METHOD FOR DETERMINATION OF A PROTEIN

Abstract

The present invention concerns a method for determining the concentration of a protein in a gastrointestinal (GI) tract sample taken from a human or an animal. The present invention is characterized by the feature that a dilution of the sample in the buffered aqueous extraction medium in a range of 1:100 to 1:10,000 is obtained. The present invention leads to a significant improvement of the technical situation, and provides a simple, sensitive and specific determination tool of proteins in GI tract samples. The determination of proteins, e.g. calprotectin, elastases or hemoglobin, in GI tract samples leads to more accurate and reproducible results.

Claims

1. A method for determining the concentration of a protein in a gastrointestinal (GI) tract sample taken from a human or an animal, comprising the steps of a) Collecting said sample and b) Mixing the sample of step a) with a determined amount of a buffered aqueous extraction medium; c) Homogenizing the mixture of step b) and d) Performing an immunoassay by using the mixture obtained in step c); e) Determining the concentration of the protein, wherein in step b) a dilution of the sample in the buffered aqueous extraction medium in a range of 1:100 to 1:10000 is obtained.

2. The method according to claim 1, wherein the GI tract sample is a watery GI tract sample or a non-watery GI tract sample.

3. The method according to claim 1, wherein the protein is present in the GI tract sample in a concentration range of 1 ng/ml of the sample to 100 mg/ml of the sample in case of a watery GI tract sample or in a concentration range of 1 ng/g of the sample to 100 mg/g of the sample in case of a non-watery GI tract sample.

4. The method according to claim 1, wherein the protein is selected from the group comprising lactoferrin, elastases (such as PMN-elastase, elastase-1, elastase-2A, elastase-2B, elastase-3A, elastase-3B), M2-pyruvate kinase, hemoglobin, haptoglobin, hemo-globin/haptoglobin complex, chymotrypsin, lysozyme, albumin, pre-albumin, beta-defensin 2, alpha-I-antitryp-sin, alpha-2-macroglobulin, carbonic anhydrase I, carbonic anhydrase II, myeloperoxidase, eosinophil-derived neurotoxin, eosinophilic peroxidase, major basic protein-1, Charcot Leiden Crystal protein (CLC/GAL10), eosinophilic protein X, C-reactive protein, mmuno globulins, secretory IgA, anti-tissue transglutaminase antibodies, anti-gliadin antibodies, anti-deamidated gliadin antibodies, interleukins (such as interleukin-1 interleukin-6, interleukin-8) tumor necrosis factor-alpha, antigens of pathogens, antibodies to pathogens, anti-H. pylori antibodies, S100 proteins, calgranulin C (S100A12; EN-RAGE), calgranulin B (S100A8; MIF-related protein 8), calgranulin C (S100A9; MIF related protein 14), and calprotectin (calgranulin A/B; S100A8/A9; MIF-related protein 8/14).

5. The method according to claim 4, wherein the protein is calprotectin, an elastase or hemoglobin.

6. The method according to claim 1, wherein the stability coefficient of the protein is in a range of 1.001 to 15.

7. The method according to claim 1, wherein the protein in the buffered aqueous extraction medium is stable at a temperature range of 2 to 42 C. for a time period of 1 to 28 days.

8. The method according to claim 1, wherein the protein concentration in the sample is higher than 300 pg/g of the sample in case of a non-watery GI tract sample and 300 pg/ml of the sample in case of a watery GI tract sample.

9. The method according to claim 1, wherein the GI tract sample is feces.

10. The method according to claim 1, wherein in step b) a dilution in a range of 1:450 to 1:550 is obtained.

11. The method according to claim 1, wherein the animal is a dog, cat, monkey, bovine, pig, horse, rat or mouse.

12. The method according to claim 1, wherein in step a) 1 to 1,000 mg of the sample are collected.

13. The method according to claim 12, wherein in step a) 2 to 100 mg of the sample are collected.

14. The method according to claim 1, wherein the immunoassay in step d) is selected from the group comprising an enzyme-linked immunosorbent assay (ELISA), an immunoturbidimetric assay, an immunochromato-graphic (lateral flow) assay and a flow-assisted cytometric assay.

15. The method according to claim 1, wherein the determination in step e) is carried out visually or by a method selected from the group comprising reflectometry, absorbance, fluorescence, chemiluminescence, electrochemiluminescence, UV/VIS spectroscopy, amperometry, magnetometry, voltametry, potentiometry, conductometry, coulometry, polarography, and electrogravimetry.

