Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies detail a standard chemiluminescent ELISA assay and a stool extraction method. There is no mention of AI, ML, or any computational analysis beyond standard quantitative measurement and statistical analysis of results.

Therapeutic

No
The device is an in vitro diagnostic assay intended to measure fecal calprotectin as an aid in diagnosing inflammatory bowel disease and differentiating it from irritable bowel syndrome. It does not directly treat or alleviate a disease, but rather provides information for diagnosis.

Diagnostic

Yes

The device is an in vitro diagnostic assay intended for the quantitative measurement of fecal calprotectin, which is a marker of intestinal mucosal inflammation. It is intended to aid in the diagnosis of inflammatory bowel disease (IBD) and differentiate it from irritable bowel syndrome (IBS), thus providing information for a medical diagnosis.

SaMD (Software as a Medical Device)

No

The device is an in vitro diagnostic (IVD) kit that involves physical components (reagents, microtiter plate, extraction device) and a laboratory procedure (ELISA) to measure a biomarker in a stool sample. It is not solely software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The intended use statement explicitly states: "The ALPCO Calprotectin Chemiluminescence ELISA is an in vitro diagnostic chemiluminescent assay intended for the quantitative measurement of fecal calprotectin... The ALPCO Calprotectin Chemiluminescence ELISA is intended for in vitro diagnostic use as an aid in the diagnosis of inflammatory bowel disease (IBD)..."

The device description also refers to its use in preparing human stool specimens for testing in the ELISA, which is an in vitro diagnostic procedure.

Furthermore, the document includes sections detailing clinical performance studies, expected values, and a comparison with a predicate device (K160447; Calprest®NG), all of which are standard components of documentation for IVD devices submitted for regulatory review.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The ALPCO Calprotectin Chemiluminescence ELISA is an in vitro diagnostic chemiluminescent assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. The ALPCO Calprotectin Chemiluminescence ELISA is intended for in vitro diagnostic use as an aid in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC), and as an aid in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other clinical and laboratory findings.

The ALPCO Easy Stool Extraction Device is intended for use in the preparation of human stool specimens for testing in the ALPCO Calprotectin Chemiluminescence ELISA.

Product codes (comma separated list FDA assigned to the subject device)

NXO

Device Description

The ALPCO Calprotectin Chemiluminescence ELISA is an in vitro diagnostic chemiluminescent assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. The ALPCO Calprotectin Chemiluminescence ELISA is intended for in vitro diagnostic use as an aid in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC), and as an aid in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other clinical and laboratory findings. The ALPCO Easy Stool Extraction Device is intended for use in the preparation of human stool specimens for testing in the ALPCO Calprotectin Chemiluminescence ELISA. The assay is performed on stool samples collected without preservatives. After an extraction procedure of the stool sample, using either the manual weighing or Easy Extraction Device procedure, the test allows the selective measurement of calprotectin-antigen by sandwich ELISA. A monoclonal capture antibody (mAb) highly specific to the calprotectin heterodimeric and polymeric complexes, respectively, is coated onto the microtiter plate. Calibrators, controls and specimen extracts are incubated. After a washing step, a biotinylated secondary monoclonal detection antibody detects the calprotectin molecules bound to the antibody coated onto the plate. After incubation and a further washing step, a Streptavidin-Horseradish Peroxidase Enzyme conjugate binds to the available biotin on the immobilized secondary antibody. A chemiluminescent substrate is added and read when the substrate glows as a result of its oxidation with the enzyme. The signal is then read on a chemiluminescent plate reader.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Fecal / Human stool

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Description of Clinical Performance Study Test Set:
Sample Size: 424 prospectively collected stool specimens.
Data Source: Clinical diagnosis made by the clinical investigator/gastroenterologist (reference standard).
Annotation Protocol:

IBD diagnosis was based on endoscopy results and/or histology of biopsies taken during the endoscopy.
IBS diagnosis was based on the Rome IV criteria and confirmed by negative endoscopy including the colon and terminal ileum.
Subjects were diagnosed with "Other Gl conditions" when they did not meet the diagnostic criteria for IBD or IBS (Rome IV).

Description of Expected Values Study Test Set:
Sample Size: 131 samples.
Data Source: Stool samples obtained from asymptomatic individuals with no abdominal complaints and no history of IBS, IBD or other chronic intestinal disorders.
Annotation Protocol: Not explicitly stated, but implies self-reported health status based on absence of symptoms and relevant medical history.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Precision/Reproducibility Studies:

