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 an elastase protein in a gastrointestinal (GI) tract sample taken from a human or an animal, comprising the steps of: a) Collecting a watery GI tract sample or a non-watery GI tract sample, and b) Mixing the sample of step a) with a determined amount of a buffered aqueous extraction medium to extract elastase from the sample wherein a dilution of the sample in the buffered aqueous extraction medium in a range of 1:400 to 1:1000 w/vol is obtained for the non-watery GI tract sample and wherein a dilution of the sample in the buffered aqueous extraction medium in a range of 1:400 to 1.1000 is obtained for the watery GI tract sample; c) Homogenizing the mixture of step b) to extract elastase from the sample; d) Performing an immunoassay by using the mixture obtained in step c) to determine the concentration of the elastase protein.

2. (canceled)

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 elastase is selected from the group comprising PMN-elastase, elastase-1, elastase-2A, elastase-2B, elastase-3A, and elastase-3B).

5. (canceled)

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 g/g of the sample in case of a non-watery GI tract sample and 300 g/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 d) is carried out visually or further 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 the 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 g/g of the sample in case of a non-watery GI tract sample and 300 g/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 g/g of the sample in case of a non-watery GI tract sample and 300 g/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 g/g of the sample in case of a non-watery GI tract sample and 300 g/ml of the sample in case of a watery GI tract sample.

Description

DESCRIPTION OF THE FIGURES

[0086] FIG. 1: 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).

[0087] FIG. 2: 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: 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).

[0089] FIG. 4: 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 BUHLMANN Calexm Cap device by pipetting-in and a manual weighing-in procedure, respectively) and analyzed in the BUHLMANN 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).

[0090] The characteristic effects and advantages of the present invention are illustrated by the following non-limiting examples.

Example 1: Extraction of Feces Samples

[0091] 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: [0092] a) Reference Method: 60 to 100 mg of feces were weighed into the sample chamber of the Smart-Prep device (B-CAL-RD, BUHLMANN 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, BUHLMANN 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). [0093] 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, BUHLMANN Laboratories AG, Schoenenbuch, Switzerland), respectively. Accordingly, approximately 4 ml, 7.5 ml, and 7.5 ml of extraction buffer (B-CAL-EX, BUHLMANN 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, BUHLMANN 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). [0094] c) Alternatively, the CALEX Cap and CALEX Valve devices (BUHLMANN 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, BUHLMANN 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 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

[0095] If not mentioned explicitly, the BUHLMANN Calprotectin sandwich ELISA (EK-CAL; BUHLMANN 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; BUHLMANN 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; BUHLMANN Laboratories AG, Schoenenbuch, Switzerland). Subsequently, 100 l of a second monoclonal anti-calprotectin antibody conjugated to horse-radish peroxidase (enzyme label B-CAL-EL; BUHLMANN 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; BUHLMANN 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

[0096] 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 BUHLMANN 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 Conc. Calprotectin Conc. Feces [g/g of the sample] [g/g of 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

[0097] 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

[0098] 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%

[0099] 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)

[0100] 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, BUHLMANN 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 BUHLMANN 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.

[0101] 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

[0102] 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, BUHLMANN 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 BUHLMANN 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

[0103] 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; BUHLMANN 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 BUHLMANN 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 Buffer 1 (BL) Extraction Buffer 2 (ID) Extraction 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 Buffer 1 (BL) Extraction 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

[0104] 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 BUHLMANN 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 Calprolabsm 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 Calprolabsm 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 BUHLMANN BL extraction buffer (B-CAL-EX; BUHLMANN 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.

[0105] 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

[0106] 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 (BUHLMANN 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 BUHLMANN Calprotectin ELISA (EK-CAL; BUHLMANN Laboratories AG, Schoenenbuch, Switzerland).

[0107] 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; BUHLMANN 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 BUHLMANN 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; BUHLMANN 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 BUHLMANN Calprotectin ELISA in the same run together with the 40 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

[0108] 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).

[0109] 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; BUHLMANN 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; BUHLMANN 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 BUHLMANN 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.

[0110] 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.

[0111] 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 [%] 1 152 188 Mean Conc. 155 171 78 Semi- 142 2 167 177 SD 11 21 110 liquid 3 146 147 CV [%] 7.1 12.6 1 93 88 Mean Conc. 93 56 79 Liquid 93 2 94 39 SD 1 27 60 3 94 43 CV [%] 0.8 48.4 1 29 26 Mean Conc. 27 27 80 Liquid 21 2 27 26 SD 1 2 99 3 27 29 CV [%] 4.9 5.7 1 2,883 2,256 Mean Conc. 2,568 2,047 81 Liquid 1,267 2 2,623 1,967 SD 345.8 182.7 80 3 2,198 1,918 CV [%] 13.5 8.9 1 69 52 Mean Conc. 63 43 82 Liquid 52 2 63 49 SD 6 14 68 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

[0112] 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 BUHLMANN 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).

[0113] 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.

