Predicate Devices

None

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a laboratory-based ELISA assay for antibody detection and does not mention any AI or ML components in the device description, intended use, or performance studies. The "Mentions AI, DNN, or ML" section is explicitly marked as "Not Found".

Therapeutic

No.
The device is used for diagnostic purposes, specifically for the qualitative detection of John Cunningham virus antibodies, not for direct treatment or therapy.

Diagnostic

Yes

Explanation: The device is intended for the qualitative detection of antibodies to John Cunningham virus, as an aid in risk stratification for progressive multifocal leukoencephalopathy development, which is a diagnostic purpose.

SaMD (Software as a Medical Device)

No

The device description explicitly states that the device consists of two ELISA tests and includes reagents. This indicates a physical, hardware-based diagnostic test, not a software-only device.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The intended use explicitly states it's for the "qualitative detection of antibodies to John Cunningham virus in human serum or plasma." This is a diagnostic test performed on biological samples taken from the human body.
Purpose: The assay is intended "as an aid in risk stratification for progressive multifocal leukoencephalopathy development" in specific patient populations. This indicates a diagnostic purpose to inform clinical decisions.
Device Description: It describes reagents and components used to perform a laboratory test on patient samples.
Performance Studies: The document details various performance studies (precision, analytical specificity, interferences, method comparison, etc.) which are standard for evaluating the performance of IVD devices.
Professional Use: While it's for professional use only and performed in a reference laboratory, this is common for many IVD tests, especially those with specific technical requirements.

The definition of an In Vitro Diagnostic (IVD) is a medical device that is used to perform tests on samples such as blood, urine, or tissues to detect diseases or other conditions. This description clearly fits the STRATIFY JCVTM Antibody ELISA testing service.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The STRATIFY JCVTM Antibody ELISA testing service provided by Focus Diagnostics is intended for the qualitative detection of antibodies to John Cunningham virus in human serum or plasma. The assay is intended for use in conjunction with other clinical data, in multiple sclerosis and Crohn's disease patients receiving natalizumab therapy, as an aid in risk stratification for progressive multifocal leukoencephalopathy development. The assay is for professional use only and is to be performed only at Focus Diagnostics' Reference Laboratory.

The assay is not intended for donor screening. The performance of this assay has not been established for use in other immunocompromised patient populations or in neonates and pediatrics patient populations.

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

OYP

Device Description

The STRATIFY JCV™ Antibody ELISA consists of two devices. The first is an initial detection ELISA and the second device is a confirmatory (inhibition) ELISA. Both tests utilize the same recombinant antigen which is used in two different formats as described in section L. The devices include the following reagents; Recombinant JC virus like particles (VLP), a JCV high and low positive controls, consisting of human sera that is positive for JCV antibodies, and JCV negative control consisting of human sera that is negative for JCV antibodies. The conjugate, substrate, wash buffers and blocking buffers needed for the test are not supplied with the device and are listed together with other reagents and consumables in the device labeling.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

The performance of this assay has not been established for use in other immunocompromised patient populations or in neonates and pediatrics patient populations.

Intended User / Care Setting

professional use only and is to be performed only at Focus Diagnostics' Reference Laboratory.

