.

The so-called blood-in-stool tests show an insufficient sensitivity of only 25% for the detection of colorectal cancer. The tumor M2-PK test shows a higher sensitivity of 80%. With the Tumor M2-PK test, polyps larger than 1 cm can be detected with a sensitivity of 60% (FOBT: 20%), polyps less than 1 cm with a sensitivity of 20% (FOBT: 0%). (All values with a specificity of 95%).

In general, immunochromatographic blood-in-stool tests show a higher sensitivity for colorectal cancer and its precursors than the normally used gFOBTs, so the gFOBTs are inferior to the iFOBTs.

The positive rates as well as the specificity and the sensitivity under these tests vary widely. The observed patterns suggest that the widely differing positive rates are due to different cut-off levels, different limits and limits of tests of different manufacturers from different countries. The manufacturing quality of the tests of different manufacturers is different.

Test kits from different manufacturers address different cut-offs (= tolerance limit denotes a tolerance value). The tolerance value determines from when a test result is to be evaluated positively or negatively. The cut-off should be distinguished from the concept of the detection limit.

Lit: Inter-test agreement and quantitative cross-validation of immunochromatographic fecal occult blond tests, Hermann Brenner et al .; Int. J. Cancer (2010)

One consequence of this is a strong variation in the test characteristics of individual tests (technical problem).

This technical problem is solved by providing the test kit containing the combination cassette containing two test strips improved according to the prior art. Cut-off levels, marginal ranges and limits for both biomarkers were found through extensive in-house measurements and established. The determination was carried out by descriptive statistical analysis and interpretation of the biomarker-value histograms (box-plot analyzes and Kruskal-Wallis calculations). The preparation of both test strips and the adjustment of the chromatographic conditions for the synchronous determination of both biomarkers to achieve better clinical sensitivities and specificities, a great challenge dar. This was technically solved by adapting all the chemical and physical conditions (see claims and examples). Thus, all conditions for the determination of the two biomarkers were chosen so that the least possible "false positive" or "false negative" results occur when performing the combined test. To achieve this goal, a technical solution was sought and found. For the technical solution, the synchronous detection of the two biomarkers resulted under coordinated chromatographic and coordinated material conditions. D. h. correct choice of membrane, running conditions, etc. (see examples and claims).

Tumor markers are gene products that are expressed in tumor tissue differentially from normal reference tissue, i. e. a tumor marker is either over- or under-expressed in tumor tissue over normal tissue. In practice, tumor markers that are overexpressed are more important because then a positive analysis result (presence of the biomarker) of a sample is indicative of the presence of the biomarker-specific tumor. Tumor markers are present in the cytoplasm of the tissue cells, but may also be found in body fluids such as blood, urine, sweat, semen, saliva, etc. but also in the stool.

A tumor marker should have the following properties: i) high specificity, i. H. undetectable in benign diseases and healthy individuals, ii) high sensitivity, d. H. it can be detected in a high percentage of tumor patients, iii) organ specificity, iv) good correlation with tumor stages or mass, v) relationship with prognosis, and vi) reliable predictive values. The criteria of 100% specificity and 100% sensitivity as well as the other criteria listed are still not met by any of the known tumor markers.

U. a. tumor markers specific for tumors of the gastrointestinal tract include CCSA-2, CCSA-3, CCSA-4, CC2, CC3, CC4, CC5, CC6a, CC6b, L1, L2, N1, N2, N3, N4, N5, and N6 (see e.g. B. WO 03/065003 A2 ; Leman ES et al., Cancer Res. 67 (12): 5600-5605 (2007) and Leman ES et al., Clinical Cancer Research 14: 1349-1354 (2008) ). However, these tumor markers are used in the prior art only in the context of blood and plasma tests, which leads to the disadvantages mentioned above.

It is desirable, as early as possible, a tumorous event in the gastrointestinal tract, in particular in a growth phase in which the tumor has not yet made contact with the vascular system of the body (for example, already in the "polyp-cancer-sequence", ie before the infiltration of Submucosa). When neoplastic events in the gastrointestinal tract are suspected, especially with regard to the so-called adenoma-carcinoma sequence in polyps, the prior art attempts to detect faecal occult blood by means of various determination methods. For this purpose, non-immunological tests (eg pseudoperoxidase activity, porphyrin detection) and immunological tests are used ( Favenne L. et al. (1992) Ann. Biol. Clin. 50: 311-313) , However, both test principles are not very specific. In addition, the non-immunological test can be disturbed by a variety of factors (false positive / false negative, eg by non-compliance with urgent dietary requirements on the part of the patient and a number of drugs and by excessive vitamin C administration (eg B. in vegetables, fruit juices etc.), Thomas L., Laboratory and Diagnosis, 5th Edition, 1998 ).