TABLE-US-00008 TABLE 8a Calprotectin Conc. [g/g of Extractive Dilution by Factor 1:500 - Recovery [%]/Time 0 (Start) Feces the sample] Storage Temperature 2-8 C. Storage Temperature 28 C. Sample Time 0 (Start) Day 1 Day 2 Day 3 Day 6 Day 1 Day 2 Day 3 Day 6 9 25 104 106 106 106 92 87 94 79 16 30 105 111 115 111 111 105 98 91 13 36 111 122 121 125 125 122 119 114 6 38 104 113 115 124 132 137 126 126 15 38 108 118 115 115 121 113 73 100 12 47 102 109 106 113 119 111 115 111 1 59 119 126 129 141 145 143 147 139 27 60 85 69 77 75 97 50 50 42 2 64 108 118 121 132 136 144 144 147 21 69 141 99 106 109 117 104 107 109 14 70 108 112 118 124 136 132 134 129 31 77 155 117 122 130 112 124 129 129 8 78 97 110 124 123 160 136 132 132 25 78 160 108 101 121 126 121 126 122 7 109 106 111 112 116 115 110 109 109 24 123 169 111 115 122 119 129 124 147 4 167 102 105 59 63 129 139 69 71 11 196 113 107 120 117 123 112 123 137 5 230 110 112 110 118 112 107 110 116 10 237 105 108 111 121 116 122 120 132 22 249 160 106 110 107 129 118 120 133 20 351 112 101 119 109 138 101 108 92 3 363 116 119 59 66 137 138 71 75 28 411 128 98 105 114 106 126 127 137 18 457 93 90 93 98 106 86 92 91 30 623 139 113 115 129 122 131 133 127 39 756 206 126 146 179 127 210 195 217 26 781 136 112 118 128 122 130 144 154 23 790 135 126 131 134 124 124 126 115 17 853 105 105 109 111 114 107 119 118 32 1,125 180 115 128 142 113 125 128 196 34 1,448 140 107 148 117 147 129 138 134 19 2,428 118 108 115 124 120 105 114 122 37 2,462 135 117 128 144 106 116 122 113 38 3,053 158 101 104 103 99 106 122 141 29 3,244 127 110 103 111 99 116 123 128 36 4,190 84 91 93 95 59 106 104 218 33 4,857 109 135 119 122 93 133 128 134 35 4,961 108 122 132 94 131 148 Mean as compared to 123 110 112 117 118 120 118 124 Time 0 Mean as compared to 100 89 91 95 100 102 100 106 Day 1 Storage 2-8 C. Storage 28 C. Day 1 Day 2 Day 3 Day 6 Day 1 Day 2 Day 3 Day 6 Number of Samples 39 39 39 38 39 39 39 38 analyzed Feces Extracts showing 0 1 3 3 1 1 4 4 <80% Recovery as compared to Time 0

TABLE-US-00009 TABLE 8b Calprotectin Conc. [g/g of Extractive Dilution by Factor 1:50 - Recovery [%]/Time 0 (Start) Feces the sample] Storage Temperature 2-8 C. Storage Temperature 28 C. Sample Time 0 (Start) Day 1 Day 2 Day 3 Day 6 Day 1 Day 2 Day 3 Day 6 9 18 96 118 108 116 108 110 101 95 16 3 103 115 104 114 97 118 104 100 13 43 119 132 57 64 121 122 54 50 6 39 80 122 59 62 114 129 58 58 15 36 112 113 98 111 99 94 89 102 12 43 96 105 95 97 93 103 90 85 1 90 98 98 91 93 92 91 74 76 27 51 107 116 108 116 104 107 99 99 2 59 100 91 86 80 65 45 57 40 21 71 102 104 104 114 98 108 101 101 14 64 103 106 91 110 100 96 99 97 31 65 93 100 100 93 91 107 105 93 8 73 72 77 69 72 61 65 61 51 25 80 97 107 107 111 119 102 94 78 7 127 106 112 101 89 89 89 123 67 24 163 185 176 88 185 118 59 4 125 95 94 73 81 76 73 70 11 175 99 95 86 75 78 67 58 53 5 238 91 64 69 61 70 67 63 45 10 213 106 85 77 89 98 84 79 67 22 253 97 91 96 98 92 82 77 62 20 283 95 82 86 79 94 82 86 79 3 348 81 97 101 98 91 81 72 63 28 326 124 115 102 94 100 105 106 80 18 376 103 116 92 94 99 98 101 85 30 565 84 88 86 81 119 89 95 90 39 731 63 96 87 73 85 48 37 28 26 711 112 85 89 77 94 80 76 74 23 886 84 73 61 58 56 76 59 62 17 795 64 80 72 88 67 72 56 32 1,028 125 116 121 115 120 131 112 88 34 1,696 95 81 80 82 79 84 96 93 19 2,329 64 87 79 71 68 71 76 56 37 1,776 65 70 68 50 60 61 64 50 38 1,704 121 131 139 115 118 137 140 128 29 2,409 71 50 54 40 52 46 50 37 36 4,319 86 93 96 90 111 71 66 58 33 2,739 72 66 69 64 49 68 62 63 35 1,417 140 115 113 127 120 123 125 132 Mean as compared to 98 97 92 87 94 89 84 74 Time 0 Mean as compared to 100 99 94 89 100 95 89 78 Day 1 Storage 2-8 C. Storage 28 C. Day 1 Day 2 Day 3 Day 6 Day 1 Day 2 Day 3 Day 6 Number of Samples 39 38 37 39 39 38 39 39 analyzed Feces Extracts showing 8 6 11 16 10 13 20 23 <180% Recovery as compared to Time 0

[0114] 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).