16. The method according to claim 3, wherein the stability coefficient of the protein is in a range of 1.001 to 15.

17. The method according to claim 16, wherein in step b) a dilution in a range of 1:450 to 1:550 is obtained.

18. The method according to claim 3, wherein the protein concentration in the sample is higher than 300 pg/g of the sample in case of a non-watery GI tract sample and 300 pg/ml of the sample in case of a watery GI tract sample.

19. The method according to claim 16, wherein the protein concentration in the sample is higher than 300 pg/g of the sample in case of a non-watery GI tract sample and 300 pg/ml of the sample in case of a watery GI tract sample.

20. The method according to claim 17, wherein the protein concentration in the sample is higher than 300 pg/g of the sample in case of a non-watery GI tract sample and 300 pg/ml of the sample in case of a watery GI tract sample.

Description

DESCRIPTION OF THE FIGURES

[0084] FIG. 1:

[0085] Comparison of 33 feces samples extracted with a dilution factor of 1:50 versus a dilution factor of 1:500. The thin line represents the regression line between the matching sample pairs (slope: y=1.5931x). The bold line represents the hypothetical identity line between the matching sample pairs (slope: y=1.00x).

[0086] FIG. 2:

[0087] Comparison of 29 feces samples with calprotectin concentrations above 300 g/g of the sample extracted with a dilution factor of 1:50 versus a dilution factor of 1:500. The fecal samples are sorted by increasing concentrations measured with the 1:50 extractive dilution. The speckled bars represent the calprotectin concentrations determined with an extractive dilution of 1:50. The solid bars represent the calprotectin concentrations determined with an extractive dilution of 1:500. Samples measuring higher than 6,000 g/g of the sample were not further diluted in the ELISA assay and are shown as 6000 g/g.

[0088] FIG. 3:

[0089] Six feces samples with calprotectin concentrations in a range of 50 to 1,800 g/g of the sample were extracted using dilution factors of 1:50 and 1:500, respectively, with 3 different commercial extraction buffers and analyzed in two different commercial ELISA tests. All data points measured for the two different extractive dilutions were summarized, and the matching data pairs were compared by regression analysis. The solid, bold line represents the hypothetical identity line between the matching sample pairs (slope: y=1.00x), whereas the dashed line represents the true regression line through all 30 matching data pairs (slope: y=2.8612x).

[0090] FIG. 4:

[0091] 40 feces samples with calprotectin concentrations in a range of 20 to 2,500 g/g of the sample were extracted with a dilution factor of 1:500 using two different extraction methods (the BHLMANN Calex Cap device by pipetting-in and a manual weighing-in procedure, respectively) and analyzed in the BHLMANN Calprotectin ELISA. The matching data pairs were compared by regression analysis. The fine line represents the hypothetical identity line between the matching sample pairs (slope: y=1.00x), whereas the solid, bold line represents the true regression line through all 40 matching data pairs (slope: y=0.9636x).

[0092] The characteristic effects and advantages of the present invention are illustrated by the following non-limiting examples.