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

Not Found

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

Precision/Reproducibility:
- The precision of the detection assay was characterized in one laboratory.
- Study design: testing a panel of serum and plasma samples by three operators, two runs per day over a period of 5 non-consecutive days spread across two weeks.
- Each sample pool was diluted in three aliquots; each individual aliquot was assayed in duplicate providing one nOD result per aliquot.
- Results:
  - Plasma pools total % CV: 7.0% to 18.3%
  - Serum pools total % CV: 9.1% to 12.9%
  - Serum indeterminate pool returned an indeterminate result 96.7% (87/90) of the time.
  - Plasma indeterminate pool returned an indeterminate result 100% of the time.
  - The confirmation (inhibition) assay was performed in batches by random operators on the indeterminate pools.
  - The indeterminate pool was confirmed as positive in the inhibition assay 100% (86/86) of the time for the serum indeterminate pool and 100% (90/90) for the plasma indeterminate pool.
Analytical specificity:
- Cross Reactivity: Evaluated in a two-part study.
  - Part one: evaluated cross reactivity with commercially available human antibodies (Antibody to Escherichia coli, Antibody to Mycobacterium tuberculosis, Antibody to Pneumocystis jiroveci) spiked into JCV negative serum and plasma.
  - Results: No observed reactivity with the three potentially cross-reacting antibodies tested.
  - Part two: A panel of up to twenty remnant specimens that previously tested positive for each of the potential cross reacting antibodies was evaluated.
  - Results: Three groups of patients (C. pneumoniae, HSV1 and T. pallidum) exhibited a positivity rate slightly above that observed in previously reported studies. One group (M. pneumoniae) exhibited a lower than observed rate. This suggests potential cross reactivity.
- Interferences: Evaluated potential interference due to endogenous substances and concomitant medications.
  - Endogenous substances: studied using a sample panel of native sera and plasma samples containing low concentration of JCV antibody spiked with potential interferent.
  - Results: For all potential interferents except y globulin, observed differences in signal did not cause changes in interpretation. Plasma spiked with triglyceride, hemogolobin, and bilirubin, and sera spiked with hemoglobin and bilirubin demonstrated a >20% shift from baseline, indicating potential interference. Commercially available y globulin is expected to react as it contains human IgG antibodies and seroprevalence of JCV antibodies is ~55% in normal population.
  - Concomitant medications: evaluated by reviewing clinical information from the AFFIRM study (585 MS patients, 557 used concomitant medications).
  - Results: The proportion of patients in each JCV antibody status category (Positive at ANY time point, Positive at ALL time points, Negative at ALL time points) was consistent between those who received and did not receive common concomitant medications (Paracetamol, Ibuprofen, Methylprednisolone, Amoxicillin, Acetylsalicylic Acid, Multi-Vitamin, Amantadine, Diclofenac). No potential interfering effect of these medications was shown.
Assay cut-off:
- Established from the distribution of serological responses of samples collected from JCV viruric patients.
- Lower cut point selected as 0.1, as none of the JC viruric subjects had reactivity below this value.
- False negative rate calculation: based on urinary JCV DNA positive (viruric) patients who were negative for serum JCV antibodies.
- Initial assessment false negative rate: 2.5% (95%CI: 0.5 to 4.9%) from 5 of 204 viruric patients from the STRATA study.
- Confirmed false negative rate: 2.7% (95%CI: 0.9 to 6.2%) using 186 of 1073 urinary JCV DNA positive subjects in the STRATIFY-1 study (5 subjects did not have detectable serum JCV antibodies).
- Seropositivity rate for CD patients was similar to MS patients.
Comparison studies:
- Method comparison with predicate device: No predicate device. Sponsor performed comparison with a laboratory developed test for anti-JCV antibodies using a panel of 100 samples.
- Sample panel composition: 62 negative, 71 low positives near the cut-point, and 67 positive samples.
- Results:
  - Positive percent agreement (PPA): 91.0% (122/134) with 95% CI: 85.0% to 94.8%.
  - Negative percent agreement (NPA): 89.4% (59/66) with 95% CI: 79.7% to 94.8%.
- Matrix comparison: Fifty paired plasma and serum samples were assayed.
- Results:
  - Slope: 0.99 with 95% CI: 0.9695 to 1.0103.
  - Intercept: 0.0 with 95% CI: -0.0034 to 0.0043.
  - Mean % difference for nOD values: 4.9%.
  - 50 out of 50 (100%) qualitative results for plasma samples matched matched serum samples.
  - Concludes equivalency of both matrices.
Clinical studies:
- Other clinical supportive data: Data from clinical trial and post-marketing reports of confirmed PML cases used to assess clinical performance for PML risk stratification.
- 37 available serum samples from confirmed PML patients collected at least 6 months prior to clinical diagnosis of PML were tested.
- All 37 pre-PML samples tested positive for JCV antibodies, with an estimated sensitivity of 100% (37/37) with 95% CI: 90.6% to 100%.
- One natalizumab treated CD patient with an available pre-PML sample also tested positive.
- 5,896 MS patients tested: 3,239 positive (54.9% positivity rate, 95% Cl: 53.7% to 56.2%).
- The 100% JCV antibody positivity in 37 natalizumab-treated PML patients was significantly different from 54.9% in MS population, representing a 1.82-fold (95% CI: 1.65 to 1.86) increased risk of PML.
- Data from three studies in MS patients analyzed to determine effect of prior immunosuppressant (IS) therapy on JCV antibody status: No dependence found.
- Data from STRATIFY-1 study analyzed for effect of natalizumab treatment duration on JCV antibody status: No dependence found.
- Conclusion: STRATIFY JCV antibody ELISA testing service results can be used with prior immunosuppressant use and natalizumab treatment duration to stratify PML risk. JCV antibody positive status results in a 1.82-fold increased risk of PML.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Sensitivity: 100% (37/37) with 95% CI: 90.6% to 100% (for pre-PML samples testing positive).
False negative rate: 2.5% (95%CI: 0.5 to 4.9%) and 2.7% (95%CI: 0.9 to 6.2%).
Positive percent agreement (PPA): 91.0% (122/134) with 95% CI: 85.0% to 94.8% (method comparison).
Negative percent agreement (NPA): 89.4% (59/66) with 95% CI: 79.7% to 94.8% (method comparison).
Risk of PML (per 1,000) treated with ≥ 18 months for Positive result: 5.09 (95% CI: 3.70 to 7.01)
Risk of PML (per 1,000) treated with ≥ 18 months for Negative result: 0.17 (95% CI: 0.03 to 0.95)
Relative risk: 30.4 (95% CI: 5.3 to 437.4)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