A positive fecal occult blood test should be checked until the source of bleeding is localized or the cause of the bleeding is found. The clinical findings justify in each case a swiftly to be carried out further diagnostics, z. B. by endoscopy, sonography, X-ray.

Technical problem of the invention

The existing prior art M2-PK lateral flow test (test strip) overlooks pathological changes. The M2-PK + test strip according to the invention also overlooks pathological changes. The iFOBT existing in the prior art overlooks pathological changes. The iFOBT + test strip according to the invention overlooks pathological changes. Bringing both improved tests M2-PK + and iFOBT + as a test strip according to the invention in a combination cassette obtained according to the object of the invention, a higher detection rate for pathological changes in the intestine. Technical problem solutions, in particular the selection of active pairings (see examples and list of the components according to the invention and active ingredients, the correct choice of the antibodies and the correct choice of the cut- offsets for both parameters, analytical sensitivity, capillary flow rates, etc.) for synchronous detection of the two biomarkers are the subject of this invention's inventive parameter selection.

The invention is therefore based on the technical problem of specifying a simple test kit (combi rapid test) which serves as a method for detecting markers, biomarkers, in particular enzyme markers in the human or animal stool, the method for detecting a malignant event in the gastrointestinal tract and the abdominal cavity (esophagus, stomach, small intestine, biliary tract, pancreas and large intestine), in particular of tumors and tumor precursors of the intestine (polyps, adenomas) is suitable. The combination test kit according to the invention (combi rapid test) should make the detection of pathological changes possible in a simple manner, so that the test can be carried out by technical employees. The invention is based in particular on the technical problem of satisfying the continuing increasing demand for specific, easily performed methods, so that a pathological event, in particular a neoplastic event, can be detected particularly early and without doubt, particularly with regard to the problem of the so-called adenoma carcinoma Sequence in polyps.

Broad features of the invention and preferred embodiments

To solve this technical problem, the invention teaches a test kit (combination rapid test) for detecting a pathological event in the gastrointestinal tract, wherein a sample of human or animal stool is applied to a carrier and thereupon detects both tumor M2-PK and hemoglobin on a carrier become. The reading is done here in a simple way visually with the naked eye (without the aid of a device). In a preferred embodiment of the invention, hemoglobin is determined in the form of the hemoglobin-haptoglobin complex (Hb-Hp). It has been found that the measurement of the abovementioned biomarkers is particularly suitable for detecting early malignant changes, in particular neoplasms. By measuring this biomarker combination tumor M2-PK + hemoglobin; Tumor M2-PK + hemoglobin-haptoglobin complex (Hb-Hp) has a marked improvement in the accuracy of reading (findings), in particular a significant reduction in false-negative but also false-positive findings.

This is particularly surprising since extensive investigations with respect to other specific analytes (proteins) in the stool did not allow any indication of their application as biomarkers, in particular as tumor markers. Both the structure and the physicochemical properties of the tumor markers mentioned are apparently not permanently impaired due to the considerable proteolytic activity and the extreme physiological conditions (eg pH, acid in the stomach, alkaline in the intestine) of the gastrointestinal tract. This also applies to the detection of biomarkers by immunological methods. It was therefore found and demonstrated that, despite the above-mentioned protein denaturation and protein digestion in the gastrointestinal tract, a specific detection of several enzyme markers in the stool of tumor patients can be carried out.

The systems necessary for the determination of the enzyme markers are not readily available commercially. To develop the combined rapid test according to the invention, both the detection limits and the composition of the reagents (according to the invention: specific antibodies for the liquid and for the solid phase optimized for both analytes for the synchronous measurement of the analytes) and buffer solutions had to be adapted in a complex manner. In a M2-PK and / or hemoglobin positive stool sample is thus an immediate indication of a pathological, possibly neoplastic, especially malignant, events in the gastrointestinal tract, ie in particular in the intestine, the esophagus, in the stomach, in the biliary ducts, in the pancreas or / and in the large intestine, thus allowing a localization of the events in the gastrointestinal tract.