EXAMPLE 1: EXTRACTION OF FECES SAMPLES

[0093] Feces were obtained from anonymized surplus stool samples kindly provided by local routine laboratories and the gastroenterology department of the University Hospital of Basel. Upon receipt, samples were either stored at 2 to 8 C. for up to one week or at <20 C. for longer periods of time (up to two years). The feces samples were equilibrated to ambient temperature (20 to 25 C.) prior to extraction and were then extracted as follows: [0094] a) Reference Method: 60 to 100 mg of feces were weighed into the sample chamber of the Smart-Prep device (B-CAL-RD, BHLMANN Laboratories AG, Schoenenbuch, Switzerland) by using either a small spoon for non-watery samples or a quantitative pipette for watery (liquid) samples. In the latter case, 60 to 100 l of watery feces was pipetted into the sample chamber as the density of an average feces sample is close to 1 g/ml. Accordingly, 3 to 5 ml of extraction buffer (B-CAL-EX, BHLMANN Laboratories AG, Schoenenbuch, Switzerland) were added resulting in an extraction factor of 1:50. The Smart-Prep device was then vortexed at maximum speed for 1 minute and the resulting homogenate was allowed to sediment for 30 minutes. Aliquots of the supernatant were then used for the respective Calprotectin ELISA assay (see Example 2). [0095] b) In order to generate alternative extraction factors of feces vs. extraction buffer of 1:100, 1:250, and 1:500, approximately 40 mg (40 l), 30 mg (30 l), and 15 mg (15 l) of feces were weighed (pipetted) into the sample chamber of the Smart-Prep device (B-CAL-RD, BHLMANN Laboratories AG, Schoenenbuch, Switzerland), respectively. Accordingly, approximately 4 ml, 7.5 ml, and 7.5 ml of extraction buffer (B-CAL-EX, BHLMANN Laboratories AG, Schoenenbuch, Switzerland), respectively, were added to obtain the respective extractive dilution factors. Extractive dilution factors of 1:1,000 and 1:5,000 were obtained by weighing (pipetting) of 20 mg (20 l) and 10 mg (10 l) of feces into 50 ml-Falcon Tubes (ThermoFisher Scientific AG, Reinach, Switzerland), and adding 20 ml and 50 ml of extraction buffer (B-CAL-EX, BHLMANN Laboratories AG, Schoenenbuch, Switzerland), respectively. All filled devices were then vortexed at maximum speed for 1 minute and the resulting homogenates were allowed to sediment for 30 minutes. Aliquots of the supernatants were then used for the respective Calprotectin ELISA assay (see Example 2). [0096] c) Alternatively, the CALEX Cap and CALEX Valve devices (BHLMANN Laboratories AG, Schoenenbuch, Switzerland), respectively, were used to obtain an extraction factor of 1:500. The devices consist of a sampling pin, an extraction chamber of approximately 8 ml of volume and either a closing screw cap (CALEX Cap) or a valve portion (CALEX Valve) at the other end to transfer the resulting feces extract to the analyzing devices. The sampling pin with 8 to 10 grooves (total volume of the grooves corresponds to the volume of 10 mg of feces) was introduced three to five times into a non-watery feces sample in order to completely fill the grooves. Then the sampling pin was introduced through a funnel located at the opening of the extraction chamber whereby excess feces is stripped off and exactly 10 mg of feces is transferred into the extraction chamber, which was previously filled with 5 ml of extraction buffer (B-CAL-EX, BHLMANN Laboratories AG, Schoenenbuch, Switzerland), thereby generating an extraction factor of 1:500. In case of watery (liquid) feces samples 10 l of feces were introduced by a precision pipette through the funnel located at the opening of the extraction chamber and the device was closed by pushing the sampling pin into its closed position. The devices were then manually and vigorously shaken for at least twice 10 seconds or until all remaining feces was removed from the grooves of the sampling pin or were vortexed for 30 to 60 seconds at maximum speed. The resulting homogenates were allowed to sediment for 30 minutes. Aliquots of the supernatants and/or the homogenates were then used for the respective Calprotectin assays (see Example 2).

EXAMPLE 2: CALPROTECTIN ASSAYS

[0097] If not mentioned explicitly, the BHLMANN Calprotectin sandwich ELISA (EK-CAL; BHLMANN Laboratories AG, Schoenenbuch, Switzerland) was used to assess the concentrations of calprotectin in extracted fecal samples. The fecal extracts obtained in Examples 3 to 10 were further diluted with incubation buffer (B-CAL-IB; BHLMANN Laboratories AG, Schoenenbuch, Switzerland) in a range of 1:5 to 1:150 and 100 l of the diluted fecal sample extract as well as 100 l of prediluted calibrators and controls were pipetted in duplicates into wells of a microtiter plate coated with a highly specific anti-calprotectin antibody (B-CAL-MP; BHLMANN Laboratories AG, Schoenenbuch, Switzerland). Subsequently, 100 l of a second monoclonal anti-calprotectin antibody conjugated to horse-radish peroxidase (enzyme label B-CAL-EL; BHLMANN Laboratories AG, Schoenenbuch, Switzerland) was added to each well and the microtiter plate was then incubated on a rotatory shaker (400-600 rpm) for 30 minutes at ambient temperature (18-28 C.). After extended washing 100 l of a TMB substrate solution (B-TMB12; BHLMANN Laboratories AG, Schoenenbuch, Switzerland) was added to each well and the microtiter plate was incubated on a rotatory shaker (400-600 rpm) for 15 minutes at ambient temperature (18-28 C.). After adding 100 l of a stopping solution the absorbance of each well was measured at 450 nm in the SpectraMax 190 microtiter plate reader (Molecular Devices, Sunnyvale, Calif., USA). The absorbance of the calibrators (on the vertical axis) was plotted vs. their respective calprotectin concentrations (on the horizontal axis), and the best fitting standard curve was drawn using a 4-parameter logistics fit. The absorbance of controls and extracted fecal samples were then intersected with the standard curve and the resulting calprotectin concentrations were read from the horizontal axis. Finally, the read calprotectin concentrations were corrected for the different dilution factors where necessary. Alternative calprotectin ELISAs were used accordingly.