None

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.3336 John Cunningham Virus serological reagents.

(a)
Identification. John Cunningham Virus serological reagents are devices that consist of antigens and antisera used in serological assays to identify antibodies to John Cunningham Virus in serum and plasma. The identification aids in the risk stratification for the development of progressive multifocal leukoencephalopathy in multiple sclerosis and Crohn's disease patients undergoing natalizumab therapy. These devices are for adjunctive use, in the context of other clinical risk factors for the development of progressive multifocal leukoencephalopathy.(b)
Classification. Class II (special controls). The special control for this device is the FDA guideline document entitled “Class II Special Controls Guideline: John Cunningham Virus Serological Reagents.” For availability of the guideline document, see § 866.1(e).

Summary

0

510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:

K112394

B. Purpose for Submission:

New Device

C. Measurand:

Anti- John Cunningham Virus (JCV) antibodies

D. Type of Test:

Enzyme Linked Immunosorbent Assay (ELISA)

E. Applicant:

Focus Diagnostics Inc.

F. Proprietary and Established Names:

STRATIFY JCV™ Antibody ELISA

G. Regulatory Information:

1. Regulation section:
  21 CFR 866.3336: John Cunningham Virus serological reagents
1. Classification:
  Class II (de novo)
1. Product code:
  OYP
1. Panel:
  Microbiology (83)

1

H. Intended Use:

1. Intended use(s):

The STRATIFY JCVTM Antibody ELISA testing service provided by Focus Diagnostics is intended for the qualitative detection of antibodies to John Cunningham virus in human serum or plasma. The assay is intended for use in conjunction with other clinical data, in multiple sclerosis and Crohn's disease patients receiving natalizumab therapy, as an aid in risk stratification for progressive multifocal leukoencephalopathy development. The assay is for professional use only and is to be performed only at Focus Diagnostics' Reference Laboratory.

The assay is not intended for donor screening. The performance of this assay has not been established for use in other immunocompromised patient populations or in neonates and pediatrics patient populations.

1. Indication(s) for use:
  Same as intended use
1. Special conditions for use statement(s):
  N/A
1. Special instrument requirements:
  N/A

I. Device Description:

The STRATIFY JCV™ Antibody ELISA consists of two devices. The first is an initial detection ELISA and the second device is a confirmatory (inhibition) ELISA. Both tests utilize the same recombinant antigen which is used in two different formats as described in section L. The devices include the following reagents; Recombinant JC virus like particles (VLP), a JCV high and low positive controls, consisting of human sera that is positive for JCV antibodies, and JCV negative control consisting of human sera that is negative for JCV antibodies. The conjugate, substrate, wash buffers and blocking buffers needed for the test are not supplied with the device and are listed together with other reagents and consumables in the device labeling.