It should be noted that a stool sample can not be considered as a "body fluid" sample because the entire gastrointestinal tract is biologically outside the body.

With the method according to the invention, it is now possible to detect a malignant tumors in the stool in a simple manner by determining a plurality of said tumor marker proteins and at the same time be able to clearly establish, in the case of a positive test procedure, that a tumor is present in the gastrointestinal tract and not at elsewhere in the body.

Not only does the primary diagnosis work with this procedure, but also a follow-up check can be carried out. Due to the individual protein distribution, an individual diagnosis is possible.

Surprisingly, it is possible with the combi rapid test according to the invention to detect the said enzyme markers as free proteins in the stool. This is also advantageous because the test can be carried out by assistant personnel, for example a medical-technical employee (MTA). The fact that the two analytes (tumor M2-PK and hemoglobin) are determined synchronously, especially reading errors (for example, the MTA) are avoided, because the test drops the two sample drops of a patient succession (naturally with a few seconds delay) applied and after a certain period of a few minutes, both results are read. This approach achieves not only greater convenience of performing the test, but also a reduction in sources of error (as compared to conventional tests).

For the test according to the invention, no isolation of cells from the stool and analysis of the proteins contained in the cells is required. Because it has been found that solid tumors donate the mentioned enzyme markers as soluble proteins into the lumen of the gastrointestinal tract and these biomarkers are not located in exfoliated gastrointestinal epithelial cells. Said enzyme markers are released by tumor cells, pass through the gastrointestinal tract as protein, and can eventually be detected as a protein in stool. This possibility, namely that these proteins after z. As the stomach or intestinal passage remain detectable in the stool, was not expected, since normally a substantial decomposition of the proteins in question would have been expected in the course of normal digestion process.

In the context of the invention, it has been found that said enzyme markers remain detectable quantitatively even in the case of intensively homogenized, longer-stored stool samples (for example, during sample shipment). Even with strong dilution of the stool, a clear reaction is obtained. In addition, the detection can be selective even without prior extraction in the stool sample. Preferably, however, an extraction procedure using, for example, a specific detergent (e.g., CHAPS) is to be used (the extraction procedure is described in more detail in Example 2).

In the method according to the invention, the malignant event in the gastrointestinal tract of a human or an animal is preferably determined. Furthermore, it is preferred to detect the tumor marker immunochemically, in particular by means of anti-tumor marker antibodies. It is possible to use monoclonal or polyclonal antibodies, preferably monoclonal antibodies. Advantageously, antibodies are used which do not cross-react with other constituents of the stool, in particular with other stool proteins.

Fragments in the sense of the invention are proteins or polypeptides having an amino acid sequence which are identical to a partial sequence of one of the tumor markers. The length of a fragment can range from 5 aa (aa = amino acids) to n - 1 aa (n = length of the tumor marker from which the fragment derives). Typically, the length will be more than 10 aa.

Antibodies which can be used according to the invention can be prepared by methods known from the prior art. Methods for producing specific monoclonal antibodies are well known to those skilled in the art. For example, for this purpose, the antigen, in the present case an enzyme mark, is used to generate antibodies. In principle, the method described for the first time by Koehler and Milstein can be used for this purpose, with the person skilled in the art also being aware of modifications and further developments of these methods. With this preparation, specific antibodies can be obtained, whereby the specificity can be determined by selection. Selection is for specific antibodies that bind to an enzyme markers but not to other stool proteins.

In a particularly preferred embodiment, the detection is carried out according to the principle of the immunoassay. An immunoassay preferred according to the invention is carried out in such a way that a) brings the sample into contact with at least two different receptors, of which the first receptor R1 is in the solid phase and is capable of binding with the tumor marker and the second receptor R2 is in the liquid phase and is also capable of binding with the same enzyme markers, wherein the receptor R2 carries a label or mediates binding to a detectable molecule, b) separates the solid from the liquid phase, and c) the label or the detectable molecule in one of the phases is preferred determined in the solid phase and quantified according to the amount of Enzymbiomarkern present in the sample, wherein as at least one of the receptors R1 or R2 uses an antibody that is specifically bindable with tumor M2-PK and in particular does not bind to any other stool protein.