EXAMPLE 3: EFFECT OF DILUTION FACTOR 1:50 VS. 1:500 DURING EXTRACTION PROCESS ON CALPROTECTIN YIELD

[0098] 33 feces samples were extracted either according to Example 1a) (extractive dilution 1:50) or according to Example 1b) (extractive dilution 1:500) and assayed in the BHLMANN Calprotectin ELISA as described in Example 2. The results are listed in Table 1 and are graphically illustrated in FIG. 1. It can clearly be seen that samples showing a calprotectin concentration above approximately 300 g/g of the sample are measuring higher at an extractive dilution of 1:500 as compared with 1:50, in particular feces samples above 1,000 g/g of the sample.

TABLE-US-00001 TABLE 1 Calprotectin Calprotectin Conc. [g/g of Conc. [g/g of Feces the sample] the sample] Sample 1:50 1:500 16 3 30 9 18 25 15 36 38 6 39 38 13 43 36 12 43 47 27 51 60 2 59 64 14 64 70 31 65 77 21 71 69 8 73 78 25 80 78 4 125 167 11 175 196 10 213 237 5 238 230 22 253 249 20 283 351 28 326 411 3 348 363 18 376 457 30 565 623 26 711 781 39 731 756 17 795 853 32 1,028 1,125 35 1,417 4,961 38 1,704 3,053 37 1,776 2,462 19 2,329 2,428 29 2,409 3,244 33 2,739 4,857

[0099] Subsequently, 29 additional samples with calprotectin concentrations higher than 300 g/g of the sample were tested in the same way as described above. The results are shown in Table 2 and FIG. 2. All samples examined exhibited a higher concentration when extracted using a dilution factor of 1:500.

TABLE-US-00002 TABLE 2 Calprotectin Conc. Feces Consistency of [g/g of the sample] Sample Feces Matrix 1:50 1:500 EYR [%] 3 not assessed 348 363 104 18 not assessed 376 457 122 20 not assessed 283 351 124 28 not assessed 326 411 126 29 liquid 2,621 3,436 131 40 normal consistency 1,333 3,579 268 41 liquid 427 472 111 42 normal consistency 1,069 2,285 214 43 liquid 2,310 6,000 260 44 dry, hard 1,177 6,000 510 45 semi-liquid, 2,803 3,792 135 not homogenous 46 normal consistency 1,546 3,781 245 47 normal consistency 1,376 6,000 436 48 normal consistency, 1,309 1,543 118 not homogenous 49 normal consistency 1,232 5,925 481 50 normal consistency 918 2,002 218 51 normal consistency 908 3,625 399 52 normal consistency 2,731 6,000 220 53 not assessed 2,207 4,403 200 54 dry, hard 663 1,518 229 55 normal consistency 597 821 137 56 semi-liquid, 2,611 6,000 230 not homogenous 57 normal consistency 896 6,000 670 58 not assessed 1,362 5,548 407 59 not assessed 1,088 4,934 453 60 not assessed 2,776 6,000 216 61 not assessed 895 2,436 272 62 not assessed 598 931 156 63 not assessed 1,206 3,531 293 Min. 283 351 104 Max. 2,803 6,000 670 Avg. 1,310 3,384 258

[0100] The extraction yield ratio (EYR) of a sample is given in % and is determined according to the following equation:

EYR=[(Concentration of the sample extracted using a dilution factor of 1:500)/(Concentration of the sample extracted using a dilution factor of 1:50)]*100%

[0101] The extraction yield ratio ranged from 104 to 670%. The average extraction yield ratio was 258%. These results were independent from the consistency of the feces sample as this effect can be reported from liquid (watery) to very hard samples. The difference between the extractive dilution at 1:500 vs. 1:50 was statistically highly significant showing a p value of <0.0001 (Wilcoxon Signed Rank test).

EXAMPLE 4: EFFECT OF STEPWISE EXTRACTIVE DILUTION FROM 1:50 UP TO 1:5,000 (COMPARATIVE EXAMPLE)

[0102] Interpreting the results of Example 3 it seems that the extraction capacity is limited at an extractive dilution of 1:50, particularly for feces samples containing a calprotectin concentration of more than approximately 500 g/g of the sample. To test this hypothesis, the resulting homogenates of a 1:50 extraction were split immediately after the extracting step as described in Example 1a) into four aliquots. Three of these aliquots were diluted further 10-fold, 20-fold and 100-fold, respectively, with further extraction buffer (B-CAL-EX, BHLMANN Laboratories AG, Schoenenbuch, Switzerland) such that a final dilution of 1:500, 1:1,000 and 1:5,000, respectively, was obtained. The diluted homogenates were extracted further by vortexing for 1 minute at maximum speed. The diluted homogenates were allowed to sediment for at least 30 minutes. All homogenates were then assayed in the BHLMANN Calprotectin ELISA according to Example 2. Six feces samples with increasing calprotectin concentrations were tested and the results are shown in Table 3. It can be seen that calprotectin from feces containing high protein concentrations above 400 g/g of the sample cannot be fully extracted out of the feces matrix at an extractive dilution of 1:50 and show a higher extraction yield when allowed to be re-extracted with 10- to 100-times higher volumes of extraction buffer.