J. Substantial Equivalence Information:

1. Predicate device name(s):
  None

2

1. Predicate 510(k) number(s): None
1. Comparison with predicate: N/A

K. Standard/Guidance Document Referenced (if applicable):

A Special Controls Guidance Document will be promulgated.

L. Test Principle:

The STRATIFY JCV™ Antibody ELISA, consists of two assays performed in 2 steps. The first test detects anti-JCV antibodies and the second test is a confirmation (inhibition) assay to confirm the indeterminate results obtained by the first assay. In the first step (or assay) JCV-VLP are coated onto 96-well microtiter plates. Samples and controls are incubated in the wells to allow the binding of JCV specific antibodies in the samples to the JCV-VLP in the solid phase. After washing away the unbound reactants the plates are incubated with horseradish peroxidase labeled donkey antihuman IgG (H+L). Then, the excess conjugate is washed and the reaction developed using tetramethylbenzidine peroxidase substrate and stopped by the addition of a diluted acid stop solution. The results are read as optical density (OD) values by spectrophotometry. Results are recorded as a normalized value (nOD). Samples with nOD values greater than 0.25 are reported as positive for detectable anti-JCV antibodies, samples with nOD values less than 0.1 are reported as negative for detectable anti-JCV antibodies. Samples with nOD values between 0.25 and 0.1 are reported as indeterminate and requiring evaluation in the confirmation (inhibition) assay.

The second step is intended to identify samples containing antibodies which cross react with denatured capture antigen or with other polyomaviruses such as BK virus (BK V). In the second step, the confirmation (inhibition) assay, samples are preincubated with the JCV-VLP in solution prior to the incubation in the JCV-VLPcoated plates, competing with the plate bound JCV-VLP. JCV specific antibodies will bind to the free VLP. The ELISA is then completed as described for the first step. False positive results are characterized by the minimum competition. The percent inhibition is calculated to confirm the presence of anti-JCV antibodies. Samples with a percent inhibition > 40% are reported as confirmed positive, and samples with percent inhibition ≤ 40% are reported as confirmed negative.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:
- a. Precision/Reproducibility:

The precision of the detection assay was characterized in one laboratory. The

3

study design included testing a panel of serum and plasma samples by three operators, two runs per day over a period of 5 non-consecutive days spread across two weeks. Each sample pool was diluted in three aliquots; each individual aliquot was assayed in duplicate providing one nOD result per aliquot. The STRATIFY JCVTM Antibody ELISA detection assay demonstrates a total % CV ranging from 7.0% to 18.3% for the plasma pools and 9.1% to 12.9% for the serum pools. The serum indeterminate pool returned an indeterminate result 96.7% (87/90) of the time, the plasma indeterminate pool returned an indeterminate result 100% of the time. The confirmation (inhibition) assay was performed in batches by random operators on the indeterminate pools. The indeterminate pool was confirmed as positive in the inhibition assay 100% (86/86) of the time for the serum indeterminate pool and 100% (90/90) for the plasma indeterminate pool.

Precision - Detection Assay (nOD values)

| Sample
Matrix | Sample
Level | *Qualitative
Result using
nOD | | | N | Mean | Between
Operators | | Between
Days | | Between
Runs | | Within-Run
(repeatability) | | Total | |
|------------------|-----------------|-------------------------------------|----|----|----|-------|----------------------|-------|-----------------|-------|-----------------|-------|-------------------------------|-------|-------|-------|
| | | D | I | ND | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Plasma | High Positive | 90 | | | 90 | 0.993 | 0.036 | 3.605 | 0.029 | 2.960 | 0.041 | 4.083 | 0.032 | 3.178 | 0.069 | 6.97 |
| | Indeterminate | | 90 | | 90 | 0.150 | 0.003 | 2.045 | 0.011 | 7.402 | 0.006 | 4.010 | 0.006 | 3.953 | 0.014 | 9.52 |
| | Low Positive | 87 | 3 | | 90 | 0.337 | 0.038 | 11.29 | 0.037 | 10.82 | 0.027 | 7.861 | 0.017 | 5.172 | 0.062 | 18.25 |
| | Negative | | 6 | 84 | 90 | 0.082 | 0.001 | 0.775 | 0.009 | 10.79 | 0.006 | 6.842 | 0.003 | 3.811 | 0.011 | 13.36 |
| Serum | High Positive | 90 | | | 90 | 0.978 | 0.061 | 6.218 | 0.091 | 9.253 | 0.039 | 3.994 | 0.046 | 4.712 | 0.125 | 12.75 |
| | Indeterminate | | 87 | 3 | 90 | 0.116 | 0.000 | 0.000 | 0.008 | 6.570 | 0.004 | 3.518 | 0.006 | 5.136 | 0.011 | 9.05 |
| | Low Positive | 86 | 4 | | 90 | 0.296 | 0.000 | 0.000 | 0.033 | 11.06 | 0.013 | 4.346 | 0.014 | 4.876 | 0.038 | 12.85 |
| | Negative | | | 90 | 90 | 0.058 | 0.000 | 0.000 | 0.004 | 6.586 | 0.003 | 4.513 | 0.003 | 4.616 | 0.005 | 9.22 |