In a particularly preferred embodiment, an antibody against tumor M2-PK is used both as receptor R1 and as receptor R2. According to the invention, tumor M2-PK is understood to mean the dimeric form of this enzyme markers, which differs from the tetrameric form. The hemoglobin is determined according to the test principle described above for tumor M2-PK. In addition to performing the method according to the invention in the form of an immunoassay and in particular an ELISA, other detection technologies can be used, for. As the quartz crystal technology, microbalance technology, lateral flow technology, candelabra technology, TRACE technology or electrochemiluminescence technology, but also the agglutination with micro or nanoparticles and multiplexing technologies on the liquid or solid phase or array Technologies, such as protein chips. These technologies are well known to those skilled in the art and therefore need not be discussed further here. Preferably, the lateral flow technology is used.

The present invention therefore relates to a combined rapid test for the detection and / or diagnosis of a malignant tumor in the gastrointestinal tract in humans or animals, which is intended in particular for carrying out the method described above, by determining a Enzymbiomarkerkombination, wherein the combination rapid test synchronous tumor M2-PK and hemoglobin or hemoglobin / haptoglobin.

The combination rapid test preferably contains an antibody for tumor M2-PK which does not cross-react with any other chair protein and recognizes an antibody that specifically harbors hemoglobin / haptoglobin and does not cross-react with any other chair protein. If appropriate, the test kit may contain further reagents necessary for carrying out an immunoassay or for carrying out the respectively provided test procedure. The test kit preferably contains an antibody bound to a solid phase and specific for at least one of said enzyme markers.

Another object of the invention are antibodies, in particular monoclonal antibodies which specifically bind one of said enzyme markers and preferably cross-react with no other stool protein. The antibodies can, for example, according to the method of Koehler and Milstein (Nature 256, 49-497 (1995) ) be generated.

Furthermore, the present invention relates to aptamers or spiegelmers and their use in place of the antibodies described above (all antibody-related versions apply analogously), which bind specifically to one of said enzyme markers and optionally cross-react with no other stool protein. Aptamers are oligonucleotide sequences which have specific binding properties. Such aptamers may, for example, according to the in US 5,270,163 or in Sumedha, Clin. Chem. 45 (1999), 1628-1650 produced or identified methods described. Spiegelmers are aptamers formed from L oligonucleotides.

The evaluation of the tests according to the invention is carried out using automatic classification, cluster analysis, pattern recognition. In particular, methods of the "maximum-likelihood method" or the cluster membership via probability distributions are used.

For the laboratory staff and the attending physician, the result is preferably presented in the form of a multi-colored representation (risk traffic light). For example, the measured parameters (M2-PK and hemoglobin) can be assigned as follows: M2-PK hb importance Level 1 (green) - - Colon cancer can be excluded with> 97% certainty Level 2 (yellow) - + Further clarification required (eg colonoscopy) Level 3 (orange) + - Further clarification required (eg colonoscopy) Level 4 (red) + + 90% chance of advanced neoplasia

In any case, a test kit of the invention includes a stool sampler. For this purpose, all customary devices in question, for example, in the DE 102 05 709 A1 described devices.

A particular challenge in achieving the optimal functional principle was the selection of the optimal active pairings. These mimics are described in the following examples, lists and claims, and more particularly in US Pat 1 - 6 specified.

Z. B 2 . 3 . 4 and 6 represent different functions. A particular challenge of this invention was in particular among the numerous different functions (combination options) to select the optimal active pairings to achieve the optimal operating principle (selection of parameters).

In a particular embodiment of the invention, the evaluation takes place automatically. For example, the test kit according to the invention can be photographed and the test result can be assigned to the corresponding risk level via an evaluation program. The present invention therefore also encompasses such devices for scanning the test kit and the evaluation programs for displaying the test result, preferably in the form of a multicolor representation (risk traffic light). An example is the assignment in 10 shown.

Examples

The invention is further illustrated by the following examples.