[0103] But when comparing such a stepwise extraction using a first dilution of 1:50 and a second higher dilution of 1:500, 1:1,000, and 1:5,000, respectively (see Table 3) to an extraction using a direct dilution of 1:500, 1:1,000, and 1:5,000 in one step (see Example 5, Table 4), it can clearly be seen, that the calprotectin concentration is significantly higher with the inventive extraction using a direct dilution of 1:500, 1:1,000, and 1:5,000 in one step. A stepwise extraction is not sufficient to fully extract calprotectin from the feces samples.

TABLE-US-00003 TABLE 3 Calprotectin Conc. [g/g of the sample] after Feces stepwise Extractive Dilutions with B-CAL-EX Sample 1:50 1:500 1:1,000 1:5,000 64 310 367 341 407 65 1,006 1,200 1,137 1,170 66 1,867 3,728 4,073 3,958 67 1,915 1,992 2,899 1,601 68 2,700 6,098 6,707 4,880 69 3,291 6,215 6,345 5,605

EXAMPLE 5: EFFECT OF THE DILUTION FACTOR DURING THE EXTRACTION PROCESS ON CALPROTECTIN YIELD

[0104] Four feces samples were extracted according to Examples 1a) and 1b) by adding 50-, 100-, 250-, 500-, 1,000-, and 5,000-times the volume of extraction buffer (B-CAL-EX, BHLMANN Laboratories AG, Schoenenbuch, Switzerland) to the weighed-in feces. After 1 minute of vigorous vortexing each sample was allowed to sediment for approximately 30 minutes and then assayed in the BHLMANN Calprotectin ELISA as described in Example 2. From the results presented in Table 4 it can be seen that the higher the extractive dilution factor the higher is the mean extraction yield of calprotectin. Furthermore, it can be seen that in all tests the concentration of calprotectin was higher using a dilution in a range of 1:100 to 1:5,000 in comparison to a dilution of 1:50. An extraction using a dilution of 1:50 is not sufficient to fully extract calprotectin from the feces samples. The optimum extractive dilution factor is peaking around 1:250 to 1:1,000.

TABLE-US-00004 TABLE 4 Feces Calprotectin Conc. [g/g of the sample] Sample 1:50 1:100 1:250 1:500 1:1,000 1:5,000 65 918 1,163 1,543 2,433 1,435 1,822 66 1,647 2,398 4,075 7,182 7,524 6,746 70 2,042 3,361 2,725 3,651 3,867 n.a. 71 3,383 3,619 5,440 4,382 6,491 n.a. Mean 1,608 2,128 2,807 3,629 4,063 n.a.

EXAMPLE 6: COMPARISON OF EXTRACTION BUFFERS

[0105] It can be learned from the state of the art literature that the constitution of the extraction buffer may significantly influence the extraction yield of calprotectin employing an extractive dilution from 1:2 up to 1:80 (U.S. Pat. No. 5,455,160; Tn et al. (Clinica Chimica Acta 292 (2000) 41-54)). Hence, the influence of three different optimized, commercial extraction buffers was tested using an extractive dilution of 1:50 vs. 1:500 with each of them. Six feces samples were extracted using a dilution of 1:50 according to Example 1a) and 1:500 according to Example 1b), respectively, using the BL extraction buffer (B-CAL-EX; BHLMANN Laboratories AG, Schoenenbuch, Switzerland), the ID extraction buffer (EXBUF; Immundiagnostik AG, Bensheim, Germany) and the CL extraction buffer (FEC EXTR BUF 2,5x; Calpro AS, Lysaker, Norway). All resulting extracts were assayed in the BHLMANN Calprotectin ELISA as described in Example 2. The results are shown in Table 5. Although the absolute values of the measured calprotectin levels are somewhat variable for the three different extraction buffers, it could be shown for each of the three optimized extraction buffers that the extraction yield ratio was significantly higher (p<0.0001 by Wilcoxon test) when extracted by an extraction factor of 1:500 as compared to 1:50 (see also FIG. 3). The average increase in the extraction yield ratio (in a range of 234% to 249%) was remarkably similar between the three extraction buffers. This clearly shows that the higher extraction yield of calprotectin from extracted feces is almost exclusively dependent on the higher extractive dilution factor of 1:500. Noteworthy that the CL extraction buffer (FEC EXTR BUF 2,5x; Calpro AS, Lysaker, Norway) is the optimized buffer described in EP 0 937 259 B1, U.S. Pat. No. 5,455,160, and Tn et al. (Clinica Chimica Acta 292 (2000) 41-54) for which it has been stated that an extractive dilution factor of 1:50 and 1:80, respectively, is entirely sufficient for a full extraction of calprotectin from feces.