D = Detected, I = Indeterminate, ND = Not Detected

4

Precision - Confirmation Assay

| Sample | Qualitative
Result using
%
Inhibition | | N | Min | 5th
Percentile | 95th
Percentile | Max | Mean | SD | %CV |
|---------------------------|-------------------------------------------------|----|----|-------|-------------------|--------------------|-------|-------|------|------|
| | D | ND | | | | | | | | |
| Indeterminate
(Plasma) | 90 | | 90 | 61.11 | 62.93 | 80.10 | 81.50 | 71.57 | 4.97 | 6.95 |
| Indeterminate
(Serum) | 86* | | 86 | 44.71 | 47.40 | 65.71 | 68.54 | 55.28 | 5.26 | 9.52 |

D = Detected, ND = Not Detected

** Three replicates of this sample were negative in the detection assay, per the laboratory procedure the confirmation assay is not performed on negative samples. One dilution was inadvertently omitted by the operator.

b. Linearity/assay reportable range:
N/A
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
N/A
d. Detection limit:
N/A
e. Analytical specificity:

Cross Reactivity

Cross reactivity was evaluated in a two part study. The first part of the study evaluated cross reactivity with commercially available human antibodies spiked into JCV negative serum and plasma as determined by the STRATIFY JCVTM Antibody ELISA. The potentially cross reacting antibodies were spiked at high concentrations. The concentrations tested and the test results are presented in the table below. Each antibody tested was not detected with the STRATIFY JCVTM Antibody ELISA. There was no observed reactivity with the three potentially cross-reacting antibodies tested.

Cross Reactivity - Part One - Spiked Antibodies

Cross Reactant	Concentration	Quantity Detected
----------------	---------------	-------------------

5

		Serum	Plasma
Antibody to Escherichia coli	0.4 µg/mL	0/3	0/3
Antibody to Mycobacterium tuberculosis	0.4 µg/mL	0/3	0/3
Antibody to Pneumocystis jiroveci	unquantified	0/3	0/3

In the second part of the study a panel of up to twenty remnant specimens that previously tested positive for each of the potential cross reacting antibodies was evaluated. Each member of the panel was evaluated using the STRATIFY JCV™ Antibody ELISA along with appropriate controls. The seroprevalence of JCV for each group of potential cross reactants in the panel was compared to the expected seroprevalence of JCV in the normal population. If a group demonstrated a higher than expected seroprevalence of JCV it was considered as an indicator of potential cross reactivity.

Three groups of patients (C. pneumoniae, HSV1 and T. pallidum) exhibited a positivity rate that was slightly above that observed in previously reported studies: one group of patients (M. pneumoniae) exhibited a positivity rate that was lower than the observed rate in previous studies. The results suggest that these groups demonstrate potential cross reactivity.