Precision:
- Study Type: Internal (ALPCO) evaluation in accordance with CLSI EP05-A3.
- Sample Size: 8 stool samples, each analyzed 80 times (in duplicate, twice per day, for 20 days).
- Key Results: Various %CV values reported for Within-run, Between-run, Within-Day, Between-day, and Total precision. For example, Sample 1: Mean 288.7 µg/g, Total %CV 6.1%. Sample 2: Mean 5323.5 µg/g, Total %CV 11.1%.
Lot-to-Lot Reproducibility:
- Study Type: Internal (ALPCO) evaluation in accordance with CLSI EP05-A3. Performed using three (3) kit lots.
- Sample Size: 7 stool samples, each analyzed 75 times (in replicates of 5, once per day, for 5 days for each lot).
- Key Results: Various %CV values reported for Within-run, Between-day, Within-lot, Between-lot, and Total reproducibility. For example, Sample 1: Mean 288.6 µg/g, Total %CV 8.8%. Sample 2: Mean 4480.2 µg/g, Total %CV 13.9%.
Site-to-Site Reproducibility:
- Study Type: Internal (ALPCO) and at two other analytical test sites in accordance with CLSI EP05-A3.
- Sample Size: 7 stool samples, each analyzed 75 times (25 replicates per site). Stool samples were extracted at ALPCO, frozen, and shipped.
- Key Results: Various %CV values reported for Within-run, Between-day, Within-site, Between-site, and Total reproducibility. For example, Sample 1: Mean 46.3 µg/g, Total %CV 9.9%. Sample 7: Mean 2353.7 µg/g, Total %CV 10.8%.
Operator-to-Operator Reproducibility:
- Study Type: Internal (ALPCO) evaluation in accordance with CLSI EP05-A3. Performed by three (3) operators.
- Sample Size: 7 stool samples, each analyzed 75 times (25 replicates per sample, per operator).
- Key Results: Various %CV values reported for Within-run, Between-run, Within-operator, Between-operator, and Total reproducibility. For example, Sample 1: Mean 4468.6 µg/g, Total %CV 13.5%. Sample 6: Mean 68.1 µg/g, Total %CV 15.3%.
Extraction Method Reproducibility:
- Study Type: Internal (ALPCO) evaluation comparing Easy Extraction method and manual weighing method.
- Sample Size: 7 stool samples, each extracted 10 times by both methods, and analyzed in duplicate (20 replicates per sample per extraction method).
- Key Results (Easy Extraction Method): Various %CV values for Within-Extraction, Between-Extraction, and Total Imprecision. For example, Sample 1: Mean 129.0 µg/g, Total Imprecision %CV 5.0%.
- Key Results (Manual Weighing Method): Various %CV values for Within-Extraction, Between-Extraction, and Total Imprecision. For example, Sample 1: Mean 4063.3 µg/g, Total Imprecision %CV 9.6%.

Analytical Sensitivity Studies:

Limit of Blank (LoB):
- Study Type: Determined in accordance with CLSI EP17-A2.
- Sample Size: Four blank stool samples, tested in replicates of six on two reagent lots across three days.
- Key Results: LoB was determined to be 3.6 µg/g.
Limit of Detection (LoD):
- Study Type: Determined in accordance with CLSI EP17-A2.
- Sample Size: Four stool samples containing a low level of calprotectin, tested in replicates of five on two reagent lots across three days.
- Key Results: LoD was determined to be 7.7 µg/g.
Limit of Quantitation (LoQ):
- Study Type: Determined in accordance with CLSI EP17-A2.
- Sample Size: Six stool samples containing a low level of calprotectin, tested in replicates of five on two reagent lots across three days.
- Key Results: LoQ was determined to be 7.9 µg/g.

Linearity:

Study Type: Matrix linearity and aqueous linearity evaluated in accordance with CLSI EPOG-A.
Sample Size: Serially diluted stool sample extract and serially diluted aqueous sample.
Key Results: Acceptably linear over the analytical measuring range.
- Matrix: Test Range 2.5 — 5780.1 µg/g, Slope 1.003, R² 1.00, Average Recovery 99.2%.
- Aqueous: Test Range 3.0 – 6221.7 µg/g, Slope 0.99, R² 0.99, Average Recovery 100.6%.

Accuracy/Recovery:

Study Type: Evaluation using spiked stool samples.
Sample Size: 7 extracted stool samples.
Key Results: Recovery of spiked samples ranged from 89.7% to 110.8%.

Interfering Substances:

Study Type: Interference testing in accordance with CLSI EP07-ED3.
Sample Size: Seven stool samples.
Key Results: Recovery of test samples ranged from 91.0 – 108.1%, indicating no interference at the concentrations tested for listed oral pharmaceuticals, nutritional supplements, HAMA, Hemoglobin, and Microorganisms (Escherichia coli, Salmonella enterica subsp., Klebsiella pneumoniae subsp., Citrobacter freundii, Shigella flexneri, Yersinia enterocolitica subsp.).

Product Stability:

Real-Time Reagent Stability Summary: 18 months at 2-8°C.
Open Vial Stability Summary: 7 days at 2-8°C once opened.
Transport Stability Summary: 14 days at 2-8°C.
Raw Stool Sample Stability: 14 days at 2-8°C; 14 days at -20°C; 3 freeze/thaw cycles.
Stool Sample Extract Stability: 3 days at 2-8°C; 14 days at -80°C; 3 freeze/thaw cycles.

Stool Sample Extraction Method Comparison:

Study Type: Internal evaluation comparing Easy Extraction method and manual weighing method.
Sample Size: 68 stool samples (61 within AMR for analysis).
Key Results:
- Passing-Bablok regression: Slope 1.014, Y-Intercept -1.296, Correlation r 0.997.
- Qualitative agreement (Equivocal considered positive): PPA 100%, NPA 100%, TPA 100%.
- Qualitative agreement (Equivocal considered negative): PPA 96.2%, NPA 100%, TPA 98.4%.

Comparison with Predicate Device:

Study Type: Method comparison study with predicate device (K160447) using clinical performance samples.
Sample Size: 400 samples for qualitative agreement analysis; 169 samples for analytical method comparison (within AMR of both assays).
Key Results:
- Qualitative agreement (Equivocal considered positive): PPA 46.8%, NPA 93.1%, TPA 75.3%.
- Qualitative agreement (Equivocal considered negative): PPA 51.4%, NPA 93.6%, TPA 85.8%.
- Analytical method comparison (Passing-Bablok): Slope 0.6919, Y-Intercept -18.35, Correlation-r 0.784.

Clinical Performance:

Study Type: Comparison of analytical test results against clinical diagnosis.
Sample Size: 424 subjects (IBD n=76, IBS n=122, Other GI conditions n=226).
Key Results (aid in diagnosis of IBD, n=424):
- Equivocal results = positive: Sensitivity 92.1%, Specificity 92.5%, PPV 72.9%, NPV 98.2%.
- Equivocal results = negative: Sensitivity 65.8%, Specificity 96.3%, PPV 79.4%, NPV 92.8%.
Key Results (aid in differentiation of IBD vs IBS, n=198):
- Equivocal results = positive: Sensitivity 92.1%, Specificity 95.1%, PPV 92.1%, NPV 95.1%.
- Equivocal results = negative: Sensitivity 65.8%, Specificity 98.4%, PPV 96.2%, NPV 82.2%.