Example 1: Test kit

The test kit based on an immuno-assay consists of two sampling and preparation devices and a test cassette. Both sampling devices contain a rod which is able to take up the required amount of stool (4-30 mg, preferably 25 mg). The sampling devices also each contain a sample receiving tube filled with buffer solution. The aqueous buffer solution for the tumor M2-PK test contains the following components:
Buffer 1 = 10-70 mM phosphate buffer (pH 6.7-7.6) or buffer 2 = 10-70 mM Hepes buffer (pH 7.6-8.2) or buffer 3 = 10-70 mM triethanolamine (pH 7, 3-7,7) preferred mixtures thereof each volume 1: 1: 1
Detergent 1 = CHAPS (3 - [(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate, 10mM-50mM (Sigma), detergent 2 = sodium dodecyl sulfate (SDS) 0.01% -0.1%), e.g. , Biochrome, detergent 3 = lauryldimethylamine oxides (10mM-50mM), e.g. Biochrom or mixtures thereof,

The aqueous buffer solution for the hemoglobin test contains the following constituents:
Buffer 1 = 10-70 mM phosphate buffer (pH 6.7-7.6) or buffer 2 = 10-70 mM Hepes buffer (pH 7.6-8.2) or buffer 3 = 10-70 mM triethanolamine (pH 7, 3-7,7) or mixtures thereof

The tumor M2-PK antibody is selected from the following list
Cell Signaling USA (polyclonal antibody # 3198),
R + D Systems, USA (polyclonal antibody 214618)
or monoclonal mouse antibodies of the company My Biosource, USA (clone AT4M3, PAT4M3AT,
the company Abnova, Taiwan (clone 5D2-3B3)
or mixtures thereof.

The tumor hemoglobin antibody is selected from the following list:
Hemoglobin antibodies from Abcam, UK (clone from 55081, clone from 119210),
LSBio, USA (clone LS-B49414, clone LS-C129648, clone LS-C83309, clone LS-C8308)
or the company Genwaybio, USA (clone B1232 M)
or mixtures thereof.

One of the important conditions for selecting the antibody is that it does not interact with other constituents of the stool, especially with other pyruvate kinase isoenzymes (eg, M1-PK, M2-PK (tetrameric form) L-PK, R-PK). allowed to cross.

The abovementioned hemoglobin antibodies are particularly suitable for binding to a membrane (solid phase), preferably nitrocellulose. The above antibodies do not cross-react with porcine, horse, sheep and bovine hemoglobin. The abovementioned hemoglobin antibodies are preferably suitable for binding to latex or gold colloids (liquid phase). They do not cross-react with sheep, horse, beef or pig hemoglobin. The abovementioned 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.

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 oxides (10mM-50mM), e.g. B. biochrome or mixtures thereof.

The sampling device for performing the hemoglobin test contains one of the above-described buffers (1.5-2.5 ml). The person to be tested pierces the stool with the dosing tip and transfers the stool sample into the tube filled with the appropriate buffer. The M2-PK test tumor delivery device provided by the physician to the person under test contains no buffer. The patient pricks the stool into the stool and transfers the stool sample to the empty tube.

The test kit according to the invention also contains a test cassette in which the two immunoassays improved according to the prior art are carried out by lateral flow technology. The test cassette contains for this purpose two, preferably round, recesses for applying the stool samples and two, preferably elongated, recesses for reading the test results. The test cassette contains in its interior as a stationary phase nitrocellulose the following preferred capillary flow rate of 135 sec / 4 cm.

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

The stationary phase = nitrocellulose (preferably with the capillary flow rate of 90 sec / 4 cm) contains hemoglobin antibody 1, hemoglobin antibody 2, preference is given to a mixing ratio as indicated above.

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

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 preferably suitable for binding to gold colloids or latex colloids, preferably 80 nm diameter gold colloids.

The hemoglobin tube and the M2-PK tube must be returned to the doctor's office within 48 hours. The dosing tip with the stool sample is transferred to a tube filled with the above-described buffer. Both the tube for the M2-PK test and the tube for the hemoglobin test are shaken vigorously.

The prepared stool specimen extracts are applied immediately after each other to the two recesses. After 5-10 minutes, it is read from the marked line whether one or both of the measured biomarkers are positive.

Crucial for the reliable determination of a malignant event in the gastrointestinal tract is the choice of the so-called "cut-off". The cut-off marks the minimum concentration of analyte in the sample that indicates a positive result. The cut-off of the hemoglobin test in the test kit according to the invention is between 15 and 40 μg hemoglobin per gram of stool, preferably between 20 and 30 μg hemoglobin per gram of stool. The cut-off in tumor M2-PK is between 4 and 6 U / ml of stool extract, preferably 4 U / ml.