Sero-positive Samples	Antibodies to JCV Detected	Quantity Tested	% Positive
C. pneumoniae	15	20	75.0%
C. trachomatis	11	20	55.0%
HSV 6	11	20	55.0%
Varicellea Zoster Virus	12	20	60.0%
Candida	13	20	65.0%
HSV 1	17	20	85.0%
HSV 2	7	20	35.0%
Treponema pallidum	4	4	100.0%
Mycoplasma pneumoniae	1	9	11.1%
CMV	10	15	66.7%
HIV 1	14	20	70.0%
Overall	115	188	61.2%

Cross Reactivity - Part Two - Sero-prevalence Comparison

Interferences

A two-fold approach was used to evaluate potential interference due to endogenous substances and concomitant medications. Endogenous substances were studied in two studies as described in the following paragraphs. The potential interference caused by concomitant medications was evaluated by reviewing clinical information provided as part of the drug trials. Potential interference due to endogenous substances were evaluated using a sample panel of native sera and plasma samples that contain a low concentration of

6

JCV antibody which are spiked with the potential interferent as compared to baseline of the same serum and plasma samples that did not contain the interferent. The interferents were spiked at the highest possible endogenous level. For all of the potential interferents with the exception of y globulin; the observed differences in signal did not cause any changes in interpretation of the final result. Plasma spiked with triglyceride, hemogolobin, and bilirubin and sera spiked with hemoglobin and bilirubin demonstrated a >20% shift from baseline, indicating potential interference. Commercially available y globulin is produced using normal human serum containing IgG antibodies: since the seroprevalence of antibodies to JCV virus is approximately 55% in the normal population y globulin is expected to react with this assay.

| | Potential
Interferent Panel | Plasma | | | Serum | | |
|--|--------------------------------|----------|---------|-----------------------------|----------|---------|-----------------------------|
| | | Mean nOD | Std Dev | %Change
from
Baseline | Mean nOD | Std Dev | %Change
from
Baseline |
| | Baseline (Part 1) | 0.385 | 0.031 | N/A | 0.440 | 0.025 | N/A |
| | Triglyceride (10
mg/mL) | 0.295 | 0.003 | -23.3% | 0.411 | 0.017 | -6.4% |
| | Hemoglobin (200
mg/mL) | 0.283 | 0.008 | -26.3% | 0.348 | 0.013 | -20.9% |
| | Bilirubin (0.2
mg/mL) | 0.289 | 0.008 | -24.9% | 0.337 | 0.005 | -23.3% |
| | Baseline (Part 2) | 0.315 | 0.0074 | N/A | 0.296 | 0.0139 | N/A |
| | Albumin (12 g/dL) | 0.276 | 0.0083 | -12.6 | 0.267 | 0.0172 | -9.6 |
| | Ascorbic Acid (3
mg/dL) | 0.284 | 0.0451 | -9.9 | 0.273 | 0.0199 | -7.9 |
| | Cholesterol (500
mg/dL) | 0.302 | 0.0175 | -4.4 | 0.289 | 0.0120 | -2.2 |
| | y globulin (6 g/dL) | 1.206 | 0.0136 | 282.4 | 1.308 | 0.0616 | 342.1 |

Interference Summary - Signal Comparison to Baseline

1 The observed decrease in signal may be in part due to a decrease in JCV antibody concentration caused by the additional volume of the spiked interferent

Interference Due To Concomitant Medications

In order to evaluate the potential effect of concomitant medications on the ability to detect JCV antibodies, results were evaluated for serum samples collected serially over a period of 30 months from a subset of 585 MS patients in the AFFIRM study using the STRATIFY JCV™ Antibody ELISA. AFFIRM is a completed clinical trial where patients with multiple sclerosis were randomized to receive placebo or natalizumab, and blood samples were collected every 6 months for 30 months. The AFFIRM study utilized an

7

ongoing Concomitant Medications Log for each patient to capture all concomitant medications used by the patient during the course of the study. A total of 557 of the 585 patient subset (95%) used concomitant medications during the 30 month AFFIRM study

In order to assess whether the presence of concomitant medications caused interference in the STRATIFY JCV™ Antibody ELISA, each of the concomitant medications used by ≥10% and ≥20% of the patients in AFFIRM study were evaluated separately. The proportion of patients who tested: JCV antibody positive at any time point, JCV antibody positive at all time points, or JCV antibody negative at all time points over the 30 month study period were evaluated in those patients who received or did not receive each individual concomitant medication. If interference occurred at any time point over the 30 month sampling period due to a particular concomitant medication, the proportion of patients who tested JCV antibody positive or negative in these categories would be expected to be different in those who received the medication compared to those patients who did not. The following table illustrates the results of the study.