Expected Values:

Study Type: Verification of clinical cut-off using normal stool samples from asymptomatic individuals.
Sample Size: 131 healthy individuals.
Key Results: With a cut-off of 50 µg/g, 130/131 of samples were normal/negative. Values ranged from

Regulation Number and Section

§ 866.5180 Fecal calprotectin immunological test system.

(a)
Identification. A fecal calprotectin immunological test system is anin vitro diagnostic device that consists of reagents used to quantitatively measure, by immunochemical techniques, fecal calprotectin in human stool specimens. The device is intended forin vitro diagnostic use as an aid in the diagnosis of inflammatory bowel diseases (IBD), specifically Crohn's disease and ulcerative colitis, and as an aid in differentiation of IBD from irritable bowel syndrome.(b)
Classification. Class II (special controls). The special control for these devices is FDA's guidance document entitled “Class II Special Controls Guidance Document: Fecal Calprotectin Immunological Test Systems.” For the availability of this guidance document, see § 866.1(e).

Summary

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Image /page/0/Picture/0 description: The image contains the logos of the Department of Health and Human Services and the Food and Drug Administration (FDA). The Department of Health and Human Services logo is on the left, and the FDA logo is on the right. The FDA logo includes the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in blue text.

October 25, 2019

ALPCO Jeffrey Freedman Regulatory Affairs Specialist 26 Keewaydin Drive, Unit G Salem, New Hampshire 03079

Re: K191807

Trade/Device Name: ALPCO Calprotectin Chemiluminescence ELISA, ALPCO Easy Stool Extraction Device Regulation Number: 21 CFR 866.5180 Regulation Name: Fecal Calprotectin Immunological Test System Regulatory Class: Class II Product Code: NXO Dated: July 3, 2019 Received: July 5, 2019

Dear Jeffrey Freedman:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

1

statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Douglas Jeffery, Ph.D. Chief Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K191807

Device Name

ALPCO Calprotectin Chemiluminescence ELISA ALPCO Easy Stool Extraction Device

Indications for Use (Describe)

The ALPCO Calprotectin Chemiluminescence ELISA is an in vitro diagnostic chemiluminescent assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. The ALPCO Calprotectin Chemiluminescence ELISA is intended for in vitro diagnostic use as an aid in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC), and as an aid in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other clinical and laboratory findings.

The ALPCO Easy Stool Extraction Device is intended for use in the preparation of human stool specimens for testing in the ALPCO Calprotectin Chemiluminescence ELISA.

Type of Use (Select one or both, as applicable)

Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)

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510(k) SUMMARY

Date of Summary:

October 22, 2019

Product Name:

ALPCO Calprotectin Chemiluminescence ELISA; ALPCO Easy Stool Extraction Device

Sponsor:

ALPCO 26 Keewaydin Drive, Unit G Salem, NH 03079

Correspondent:

Jeffrey Freedman, Regulatory Affairs Specialist 26 Keewaydin Drive, Unit G Salem, NH 03079 Phone: (603)893-8914 Fax: (603)898-6854

Common Name:

Fecal calprotectin immunological test system

Regulation Number: 21 CFR 866.5180

Classification: NXO, Class II

Predicate Device:

Calprest®NG (QUANTA Lite® Calprotectin Extended Range ELISA), 510(k) number: K160447

Intended Use

The ALPCO Calprotectin Chemiluminescence ELISA is an in vitro diagnostic chemiluminescent assay intended for the quantitative measurement of fecal calprotectin, a neutrophilic protein that is a marker of intestinal mucosal inflammation, in human stool. The ALPCO Calprotectin Chemiluminescence ELISA is intended for in vitro diagnostic use as an aid in the diagnosis of inflammatory bowel disease (IBD), specifically Crohn's disease (CD) and ulcerative colitis (UC), and as an aid in the differentiation of IBD from irritable bowel syndrome (IBS) in conjunction with other clinical and laboratory findings.

The ALPCO Easy Stool Extraction Device is intended for use in the preparation of human stool specimens for testing in the ALPCO Calprotectin Chemiluminescence ELISA.

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Methodology

The ALPCO Calprotectin Chemiluminescence ELISA is performed on stool samples, collected without preservatives. After an extraction procedure of the stool sample, using either the manual weighing or Easy Extraction Device procedure, the test allows the selective measurement of calprotectin-antigen by sandwich ELISA. A monoclonal capture antibody (mAb) highly specific to the calprotectin heterodimeric and polymeric complexes, respectively, is coated onto the microtiter plate. Calibrators, controls and specimen extracts are incubated. After a washing step, a biotinylated secondary monoclonal detection antibody detects the calprotectin molecules bound to the antibody coated onto the plate. After incubation and a further washing step, a Streptavidin-Horseradish Peroxidase Enzyme conjugate binds to the available biotin on the immobilized secondary antibody. A chemiluminescent substrate is added and read when the substrate glows as a result of its oxidation with the enzyme. The signal is then read on a chemiluminescent plate reader.