The detection limit is 150 nanograms of hemoglobin / ml of stool extract.

The evaluation of the test result and assignment to the risk levels, as exemplified in 10 is shown can be done manually, semi-automatically or fully automatically. For example, a test cassette according to the invention can be manually clamped in a read-out device, which carries out an evaluation by means of a photocell or camera and then displays the result on a monitor. The representation preferably takes place in color (risk light). For safety reasons, a parallel representation in a different form is particularly preferred (for example: risk level 1-4, or the like).

Of course, the test cassette can also be designed so that a fully automatic evaluation is possible, for. B. by detection of the gold particles by electrochemical means ..

Example 2:

The combi rapid test cassette according to the invention does not contain the lateral flow test strip on the market for the determination of the M2-PK, but a new, improved lateral flow test strip according to the invention for the determination of the M2-PK (tumor M2-PK +).

Furthermore, the combination rapid test cassette contains a non-market lateral flow test strip for the determination of hemoglobin but a new, improved lateral flow test strip according to the invention for the determination of hemoglobin (iFOB +).

In particular, the invention relates to the combination rapid test and components as well as all reagents necessary for the synchronous implementation of the immunochromatographic detection method. Synchronous in the linguistic meaning of both simultaneously and synchronously in the technical meaning of the joint, optimal coordination of the chromatographic conditions for the detection of the two biomarkers M2-PK and hemoglobin in a combination rapid test cassette.

Detection Reagents:

Various detection reagents can be used: latex spheres, 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. Gold particles are preferred according to the invention.

Polymer composition and protein binding

Nitrocellulose, polyvinylidene fluoride, charge-modified nylon, polyethersulfone are used to make lateral flow tests. The polymer surface area depends on pore size, porosity, thickness and other structural characteristics. The surface area increases non-linearly with the pore size but increases linearly with the thickness and increases non-linearly with the porosity. 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. In the combi rapid test according to the invention, preference was given to using glass fiber for the so-called sample pad, cellulose 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)
Absorbant pad = 16.5 mm (+/- 0.2 mm) × 5 mm (+/- 0.2 mm)
The width of the test membrane = 4 mm (+/- 0.2 mm)
Capillary flow rate: usually 1-6 cm / min .; According to the invention 3.74 cm / min.

detection limit

The cut-off of 4 U M2-PK / ml was found due to different studies that examined the M2-PK stool test ELISA. The inventive chromatographic conditions z. B. the cut-off value of the combi rapid test according to the invention ELISA was determined due to the previously determined cut-off value of the M2-PK stool test.

The capillary flow rates for nitrocellulose membranes

The analytical sensitivity decreases with the capillary flow time ie the nitrocellulose with the slowest capillary flow time gives the highest analytical sensitivity. Capillary flow times are between 240-75 sec / 4 cm. Capillary flow rate according to the invention 120 sec / 4 cm. List of components and active ingredients of the invention component reagents amount Capture line 1. Antibody 2. Antibody Monoclonal anti-mouse anti-M2-PK antibody Mouse anti-hemoglobin antibody 0.20-130 μg protein; 0.5-3 μg / cm protein Capture conjugate Colloidal gold particles to which the anti-M2 antibodies or anti-hemoglobin antibodies according to the invention were coupled. 0.01-0.07 μg protein Control lines antibody Special anti-mouse IgG antibodies 0.20-1.30 μg protein 0.05-1 μg / cm protein Extraction / running buffer reagents amount Triton X-100 p. A. <0.50% Sodium azide <0.05%

Description of the figures

Fig. 1

• Schematic view of a test strip in the plastic cassette of a lateral flow rapid test.