Concomitant Medications Used	AFFIRM Who Also Provided Longitudinal Samples
Tested for JCV Antibody Status

| MEDICATIO
N | Total Number
of Patients
Who Received
a Concomitant
Medication
(%)* | JCV
ANTIBODY
STATUS* | PATIENTS
REPORTING
MEDICATION USE | | PATIENTS NOT
REPORTING
MEDICATION USE | |
|----------------------|------------------------------------------------------------------------------------|-----------------------------------|-----------------------------------------|----------------------|---------------------------------------------|-------------------|
| | | | Sample
Size | Percent | Sample
Size | Percent |
| Paracetamol | 238 (41%) | Positive at
ANY time
point | 151 | 63
(57.0to69.6) | 209 | 60 (54.9to65.4) |
| | | Positive at
ALL time
points | 122 | 51 (44.7 to
57.8) | 167 | 48 (42.8 to 53.5) |
| | | Negative at
ALL time
points | 87 | 37 (30.4-
43.0) | 138 | 40 (34.6 to 45.1) |
| Ibuprofen | 211 (36%) | Positive at
ANY time
point | 125 | 59 (52.3 to
65.9) | 235 | 63 (57.7 to 67.7) |
| | | Positive at
ALL time
points | 96 | 45 (38.6 to
52.5) | 193 | 52 (46.4 to 56.8) |
| | | Negative at
ALL time
points | 86 | 41 (34.1 to
47.7) | 139 | 37 (32.3 to 42.3) |
| MEDICATIO
N | Total Number
of Patients
Who Received
a Concomitant
Medication
(%)* | JCV
ANTIBODY
STATUS* | PATIENTS
REPORTING
MEDICATION USE | | PATIENTS NOT
REPORTING
MEDICATION USE | |
| Methylprednisolone | 167 (29%) | Positive at
ANY time
point | 107 | 64 (56.3 to
71.3) | 253 | 61 (55.7 to 65.2) |
| | | Positive at
ALL time
points | 91 | 54 (46.6 to
62.2) | 198 | 47 (42.5 to 52.3) |
| | | Negative at
ALL time
points | 60 | 36 (28.7 to
43.7) | 165 | 39 (34.8 to 44.3) |
| Amoxicillin | 115 (20%) | Positive at
ANY time
point | 71 | 62 (52.2 to
70.6) | 289 | 61 (56.9 to 65.9) |
| | | Positive at
ALL time
points | 53 | 46 (36.8 to
55.6) | 236 | 50 (45.6 to 54.8) |
| | | Negative at
ALL time
points | 44 | 38 (29.4 to
47.8) | 181 | 39 (34.1 to 43.1) |
| Acetylsalicylic Acid | 78 (13%) | Positive at
ANY time
point | 51 | 65 (53.8 to
75.8) | 309 | 61 (56.5 to 65.2) |
| | | Positive at
ALL time
points | 45 | 58 (46.0 to
68.8) | 244 | 48 (43.7 to 52.6) |
| | | Negative at
ALL time
points | 27 | 35 (24.2 to
46.2) | 198 | 39 (34.8 to 43.5) |
| Multi -Vitamin | 69 (12%) | Positive at
ANY time
point | 39 | 57 (44.0 to
68.4) | 321 | 62 (57.9 to 66.4) |
| | | Positive at
ALL time
points | 27 | 39 (27.6 to
51.6) | 262 | 51 (46.4 to 55.2) |
| | | Negative at
ALL time
points | 30 | 43 (31.6 to
56.0) | 195 | 38 (33.6 to 42.1) |
| Amantadine | 73 (12%) | Positive at
ANY time
point | 46 | 63 (50.9 to
74.0) | 314 | 61 (57.0 to 65.6) |
| MEDICATIO
N | Total Number
of Patients
Who Received
a Concomitant
Medication
(%)* | JCV
ANTIBODY
STATUS* | PATIENTS
REPORTING
MEDICATION USE | | PATIENTS NOT
REPORTING
MEDICATION USE | |
| | | | Sample
Size | Percent | Sample
Size | Percent |
| Diclofenac | 66 (11%) | Positive at
ALL time
points | 36 | 49 (37.4 to
61.3) | 253 | 49 (45.0 to 53.8) |
| | | Negative at
ALL time
points | 27 | 37 (26.0 to
49.1) | 198 | 39 (34.4 to 43.0) |
| | | Positive at
ANY time
point | 34 | 52 (38.9 to
64.0) | 326 | 63 (58.5 to 67.0) |
| | | Positive at
ALL time
points | 26 | 39 (27.6 to
52.2) | 263 | 51 (46.3 to 55.1) |
| | | Negative at
ALL time
points | 32 | 48 (36.0 to
61.1) | 193 | 37 (33.0 to 41.5) |