Substantial Equivalency & Comparison with Predicate

The ALPCO Calprotectin Chemiluminescence ELISA has the same intended use and assay principle as the predicate device. A comparison of the similarities and differences between the devices are provided in the following tables:

| Characteristic | ALPCO
ALPCO Calprotectin Chemiluminescence
ELISA
(New Device) | Eurospital S.p.A.
Calprest®NG
K160447
(Predicate Device) |
|----------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | Similarities | |
| Intended Use | The ALPCO Calprotectin Chemiluminescence
ELISA is an in vitro diagnostic
chemiluminescent assay intended for the
quantitative measurement of fecal
calprotectin, a neutrophilic protein that is a
marker of intestinal mucosal inflammation, in
human stool. The ALPCO Calprotectin
Chemiluminescence ELISA is intended for in
vitro diagnostic use as an aid in the diagnosis
of inflammatory bowel disease (IBD),
specifically Crohn's disease (CD) and
ulcerative colitis (UC), and as an aid in the
differentiation of IBD from irritable bowel
syndrome (IBS) in conjunction with other
clinical and laboratory findings. | Calprest® NG is a quantitative ELISA
for detecting concentration of fecal
calprotectin, which can be used as an
in vitro diagnostic to aid in the
diagnosis of Inflammatory Bowel
Diseases (IBD), specifically Crohn's
disease and ulcerative colitis, and to
differentiate IBD from Irritable Bowel
Syndrome (IBS) in conjunction with
other clinical and laboratory findings. |
| Analyte | Fecal Calprotectin | Same |
| Assay Format | Quantitative | Same |
| Platform | 96 well microtiter plate | Same |
| Characteristic | ALPCO
ALPCO Calprotectin Chemiluminescence
ELISA
(New Device) | Eurospital S.p.A.
Calprest®NG
K160447
(Predicate Device) |
| Detection antibody | Monoclonal anti-calprotectin antibody | Same |
| Assay process | Manual | Same |
| | Differences | |
| Capture Antibody | Monoclonal anti-calprotectin antibody | Polyclonal anti-calprotectin antibody |
| Analytical
Measuring Range | $7.9 - 6000 \mu g/g$ | $27.1-3000.0 mg/kg$ |
| Units | $\mu g/g$ | $mg/kg$ |
| Method | Chemiluminescent | Colorimetric |
| Primary
Measurement Units | Relative Light Units (RLU) | Optical Density (OD) |
| Calibrators | 8 levels:
0, 5, 20, 40, 156, 625, 2500, 10000 µg/g | 6 levels:
0, 2.5, 12.5, 25, 50, 150 ng/mL |
| Controls | 3 levels | 2 levels |
| Calibrator/Control
Analyte | Native human calprotectin | Recombinant calprotectin antigen
(rAg) |
| Calibrator/Control
Physio-chemical
Characteristics | Lyophilized | Ready to use |
| Sample Dilution | 1:25000 | 1:20000 |
| Stop Solution | None | H2SO4 |
| Substrate | Luminol | TMB |
| Incubation Time | 30-30-30-5 minutes at room temp | 60-30-15 minutes at room temp |
| Pre-analytical
sample processing | Manual weighing extraction method or Easy
Extraction Device method | Manual weighing extraction method |
| Results
Interpretation | Normal: $ 100 \mu g/g$ | Normal: $ 120 mg/kg$ |

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Standard Documents Referenced:

CLSI EP05-A3 Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition
CLSI EP06-A Evaluation of The Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline
CLSI EP07-ED3 Interference Testing in Clinical Chemistry, Third Edition .

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CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition
CLSI EP28-A3c Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory - Third Edition

Performance Data

Precision/Reproducibility Studies

Precision

The precision of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO in accordance with CLSI EP05-A3, Evaluation of Quantitative Measurement Procedures. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA using one (1) ELISA reader by one (1) operator. Eight (8) stool samples containing various fecal calprotectin concentrations covering a significant portion of the reportable range of the ALPCO Calprotectin Chemiluminescence ELISA were extracted using the Easy Extraction method and the resultant extracts were analyzed in duplicate, twice per day, for 20 days to generate a total of 80 replicates per sample. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:

		Within-run		Between-run		Within-Day		Between-day		Total
		Mean
(µg/g)	SD
(µg/g)	%CV	SD
(µg/g)	%CV	SD
(µg/g)	%CV	SD
(µg/g)	%CV	SD
(µg/g)	%CV
1	80	288.7	9.5	3.3%	12.3	4.2%	15.5	5.4%	8.6	3.0%	17.7	6.1%
2	80	5323.5	220.0	4.1%	359.8	6.8%	421.7	7.9%	410.8	7.7%	588.8	11.1%
3	80	680.3	19.7	2.9%	26.2	3.8%	32.8	4.8%	29.2	4.3%	43.9	6.4%
4	80	908.3	25.7	2.8%	29.2	3.2%	38.9	4.3%	56.6	6.2%	68.7	7.6%
5	80	116.6	3.0	2.6%	4.3	3.6%	5.2	4.5%	4.2	3.6%	6.7	5.7%
6	80	32.9	1.3	3.9%	1.2	3.7%	1.8	5.3%	2.9	8.9%	3.4	10.3%
7	80	82.2	3.7	4.5%	3.3	4.0%	4.9	6.0%	3.8	4.6%	6.2	7.6%
8	80	39.1	1.8	4.7%	1.6	4.0%	2.4	6.1%	2.8	7.2%	3.7	9.5%

Lot-to-Lot Reproducibility

The lot-to-lot reproducibility of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO in accordance with CLSI EPO5-A3, Evaluation of Quantitative Measurement Procedures. The study was performed using three (3) kit lots of ALPCO Calprotectin Chemiluminescence ELISA using one (1) ELISA reader by one (1) operator. Seven (7) stool samples containing various fecal calprotectin concentrations covering a fraction of the

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reportable range of the ALPCO Calprotectin Chemiluminescence ELISA were extracted using the Easy Extraction method and analyzed in replicates of 5, once per day, for 5 days using each of the kit lots to generate a total of 75 replicates per sample. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:

Sample	N	Within-run		Between-day		Within-lot		Between-lot		Total
		Mean
(µg/g)	SD
(µg/g)	%CV	SD
(µg/g)	%CV	SD
(µg/g)	%CV	SD
(µg/g)	%CV	SD
(µg/g)	%CV
1	75	288.6	8.5	2.9%	16.5	5.7%	18.5	6.4%	17.2	5.9%	25.3	8.8%
2	75	4480.2	321.3	7.2%	445.1	9.9%	548.9	12.3%	298.0	6.7%	624.6	13.9%
3	75	684.2	23.3	3.4%	45.1	6.6%	50.8	7.4%	49.6	7.2%	71.0	10.4%
4	75	919.2	38.1	4.1%	72.3	7.9%	81.7	8.9%	60.8	6.6%	101.9	11.1%
5	75	110.8	7.7	6.9%	7.3	6.6%	10.6	9.5%	7.2	6.5%	12.8	11.5%
6	75	35.4	1.8	5.2%	2.1	6.0%	2.8	8.0%	1.8	5.2%	3.4	9.5%
7	75	76.7	3.4	4.4%	4.5	5.9%	5.6	7.3%	1.7	2.2%	5.9	7.7%

Site-to-Site Reproducibility

The site-to-site reproducibility of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO and at two other analytical test sites in accordance with CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA by one (1) operator per test site. Seven (7) stool samples containing various fecal calprotectin concentrations of the reportable range of the ALPCO Calprotectin Chemiluminescence ELISA were extracted using the Easy Extraction method at ALPCO, frozen, and then shipped to the two additional sites. Each extract was analyzed in replicates of 5, once per day, for 5 days at each site to generate 25 replicates per site and a total of 75 replicates per sample. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:

		Within-run		Between-day		Within-site		Between-site		Total
Sample	N	Mean
(μg/g)	SD (μg/g)	%CV	SD
(μg/g)	%CV	SD
(μg/g)	%CV	SD
(μg/g)	%CV	SD
(μg/g)	%CV
1	75	46.3	2.6	5.6%	3.7	8.1%	4.5	9.8%	0.7	1.6%	4.6	9.9%
2	75	195.2	8.9	4.6%	21.6	11.0%	23.3	12.0%	0.0	0.0%	23.3	12.0%
3	75	20.0	2.4	12.1%	1.7	8.6%	3.0	14.9%	0.3	1.4%	3.0	15.0%
4	75	446.5	21.5	4.8%	27.6	6.2%	35.0	7.8%	0.0	0.0%	35.0	7.8%
5	75	138.2	10.1	7.3%	15.8	11.4%	18.7	13.5%	0.0	0.0%	18.7	13.5%
6	75	21.1	2.2	10.4%	1.7	7.9%	2.8	13.1%	0.0	0.0%	2.8	13.1%
7	75	2353.7	125.3	5.3%	172.9	7.3%	213.5	9.1%	137.1	5.8%	253.7	10.8%

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Operator-to-Operator Reproducibility

The operator-to-operator reproducibility of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO in accordance with CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA by three (3) operators. Seven (7) stool samples containing various fecal calprotectin concentrations covering a significant portion of the reportable range of the ALPCO Calprotectin Chemiluminescence ELISA were extracted using the Easy Extraction method and analyzed by each operator independently in replicates of 5, once per day, for 5 days to generate 25 replicates per sample, per operator and a total of 75 replicates per sample. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:

| Sample | N | Mean
(µg/g) | Within-run | | Between-run | | Within-operator | | Between-operator | | Total | |
|--------|----|----------------|------------|------|-------------|-------|-----------------|-------|------------------|-------|-----------|-------|
| | | | SD (µg/g) | %CV | SD (µg/g) | %CV | SD (µg/g) | %CV | SD (µg/g) | %CV | SD (µg/g) | %CV |
| 1 | 75 | 4468.6 | 299.8 | 6.7% | 470.4 | 10.5% | 557.9 | 12.5% | 225.5 | 5.0% | 601.7 | 13.5% |
| 2 | 75 | 555.8 | 22.2 | 4.0% | 26.2 | 4.7% | 34.4 | 6.2% | 70.9 | 12.8% | 78.8 | 14.2% |
| 3 | 75 | 765.0 | 29.5 | 3.9% | 81.3 | 10.6% | 86.5 | 11.3% | 42.5 | 5.6% | 96.4 | 12.6% |
| 4 | 75 | 98.8 | 3.9 | 3.9% | 4.8 | 4.8% | 6.1 | 6.2% | 5.8 | 5.8% | 8.4 | 8.5% |
| 5 | 75 | 26.7 | 0.9 | 3.5% | 2.1 | 8.0% | 2.3 | 8.7% | 2.3 | 8.6% | 3.3 | 12.3% |
| 6 | 75 | 68.1 | 2.1 | 3.1% | 5.8 | 8.5% | 6.1 | 9.0% | 8.4 | 12.3% | 10.4 | 15.3% |
| 7 | 75 | 32.5 | 1.6 | 5.0% | 3.2 | 9.7% | 3.6 | 10.9% | 3.6 | 11.0% | 5.0 | 15.5% |

Extraction Method Reproducibility

The extraction reproducibility of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA by one (1) operator. Sets of seven (7) stool samples containing various fecal calprotectin concentrations, including stool samples that are near the clinical cut-offs of the ALPCO Calprotectin Chemiluminescence ELISA were extracted 10 times using both the Easy Extraction method and manual weighing method. Each stool sample extract was analyzed in duplicate to generate 20 replicates per sample, per extraction method. Data were analyzed using Analyse-it Method Validation edition and acceptance criteria were met. The results are as follows:

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| | | | Within-
Extraction | | Between
Extraction | | Total
Imprecision | |
|--------|----|----------------|-----------------------|-------|-----------------------|------|----------------------|-------|
| Sample | N | Mean
(µg/g) | SD
(µg/g) | %CV | SD
(µg/g) | %CV | SD
(µg/g) | %CV |
| 1 | 20 | 129.0 | 1.8 | 1.4% | 6.2 | 4.8% | 6.5 | 5.0% |
| 2 | 20 | 45.9 | 4.6 | 9.9% | 2.8 | 6.1% | 5.3 | 11.6% |
| 3 | 20 | 4298.5 | 317.2 | 7.4% | 426.9 | 9.9% | 531.8 | 12.4% |
| 4 | 20 | 123.7 | 6.9 | 5.6% | 3.9 | 3.1% | 7.9 | 6.4% |
| 5 | 20 | 1748.5 | 186.5 | 10.7% | 121.4 | 6.9% | 222.6 | 12.7% |
| 6 | 20 | 68.3 | 3.5 | 5.1% | 3.2 | 4.7% | 4.8 | 7.0% |
| 7 | 20 | 33.4 | 1.7 | 5.1% | 0.0 | 0.0% | 1.7 | 5.1% |

Easy Extraction method reproducibility using the Easy Extraction Device:

Extraction reproducibility using the manual weighing method:

| Sample | N | Mean
(µg/g) | Within-
Extraction | | | Between
Extraction | | Total
Imprecision | |
|--------|----|----------------|-----------------------|------|--|-----------------------|------|----------------------|------|
| | | | SD
(µg/g) | %CV | | SD
(µg/g) | %CV | SD
(µg/g) | %CV |
| 1 | 20 | 4063.3 | 389.2 | 9.6% | | 0.0 | 0.0% | 389.2 | 9.6% |
| 2 | 20 | 1336.1 | 42.4 | 3.2% | | 113.5 | 8.5% | 121.2 | 9.1% |
| 3 | 20 | 122.0 | 5.1 | 4.1% | | 8.9 | 7.3% | 10.2 | 8.4% |
| 4 | 20 | 25.7 | 1.1 | 4.5% | | 1.6 | 6.4% | 2.0 | 7.8% |
| 5 | 20 | 103.7 | 2.5 | 2.4% | | 3.7 | 3.6% | 4.5 | 4.3% |
| 6 | 20 | 69.6 | 3.2 | 4.6% | | 0.3 | 0.5% | 3.2 | 4.6% |
| 7 | 20 | 298.6 | 13.5 | 4.5% | | 26.2 | 8.8% | 29.5 | 9.9% |

Analytical Sensitivity Studies

Limit of Blank (LoB)

The limit of blank of the ALPCO Calprotectin Chemiluminescence ELISA was determined in accordance with CLSI EP17-A2. Four blank stool samples were extracted and tested in replicates of six on two reagent lots across three days. The LoB was determined to be 3.6 µg/g by the nonparametric method.

Limit of Detection (LoD)

The limit of detection of the ALPCO Calprotectin Chemiluminescence ELISA was determined in accordance with CLSI EP17-A2. Four stool samples containing a low level of calprotectin were extracted and tested in replicates of five on two reagent lots across three days. The LoD was determined to be 7.7 µg/g by the parametric method.

Limit of Quantitation (LoQ)

The limit of quantitation of the ALPCO Calprotectin Chemiluminescence ELISA was determined in accordance with CLSI EP17-A2. Six stool samples containing a low level of calprotectin were extracted and tested in replicates of five on two reagent lots across three days. The mean and

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within laboratory precision were calculated for each reagent lot. For each reagent lot, the observed precision (%CV) (Y-axis) was plotted vs. the sample calprotectin concentration (X-axis) to give a precision profile, and fit by a constant variance function using Analyse-it Method Validation edition. The LoQ estimate for each reagent lot was determined as the measurand concentration at the intersection of the precision profile curve with the accuracy goal of 20 %CV. The LoQ was determined to be 7.9 µg/g.

Linearity

The matrix linearity and aqueous linearity of the analytical measuring range of the ALPCO Calprotectin Chemiluminescence ELISA were evaluated internally at ALPCO in accordance with CLSI EPOG-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach. To evaluate matrix linearity, a stool sample extract containing a high concentration of fecal calprotectin was serially diluted 1:2 with a stool sample extract containing a low concentration of fecal calprotectin to obtain dilution levels with values that cover the entire AMR. Stool sample extracts were obtained using the Easy Extraction method. Each stool sample extract combination was assayed in duplicate in a single analysis using one reagent lot of the ALPCO Calprotectin Chemiluminescence ELISA. To evaluate aqueous linearity, a sample made by diluting native antigen in standard diluent buffer containing stabilizers and preservatives (the same buffer used to make the controls and calibrators) was serially diluted 1:2 with standard diluent buffer to obtain dilution levels with values that cover the entire AMR. Each aqueous sample dilution was assayed in duplicate in a single analysis using one reagent lot of the ALPCO Calprotectin Chemiluminescence ELISA. Results were analyzed using Analyse-it Method Validation edition to determine the best fitting polynomial. For linearity to be claimed, the best fitting polynomial had to be linear (first order) or the difference between the best fitting nonlinear polynomial (second or third order) and the linear polynomial could not exceed ±15%. The plots were further analyzed by linear regression. The slope and y-intercept, and the 95% confidence intervals thereof, and Rof the regression analysis were calculated. In addition, the recovery of each replicate value included in the regression analysis for the linearity dilutions was calculated and the average recovery was calculated. Acceptance criteria were met, the ALPCO Calprotectin Chemiluminescence ELISA is acceptably linear over the analytical measuring range. The results are as follows:

| Sample Type | Test Range (μg/g) | Slope
(95% CI) | Y-Intercept
(95% CI) | R² | Average Recovery
(%) |
|-------------|-------------------|---------------------------|---------------------------|------|-------------------------|
| Matrix | 2.5 — 5780.1 | 1.003
(0.994 to 1.013) | 1.77
(-15.87 to 19.42) | 1.00 | 99.2 |
| Aqueous | 3.0 – 6221.7 | 0.99
(0.98 to 1.00) | 0.03
(-22.42 to 22.47) | 0.99 | 100.6 |