1 Sample port sample receipt 2 Test Line test line 3 Control Line line of control 4 Housing packaging 5 Sample Pad sample pads 6 Conjugate Pad bonding pad 7 membrane membrane 8th Absorbent pad absorbent pad

Fig. 2

• Relationship between bubble point and pore size

The "bubble point" of a membrane is the pressure needed to force the air through a wet membrane

Fig. 3

• Effect of capillary flow rate on the analytical sensitivity of a lateral flow rapid test

Examples:

• Flow rate = 1.00 cm / min → effective analyte concentration = 1.00 x
• Flow rate = 1.25 cm / min → effective analyte concentration = 0.65x

Fig. 4

• Effect of detergent or wetting agent concentration on different performance characteristics of a membrane

1 Protein binding protein binding 2 Capillary flow rate Capillary flow rate 3 Striping consistency strip consistency 4 Stripe Width strip width

Fig. 5

• Calculation of the bandwidth as a function of the distribution rate

• Diaphragm bed volume = 10 μL / cm ²
• Reagent dispensing rate = 1 μL / cm
  

Fig. 6

• Typical relationships between flow rate of a membrane and function of an immunochromatographic assay

1 Surface quality surface quality 2 specificity specificity 3 Sensitivity sensitivity 4 Total Assay Time Total test time 5 Reagent Costs reagent costs
Sensitivity = analytical sensitivity = detection in μg = mass

Fig. 7-Fig. 9

The 7 - 9 show in exemplary form a test kit according to the invention. In the 7 the sampling devices can be recognized. The 9 shows the actual test cassette.

Fig. 10

10 shows an example of the assignment of the different measurement results to risk levels. To simplify the reading, the result is advantageously displayed in color. To avoid misreadings of z. B. color-blind persons, additional distinguishing features can be applied (eg different shapes, numbers and / or letters.

Objects of the invention

• 1. Hilfsmittel = Klassifikator zur Befundung (Diagnose) und Zuordnung in Wahrheitsmatrix von Befunden: richtige und falsche Klassifikationen. Da es sich um eine Ja/Nein Frage handelt, sagt man auch, der Test fällt positiv (Einordnung in auffällig) oder negativ (Einordnung nicht auffällig) aus. Fällt der Test für nur einen Biomarker positiv aus, so ist eine weitere Abklärung des positiven Befundes durch eine Koloskopie indiziert.
• 2. Hilfsmittel zur Klassifizierung, anhand bestimmter Merkmale (Bestimmung von M2-PK/Haemoglobin)
• 3. Hilfsmittel zur Entscheidung: soll Koloskopie zur weiteren Befundung durchgeführt werden: ja/nein
• 4. Hilfsmittel zur Erleichterung von ärztlichen Handlungsanweisungen durch verbesserte Einstiegsbefundung (erheblicher Zusatznutzen)
• 5. Hilfsmittel zur Erleichterung von ärztlichen Entscheidungen über Maßnahmen, z. B. Anwendung weiterer diagnostischer Verfahren z. B. Koloskopie usw. oder Therapiemaßnahmen, z. B. Chirurgie, Chemotherapie usw.
• 6. Hilfsmittel zur Verbesserung der Aussagegenauigkeit, Befundung auf Grund von Probandenstuhlproben
• 7. Testkit mit hoher Gesamt-Sensitivität und Gesamt-Spezifität für Hämoglobin und M2-PK. Zur Erkennung von Darmkrebs. Der Testkit dient als „Doppelvorfilter” im Rahmen des Darmkrebsvorsorgescreenings mittels Koloskopie.
• 8. Hilfsmittel zur Vorhersage von Therapieerfolgen
• 9. Hilfsmittel zur Verbesserung des Therapiemonitorings
• 10. Hilfsmittel zur Visualisierung des Testergebnisses (Risikoampel)

Objects of the invention are thus:

• 1. Aids = classifier for diagnosis (diagnosis) and assignment in truth matrix of findings: right and wrong classifications. Since it is a yes / no question, it is also said that the test is positive (classification in conspicuous) or negative (classification not conspicuous). If the test is positive for only one biomarker, a further clarification of the positive findings by a colonoscopy is indicated.
• 2. Classification aid based on specific characteristics (determination of M2-PK / hemoglobin)
• 3. Aid for decision: colonoscopy should be carried out for further diagnosis: yes / no
• 4. Aid to facilitate medical procedures by improved boarding (significant added value)
• 5. Aid to facilitate medical decisions on measures, eg. B. application of further diagnostic methods z. B. colonoscopy, etc. or therapeutic measures, eg. Surgery, chemotherapy etc.
• 6. aids to improve the accuracy, findings based on proband chair samples
• 7. Test kit with high overall sensitivity and overall specificity for hemoglobin and M2-PK. For the detection of colon cancer. The test kit serves as a "double pre-filter" in colonoscopy screening by colonoscopy.
• 8. Tools for predicting therapeutic success
• 9. Tools for improving therapy monitoring
• 10. Aid for visualization of the test result (risk light)

This aid is achieved according to the invention by providing a combined rapid test.