TABLE-US-00005 TABLE 5 Extraction Extraction Extraction Buffer 1 (BL) Buffer 2 (ID) Buffer 3 (CL) BHLMANN ELISA BHLMANN ELISA BHLMANN ELISA Calprotectin Conc. Calprotectin Conc. Calprotectin Conc. Feces [g/g of the sample] EYR [g/g of the sample] EYR [g/g of the sample] EYR Sample 1:50 1:500 [%] 1:50 1:500 [%] 1:50 1:500 [%] 72 79 102 129 41 87 212 67 95 142 73 411 454 110 216 278 129 335 562 168 74 633 791 125 456 786 172 773 1,526 197 75 1,245 5,459 438 856 3,599 420 1,839 5,716 311 76 1,553 4,515 291 824 2,356 286 1,477 4,013 272 77 1,688 5,399 320 999 2,723 273 1,932 6,125 317 Mean 236 249 234

TABLE-US-00006 TABLE 6 Extraction Extraction Buffer 1 (BL) Buffer 3 (CL) CALPRO ELISA CALPRO ELISA Calprotectin Conc. Calprotectin Conc. Feces [g/g of the sample] EYR [g/g of the sample] EYR Sample 1:50 1:500 [%] 1:50 1:500 [%] 72 53 60 113 64 84 131 73 258 319 124 274 413 151 74 509 538 106 494 1,021 207 75 1,004 3,357 334 982 3,733 380 76 1,030 2,187 212 925 2,722 294 77 1,738 3,041 175 2,166 3,956 183 Mean 177 224

EXAMPLE 7: COMPARISON/INFLUENCE OF CALPROTECTIN ELISA METHODS

[0106] It could be argued that the results generated in Example 6 are biased because all fecal extracts were measured in the same calprotectin assay, the BHLMANN Calprotectin ELISA, which is based on a sandwich assay technology using a set of two highly specific monoclonal antibodies. Hence the BL extracts and the CL extracts were also measured in an alternative ELISA assay, the Calprolabs Calprotectin ELISA (ALP) (Calpro AS, Lysaker, Norway) which is based on a sandwich assay technology using a monoclonal antibody for catching the calprotectin and polyclonal antibodies for detecting the bound calprotectin. Although the absolute values of the measured calprotectin concentrations are slightly different for the two extraction buffers, it could be shown also with the Calprolabs ELISA that the extraction yield was significantly higher (p=0.0024 by Wilcoxon test) when extracted using a dilution of 1:500 as compared to a dilution of 1:50 (Table 6). The average increase in extraction yield was 177% for the BHLMANN BL extraction buffer (B-CAL-EX; BHLMANN Laboratories AG, Schoenenbuch, Switzerland) and 224% for the Calpro CL extraction buffer (FEC EXTR BUF 2,5x; Calpro AS, Lysaker, Norway), respectively. Hence, the effect of a higher extraction yield of calprotectin from feces with an extractive dilution of 1:500 was independent from the ELISA assay used.

[0107] The results from Examples 6 and 7 are summarized by a regression analysis and presented in FIG. 3. From the slope of the regression line (dashed line) it can be read that the extraction yield of calprotectin is in average approximately 200% higher when the feces sample was extracted with a dilution of 1:500 as compared to a dilution of 1:50. The graph also illustrates that almost any of the 1:500 feces extracts was reading above the identity line (solid, bold line) showing again the superior performance of the 1:500 extractive dilution.

EXAMPLE 8: CALEX DEVICES PROVIDING A DIRECT 1:500 EXTRACTION METHODOLOGY

[0108] In order to obtain an easy, proper, reliable and hygienic collection and extraction of feces with a direct extraction factor of 1:500, the CALEX Cap and CALEX Valve devices were developed (BHLMANN Laboratories AG, Schoenenbuch, Switzerland). These devices demand neither for a weighing-in step nor for any pipetting step prior to use of the final, homogenized fecal extract for the respective calprotectin assay, i.e. the BHLMANN Calprotectin ELISA (EK-CAL; BHLMANN Laboratories AG, Schoenenbuch, Switzerland).