Accuracy/Recovery

The accuracy/recovery of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated using seven (7) extracted stool samples containing various concentrations of calprotectin across the analytical measuring range of the assay, samples were extracted using the Easy Extraction

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method. Native calprotectin diluted in standard diluent was used as the spiking material. The stool sample extracts were mixed with the spiking material in a proportion of 9:1 (9 parts sample:1 part spiking material) to calculate recovery: Samples with calprotectin concentrations lower than 200 µg/g were spiked with 42.8 µg/g. Samples with calprotectin concentrations higher than 200 µg/g were spiked with 129.3 µg/g. Each baseline sample extract, control spike and spiked sample were tested in duplicate in the same assay run. Recovery was calculated compared to the baseline result and acceptance criteria were met. The results are as follows:

| Sample | Mean
Baseline
Result (µg/g) | Spike Value
(µg/g) | Theoretical
Post-Spike
Result (µg/g) | Observed
Post-Spike
Result (µg/g) | Recovery
(%) |
|--------|-----------------------------------|-----------------------|--------------------------------------------|-----------------------------------------|-----------------|
| 1 | 248.5 | 129.3 | 353.0 | 391.0 | 110.8 |
| 2 | 5392.3 | 129.3 | 4982.4 | 4469.3 | 89.7 |
| 3 | 615.5 | 129.3 | 683.2 | 754.1 | 110.4 |
| 4 | 94.8 | 42.8 | 128.1 | 123.5 | 96.4 |
| 5 | 28.1 | 42.8 | 68.1 | 72.1 | 105.9 |
| 6 | 77.7 | 42.8 | 112.7 | 123.6 | 109.7 |
| 7 | 36.2 | 42.8 | 75.3 | 79.0 | 104.9 |

Interfering Substances

The interference testing of the ALPCO Calprotectin Chemiluminescence ELISA was evaluated internally at ALPCO using CLSI EP07-ED3, Interference Testing in Clinical Chemistry as a guideline. The study was performed using seven stool samples containing various concentrations of fecal calprotectin. Interfering substances were spiked into each stool sample at 10% of the total specimen volume. The solvent used to create the interferent stock was spiked into each stool sample at 10% of the total specimen volume to generate a control sample. The stool sample containing the interferents and the control stool sample were extracted using the Easy Extraction method. Recovery of the test samples ranged from 91.0 – 108.1%, no interference was observed at the concentrations tested. The list of interferents and the concentrations tested are as follows:

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Oral Pharmaceuticals
Trade Name	Active Component	Spiking Concentration
Ferro-Gradumet	Iron (II) sulfate	0.02 mg/50 mg stool
Prednisone	Prednisone	0.065 mg/50 mg stool
Imurek	Azathioprine	0.035 mg/50 mg stool
Pentasa/Asacol	Mesalamine; 5-ASA	1.0 mg/50 mg stool
Prevacid	Lansoprazol	0.035 mg/50 mg stool
Vancocin	Vancomycin	0.40 mg/50 mg stool
Sulfamethoxazole	Sulfamethoxazole	0.32 mg/50 mg stool
Trimethoprim	Trimethoprim lactate	0.065 mg/50 mg stool
Cipro	Ciprofloxacin	0.04 mg/50 mg stool
Nutritional Supplements
Trade Name	Active Component	Spiking Concentration
Vitamin E	DL-α Tocopherol Acetate	0.06 mg/50 mg stool
Multiple Vitamin	A, B1, B2, B3, B5, B6, B8, B9, B12,
C, D, E, and minerals	0.215 mg/50 mg stool
Biotin	B7	1750 ng/50 mg stool
Other
Human anti-mouse antibody (HAMA)		0.5 µg/50 mg stool
Hemoglobin		0.25 mg/50 mg stool
Microorganisms
Escherichia coli	1.5 x $10^7$ cfu/mL
Salmonella enterica subsp. enterica	1.5 x $10^7$ cfu/mL
Klebsiella pneumoniae subsp. pneumonia	1.5 x $10^7$ cfu/mL
Citrobacter freundii	1.5 x $10^7$ cfu/mL
Shigella flexneri	1.5 x $10^7$ cfu/mL
Yersinia enterocolitica subsp. enterocolitica	1.5 x $10^7$ cfu/mL

Product Stability

Real-Time Reagent Stability Summary - The ALPCO Calprotectin Chemiluminescence ELISA reagents are stable for 18 months at 2-8°C.

Open Vial Stability Summary – The ALPCO Calprotectin Chemiluminescence ELISA reagents are stable for 7 days at 2-8°C once opened.

Transport Stability Summary - The ALPCO Calprotectin Chemiluminescence ELISA reagents are stable at 28°C for 14 days.

Raw Stool Sample Stability

Storage Conditions	Stability Duration
2-8°C	14 days
-20°C	14 days
Freeze/Thaw Cycles	3 cycles

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Stool Sample Extract Stability

Storage Conditions	Stability Duration
2-8°C	3 days
-80°C	14 days
Freeze/Thaw Cycles	3 cycles

Stool Sample Extraction Method Comparison

The extraction methods of the ALPCO Calprotectin Chemiluminescence ELISA were evaluated internally at ALPCO to determine if the extraction methods provide similar results. The study was performed using one (1) kit lot of ALPCO Calprotectin Chemiluminescence ELISA by one (1) operator. Sixty-eight (68) stool samples varying in consistency and containing various fecal calprotectin concentrations over a significant fraction of reportable range and near the clinical cut-offs of the ALPCO Calprotectin Chemiluminescence ELISA were extracted in parallel using both the Easy Extraction method and manual weighing method. Each resultant stool sample extract was analyzed in singlicate. 61 of the samples were within the AMR of the ALPCO Calprotectin Chemiluminescence ELISA and included in the analysis. A scatter plot was created by plotting the values obtained from the Easy Extraction method extracts (Y-axis) against the manual weighing method extracts (X-axis) and analyzed by Passing-Bablok regression analysis using Analyse-it Method Validation Edition. The Y-Intercept, slope, and bootstrap 95% confidence intervals thereof, and correlation r were calculated. The results are as follows:

Stool Sample Extraction Analytical Method Comparison
N	61
Slope (95% CI)	1.014 (1.004 to 1.031)
Y-Intercept (95% CI)	-1.296 (-1.986 to -0.3179)
Correlation r	0.997

Qualitative agreement analysis between the Easy Extraction method and manual weighing method was also conducted to calculate the positive percent agreement, negative percent agreement, and total percent and Wilson 95% Cl considering equivocal results as both positive and negative. The results are as follows:

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	Manual weighing method
Easy
Extraction
method		Positive	Equivocal	Negative	Totals
Positive		25	0	0	25
Equivocal		1	13	0	14
Negative		0	0	22	22
Totals		26	13	22	61
Equivocal results considered positive (95% CI)
	PPA	39/39	100%	(91.0 - 100%)
	NPA	22/22	100%	(85.1 - 100%)
	TPA	61/61	100%	(94.1 - 100%)
Equivocal results considered negative (95% CI)
	PPA	25/26	96.2%	(81.1 - 99.3%)
	NPA	35/35	100%	(90.1 - 100%)
	TPA	60/61	98.4%	(91.3 - 99.7%)

Stool Sample Extraction Qualitative Method Comparison

Comparison with Predicate Device

method comparison study was performed comparing the ALPCO Calprotectin A Chemiluminescence ELISA and the predicate device. 400 samples that were used to determine the clinical performance characteristics of the ALPCO Calprotectin Chemiluminescence ELISA were tested on the predicate device according to the manufacturer-supplied labeling.

A qualitative agreement analysis between the ALPCO Calprotectin Chemiluminescence ELISA and the predicate device was conducted including all samples that were tested on both devices (N=400) using the cut-offs of both products to calculate the positive percent agreement (PPA), negative percent agreement (NPA), and total percent agreement (TPA), and 95% Cls (Wilson score method) thereof considering equivocal results as both positive and negative. The results are as follows:

	ALPCO Calprotectin Chemiluminescence ELISA
Predicate	Normal
( 100 μg/g)	Total
Normal ( 120 mg/kg)	22	14	38	74
Total	311	30	59	400

Equivocal results considered positive (95% CI)
PPA	72/154	46.8%	(39.0 – 54.6%)
NPA	229/246	93.1%	(89.2 – 95.6%)
TPA	301/400	75.3%	(70.8 – 79.2%)
Equivocal results considered negative (95% CI)
PPA	38/74	51.4%	(40.2 – 62.4%)
NPA	305/326	93.6%	(90.4 – 95.7%)
TPA	343/400	85.8%	(82.0 – 88.8%)

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An analytical method comparison was conducted including the samples that were within the analytical measuring range of both assays (N=169); results had to measure within 27.1 – 3000 mg/kg on the predicate device and 7.9 - 6000 µg/g on the ALPCO Calprotectin Chemiluminescence ELISA. A scatter plot was created by plotting the values obtained with the ALPCO Calprotectin Chemiluminescence ELISA (Y-axis) against the predicate device (X-axis) and analyzed by Passing-Bablok regression analysis using Analyse-it Method Validation Edition. The slope, y-intercept, and correlation-r were determined. The 95% Cl of the slope and intercept was determined using the bootstrap technique. The results are as follows:

Analytical Method Comparison with Predicate Device
N	169
Slope (95% CI)	0.6919 (0.5760 – 0.8777)
Y-Intercept (95% CI)	-18.35 (-29.54 - -11.13)
Correlation-r	0.784

Clinical Performance

The estimates of clinical sensitivity, clinical specificity, positive predictive value (PPV), and negative predictive value (NPV) of the ALPCO Calprotectin Chemiluminescence ELISA were determined by comparing analytical test results of the prospectively collected stool specimens against the clinical diagnosis made by the clinical investigator/gastroenterologist (reference standard):

IBD diagnosis was based on endoscopy results and/or histology of biopsies taken during the endoscopy.
IBS diagnosis was based on the Rome IV criteria and confirmed by negative endoscopy including the colon and terminal ileum.
Subjects were diagnosed with "Other Gl conditions" when they did not meet the diagnostic criteria for IBD or IBS (Rome IV).

Clinical Diagnosis	Number of Subjects
IBD	76
Ulcerative Colitis (UC)	34
Crohn's Disease (CD)	30
Indeterminant/Undefined	12
IBS	122
Other Gl conditions	226
Total	424

| | Number of Results in ALPCO Calprotectin Chemiluminescence
ELISA Range | | | |
|--------------------|--------------------------------------------------------------------------|---------------|-----------|-------|
| Clinical Diagnosis | 100 μg/g | Total |
| IBD | 6 | 20 | 50 | 76 |
| IBS | 116 | 4 | 2 | 122 |
| GI Other | 206 | 9 | 11 | 226 |
| Total | 328 | 33 | 63 | 424 |

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Estimates of sensitivity, specificity, PPV, and NPV, along with 95% confidence intervals (Wilson score method for sensitivity/specificity and Mercaldo-Wald logit method for predictive value) were calculated for the ALPCO Calprotectin Chemiluminescence ELISA as an aid in the diagnosis of IBD (n = 424). The estimates of sensitivity, specificity, PPV, and NPV were calculated considering equivocal results as both positive and negative:

	ALPCO Test Result
Equivocal results = positive	> 50	50	100