8

9

The proportion of patients in each category was consistent between those patients who received the medications and those who did not. The results did not show any potential interfering effect of these commonly used medications on the performance of the assay.

f. Assay cut-off:

To characterize antibody responses against infectious agents in humans, it is critical to have reference sera from both infected and non-infected individuals However, JCV infection is clinically asymptomatic, thereby making it difficult to generally distinguish JCV infected from non-infected individuals. Therefore it is difficult to obtain samples from confirmed infected and noninfected individuals. Even though about 20-30% of JCV infected individuals shed viral DNA in the urine, JCV DNA is often not detected in the blood or urine of infected individuals, even when the infection results in the development of PML. There is no evidence that testing for JCV DNA in blood or urine can identify all JCV infected individuals, however, those who shed virus in the urine are confirmed to be infected with JCV. Therefore, sera from viruric patients were used to establish the positive reference sera for the ELISA. The assay cut point was established from the distribution of the serological responses of samples collected from JCV viruric patients in the JCV antibody ELISA. None of the JC viruric subjects had a reactivity below 0.1, which was therefore selected as the lower cut point in the JCV antibody

10

ELISA.

The false negative rate of the assay was calculated from the number of subjects who were urinary JCV DNA positive (viruric), but negative for serum JCV antibodies in the STRATIFY JCV™ Antibody ELISA. An initial assessment of the STRATIFY JCV antibody ELISA false negative rate of 2.5% (95%CI: 0.5 to 4.9%) resulted from 5 of 204 viruric patients from the STRATA study whom tested seronegative. The false negative rate of the JCV antibody assay was confirmed using serum and urine samples collected at enrollment from 1073 subjects in the STRATIFY-1 study. Of the 1073 subjects, 186 (17%) were found to be urinary JCV DNA positive. The false negative rate of the assay in STRATIFY-1 was determined to be 2.7% (95%CI: 0.9 to 6.2%) as 5 out of the 186 urinary JCV DNA positive subjects did not have detectable serum JCV antibodies. The seropositivity rate for CD patients was assessed using the same cut-off points and found to be similar to that of MS patients. No matrix differences were found between MS patients, CD patients or healthy volunteers. The false negative rate for CD population was not determined; however, the data supports the applicability of the cut-off and the false negative rate established for MS population to the CD population.

2. Comparison studies:

a. Method comparison with predicate device:

There is no predicate device for this assay. However, the sponsor performed a method comparison study with a laboratory developed test for the detection of anti-JCV antibodies using a panel of 100 samples that cover the detection range of their assay. The purpose of this study was to evaluated the comparative performance with a different anti-JCV antibody detection test particularly in samples close to the cut-off of the STRATIFY Antibody ELISA. The sample panel consisted of 62 negative, 71 low positives near the cut-point and 67 positive samples. Low positives include samples between two cut points (0.1 and 0.25) and a small group of samples just above the upper cut point (the range is from nOD ≤ 0.1 to ≤0.35). The Positive percent agreement (PPA) was 91.0% (122/134) with 95% CI: 85.0% to 94.8%, the Negative percent agreement (NPA) was 89.4% (59/66) with 95% CI: 79.7% to 94.8%. The results of the analytical comparison are shown below.

	Comparative Assay
	Detected	Not Detected
	Sample Category based on initial nOD	Sample Category based on initial nOD
	Positive (nOD>0.35)
Low Positive* (0.1 ≤ nOD ≤ 0.35)
Negative ( nOD 0.35)	Low Positive* (0.1 ≤ nOD ≤ 0.35)
Negative ( nOD