[0109] The performance of the CALEX Cap Device, described and used as illustrated in Example 1c), was compared to a quantitative reference weighing-in procedure using a fecal sample preparation kit (Cat.No. 10745804; Roche Diagnostics GmbH, Mannheim) according to Example 1b). The dosing tip of the sampling pin of the CALEX Cap device was introduced 5-times into a fecal sample at different locations to collect enough feces material within the grooves of the dosing tip. The sampling pin was then re-introduced into the extraction chamber of the CALEX Cap device through a funnel equipped with a transversal septum so that excess fecal material was stripped off the dosing tip of the sampling pin, and exactly 101 mg of fecal material was introduced into the extraction chamber filled with 5 ml of extraction buffer (B-CAL-EX; BHLMANN Laboratories AG, Schoenenbuch, Switzerland). The CALEX device was then vortexed at maximum speed for 30 seconds, left for 30 minutes and vortexed again for 30 seconds at maximum speed. The homogenized fecal extract was then allowed to sediment for 1 hour at ambient temperature (approximately 23 C.) and finally assayed in the BHLMANN Calprotectin ELISA. For the reference procedure, 8 to 12 mg of 40 different feces samples were weighed into the sample chamber of the base cap of the Roche device (Cat.No. 10745804; Roche Diagnostics GmbH, Mannheim). The body of the extraction tube was then pressed onto the filled base cap and 4 to 6 ml of extraction buffer (B-CAL-EX; BHLMANN Laboratories AG, Schoenenbuch, Switzerland) were added. The Roche tubes were then capped, vortexed for 1 minute at maximum speed, allowed to sediment for 1 hour at ambient temperature (approximately 23 C.) and finally assayed in the BHLMANN Calprotectin ELISA in the same run together with the fecal extracts processed by the CALEX Cap devices. The comparison data are shown in FIG. 4. There was no significant difference between the two collection and extraction methods and devices, respectively, observed neither in accuracy (slope y=0.9636x) nor in precision/reliability (R.sup.2=0.9417).

EXAMPLE 9: HANDLING AND PERFORMANCE OF LIQUID (WATERY) FECES

[0110] Liquid feces are very delicate to handle as they cannot be easily collected with the dosing tip of the CALEX devices (cf. Examples 1c) and 8) or of any other state of the art device using the same or similar sample collecting principles. This was also observed by Whitehead et al. (liquid sample 5 in FIG. 3 on page 58 of Ann. Clin. Biochem. 50 (2013) 53-61).

[0111] In this example we provide a very simple and reliable method to circumvent the problem of the inferior performance (particularly the greatly diminished extraction yield) of liquid (watery) feces samples. Exactly 10 l of five watery feces samples were pipetted through the funnel into the extraction chamber of the CALEX Cap device pre-filled with 5 ml of extraction buffer (B-CAL-EX; BHLMANN Laboratories AG, Schoenenbuch, Switzerland) using a precision pipette leading to an extractive dilution of 1:500 (v/v). The CALEX device was then closed with the sampling pin and processed as described in Example 1c). Alternatively, a small portion (approximately 5 to 15 mg) of the five watery feces samples was transferred with a small spoon into a pre-weighed TPP plastic centrifuge tube (Techno Plastic Products AG, Trasadingen, Switzerland) and the net weight of the feces samples was determined with a precision balance. Then a 500-fold amount of extraction buffer (B-CAL-EX; BHLMANN Laboratories AG, Schoenenbuch, Switzerland) was added, the TTP tube tightly closed and the extraction was processed as described in Examples 1b) and 8 also leading to an extractive dilution of 1:500 (w/v). The calprotectin concentrations of the fecal extracts obtained by both methods were then measured in the BHLMANN Calprotectin ELISA. The calprotectin extraction yields and variability (CVs, coefficients of variation) of triplicate extractions for both methodological approaches were compared and are illustrated in Table 7.

[0112] It can be seen that for a semiliquid sample the CALEX pipetting method provides higher extraction yields, whereas for liquid samples the weighing-in method provides somewhat higher extraction yields.

[0113] However, both methods using a dilution of the sample in the buffered aqueous extraction medium of 1:500 (w/v or v/v) provide acceptable extraction yields, particularly when compared to other extraction tools which show much inferior extraction yields around 5% only, as reported in the state of the art literature (e.g. Whitehead et al., Ann. Clin. Biochem. 50 (2013) 53-61). The variability (CVs) between extractions of the same fecal sample is higher with the CALEX pipetting method, but still acceptable. This is due to the inhomogeneous nature of watery feces samples as they contain a lot of liquid, but also small solid portions. These small solid portions do contain much more of the calprotectin than the liquid portion. The liquid portion usually contains less than 10% of the total calprotectin of a watery fecal sample (data not shown). Therefore, it is very important, but sometimes not very easy, to mix the liquid (watery) fecal sample well before or while pipetting it.