Combination rapid test for synchronous analytical determination of the enzyme biomarker Tumor M2-PK and the biomarker blood (hemoglobin).
Combi rapid test containing the test strip (tumor M2-PK +) + the test strip (iFOB +) on a test cassette

Combination rapid test for synchronous analytical determination of the enzyme biomarker Tumor M2-PK and the biomarker Hemo-Haptoglobin complex (Hb-Hp complex).
Combi rapid test containing the test strip (tumor M2-PK +) + the test strip (Hb-Hp complex +) on a test cassette

Preferred detection method is the immunochromatographic method. In a particular embodiment, immunochemical methods in the array, miniarray format, and turbidimetric methods are also possible.

The invention also relates to monoclonal antibodies

Use of special antibodies in the combination rapid test, which are both specific in their binding properties for tumor M2-PK or hemoglobin and are useful in immunochromatographic methods (eg "membrane-friendly", "detergent-suitable").

Use of a specific antibody (clone P1F3 AK specifically recognizes the spatial, dimeric conformation of the M2-PK) to provide the combination rapid test according to the invention. This tumor M2-PK specific antibody preferentially binds to one of the following epitopes or fragments thereof or combinations (epitopes or fragments thereof) of at least 4 amino acids in length:
LAPITSDP
VEASFKCC
CSGAIIVLT
TEATAVGA
LRRLAPITSDPTEATA
KCCSGAIIV
EAEAAIYH
SGAIIVLT
LQLFEE
QLFEELRR
VEASFKC
KSGRSAHG

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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• Sumedha, Clin. Chem. 45 (1999), 1628-1650 [0071]

Claims (6)

Test kit for detecting a malignant event, in particular a tumor, in the gastrointestinal tract, consisting of a first sample tube containing a first stool sampling device, a first buffer solution containing buffer 1 = 10-70 mM phosphate buffer (pH 6.7-7.6) or buffer 2 = 10-70 mM Hepes buffer (pH 7.6-8.2) or buffer 3 = 10-70 mM Triethanolamine (pH 7.3-7.7) or mixtures thereof, a second sample tube containing a second stool sampling device, a second buffer solution, buffer 1 = 10-70 mM phosphate buffer (pH 6.7-7.6) or buffer 2 = 10-70 mM Hepes buffer (pH 7.6-8.2) or buffer 3 = 10-70 mM triethanolamine (pH 7.3-7.7) or mixtures thereof, a test cassette containing a lateral flow test system with a nitrocellulose membrane as the stationary phase, the nitrocellulose membrane in turn containing tumor M2-PK antibody selected from polyclonal antibody # 3198, polyclonal antibody 214618 or monoclonal antibody clone AT4M3, clone PAT4M3AT, clone 5D2-3B3 or mixtures thereof and hemoblobin antibodies, preferably monoclonal hemoglobin antibody clone from 55081, clone from 119210, clone LS-B49414, clone LS-C129648, clone LS-C83309, clone LS-C8308 or clone B1232 M or mixtures thereof 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 content of tumor M2-PK is greater than 4-6 units / ml of stool extract and the analysis result of the second stool sample is positive if the content of hemoglobin is between 15 and 40 μg hemoglobin per gram of stool lies and wherein the four possible test results are represented by four different colors, letters, numbers, characters and / or geometric shapes. A 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 interact with other constituents of the stool, in particular not with other pyruvate kinase isoenzymes (eg M1-PK, M2 -PK (tetrameric form) L-PK, R-PK). Test kit according to claim 1, characterized in that the hemoglobin determination of the second stool sample is based on the hemoglobin-haptoglobin complex. Test kit according to claim 1, characterized in that for the determination of tumor M2-PK an antibody is used, selected from the aforementioned tumor M2-PK antibody. Test kit according to claim 1, characterized in that an antibody is used for the determination of hemoglobin selected from the aforementioned hemoglobin antibodies. Test kit according to claim 1, characterized in that the capture antibodies are bound to gold colloids.

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