TABLE-US-00007 TABLE 7 Calprotectin Conc. [g/g of the sample] Feces Manual Manual CALEX Manual CALEX Recovery Sample Consistency 1:50 Replicate 1:500 1:500 Statistics 1:500 1:500 [%] 78 Semi- 142 1 152 188 Mean Conc. 155 171 110 liquid 2 167 177 SD 11 21 3 146 147 CV [%] 7.1 12.6 79 Liquid 93 1 93 88 Mean Conc. 93 56 60 2 94 39 SD 1 27 3 94 43 CV [%] 0.8 48.4 80 Liquid 21 1 29 26 Mean Conc. 27 27 99 2 27 26 SD 1 2 3 27 29 CV [%] 4.9 5.7 81 Liquid 1,267 1 2,883 2,256 Mean Conc. 2,568 2,047 80 2 2,623 1,967 SD 345.8 182.7 3 2,198 1,918 CV [%] 13.5 8.9 82 Liquid 52 1 69 52 Mean Conc. 63 43 68 2 63 49 SD 6 14 3 56 27 CV [%] 10.1 31.8 Average (CV; 7.3 21.5 83 Rec.) [%]

EXAMPLE 10: STABILITY OF 1:50 VS. 1:500 EXTRACTS

[0114] In most cases fecal extracts are not immediately analyzed in the testing laboratory, being it because the sample has to be shipped from the patient's home or the doctor's office to the testing laboratory first or being it because the testing laboratory analyzes the fecal sample extracts batchwise, i.e. once a week. Hence, we have also tested the stability of the fecal extracts when refrigerated (at 2 to 8 C.) and at a temperature a few degrees above room temperature, namely at 28 C. 39 feces samples were extracted 1:50 according to Example 1a) and 1:500 according to Example 1b) and then incubated for 1, 2, 3, and 6 days either at 2 to 8 C. or at 28 C. After the corresponding incubations the extracts were tested in the BHLMANN Calprotectin ELISA according to Example 2. Surprisingly, the inventors have found that the fecal extracts were much more stable, particularly at elevated temperature, when extracted and stored using an extractive dilution of 1:500 (i.e. using the CALEX devices from Examples 1c) and 8) as compared to an extractive dilution of 1:50 using a conventional state-of-the-art device. The summarized results in Tables 8a and 8b show that the fecal samples extracted with a dilution factor of 1:500 were perfectly stable at both temperatures for up to 6 days, whereas fecal samples extracted with a dilution factor of 1:50 show a gradual degradation over time. More detailed, the inventors have observed that a maximum of 3 and 4 fecal extracts were not entirely stable at 2 to 8 C. and 28 C., respectively, when extracted and stored at a dilution of 1:500 for up to 6 days (see Table 8a), whereas up to 16 and 23 fecal extracts were not stable at a temperature range of 2 to 8 C. and 28 C., respectively, when extracted and stored at a dilution of 1:50 for up to 6 days (see Table 8b). The criterion for instability in the context of this example is a recovery of calprotectin of less than 80% as compared to Time 0 (t.sub.0). All unstable samples are marked in Tables 8a and 8b by a speckled background.

[0115] After 24 hours up to 6 days the protein is degraded in average in a range of 2% to 13% when the extracted protein is kept at a temperature range of 2 C. to 8 C., and in a range of 6% to 26% when the extracted protein is kept at a temperature of 28 C., respectively, using an extractive dilution of 1:50 as applied in the state of the art. No degradation is visible at an extractive dilution of 1:500. The mean stability coefficient is in a range of 1.134 to 1.676 when calculated for the mean recoveries as compared to t.sub.0.

[0116] The present invention provides a simple, sensitive and specific determination tool of proteins in GI tract samples. The determination of proteins, e.g. calprotectin, elastases or hemoglobin, in GI tract samples leads to more accurate and reproducible results than with state of the art methods. The measured concentrations, particularly in GI tract samples containing high levels of the protein, are significantly higher. Therefore the protein determination yields more accurate levels which are particularly important during therapy follow-up of affected patients with inflammations of the gastrointestinal tract. The present invention also leads to more stable protein extracts which simplifies sample collection by and transportation from the affected patient to the doctor's or testing laboratory in terms of efficacy, storage conditions (no cooling chain needed) and shipping time (no express services needed).