(136 days)
STRATIFY JCV™ Antibody ELISA
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No
The device description details a standard ELISA assay with spectrophotometric reading and comparison to cut-off values. There is no mention of AI or ML in the device description, intended use, or performance studies. The analysis of results is based on simple comparisons to predefined thresholds.
No
The device aids in risk stratification for PML development, which is a diagnostic purpose, not a therapeutic intervention. It does not treat or cure a disease.
Yes
This device is intended for the qualitative detection of antibodies to JC virus in human serum or plasma and is used as an aid in risk stratification for progressive multifocal leukoencephalopathy development in multiple sclerosis patients, directly supporting clinical decision-making.
No
The device description clearly outlines a laboratory-based ELISA assay involving physical reagents, microtiter plates, and spectrophotometric readings. While software is likely used for data analysis and result reporting, the core of the device is a chemical and biological assay kit, not solely software.
Based on the provided information, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states the assay is for the "qualitative detection of antibodies to JC virus in human serum or plasma." This involves testing biological specimens in vitro (outside the body).
- Device Description: The description details an ELISA assay, a common laboratory technique used for in vitro testing of biological samples. It describes the process of reacting antibodies in the specimen with antigens on a plate and measuring the resulting color change.
- Professional Use Only: The indication for "professional use only" is typical for IVD devices used in clinical laboratories.
- Performance Studies: The document describes various performance studies conducted on human serum and plasma samples, which are standard for validating IVD devices.
Therefore, the Focus Diagnostics' STRATIFY JCV® DxSelect™ assay fits the definition of an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
The Focus Diagnostics' STRATIFY JCV® DxSelect™ assay is intended for the qualitative detection of antibodies to JC virus in human serum or plasma. The assay is intended for use in conjunction with other clinical data, in multiple sclerosis patients receiving or considering natalizumab therapy, as an aid in risk stratification for progressive multifocal leukoencephalopathy development. The assay is for professional use only.
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 patients with different disease conditions or undergoing other treatments or in neonates and pediatric patient populations.
Product codes (comma separated list FDA assigned to the subject device)
OYP
Device Description
In the Focus Diagnostics' STRATIFY JCVg DxSelect™ test, JC virus-like particles (VLP) are pre-coated onto 96-well microiter plates. Diluted serum or plasma specimens and controls are incubated in the wells to allow JCV-specific antibodies present in the specimens to react with the JC VLP antigen. Nonspecific reactants are removed by washing. Peroxidase-conjugated anti-human antibodies are added to react with JCV-specific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromogen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of optical density (OD). Specimen OD readings are compared with Cut-Off Calibrator OD readings to determine results. Each specimen result is reported as an index value. A specimen with an index value that is greater than a specified upper cut-off is reported as positive for detectable JCV-specific antibodies, whereas a specimen with an index value less than the specified lower cut-off is reported as negative for detectable JCV-specific antibodies. A specimen with an index value that is equal to or between the upper and lower cut-off values is reported as indeterminate. An indeterminate result requires further evaluation in the confirmation (inhibition) assay.
In the confirmation assay, soluble JC VLP antigen will compete with plate bound JC VLP antigen for the JCV-specific antibodies present in the serum or plasma specimens. After washing away the unbound antibodies, peroxidase-conjugated anti-human antibodies are added and bind to any captured JCVspecific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromagen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of OD. The percent inhibition is calculated to confirm presence of JCV-specific antibodies in the specimens with a percent inhibition value that is greater than the specified cut-off are reported as positive for detectable JCV-specific antibodies, whereas specimens with percent inhibition values less than or equal to the cut-off are reported as negative for detectable JCV-specific antibodies.
Mentions image processing
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Mentions AI, DNN, or ML
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Input Imaging Modality
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Anatomical Site
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Indicated Patient Age Range
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 patients with different disease conditions or undergoing other treatments or in neonates and pediatric patient populations.
Intended User / Care Setting
professional use only.
Description of the training set, sample size, data source, and annotation protocol
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Description of the test set, sample size, data source, and annotation protocol
A clinical plan was developed for collection of serum samples obtained from natalizumab-treated patients prior to the onset of PML for JCV antibody testing. A total of 31 available serum samples from confirmed PML patients collected at least 6 months prior to clinical diagnosis of PML were tested for JCV antibody status using the STRATIFY JCV6 DxSelect™ assay. In addition to the pre-PML samples, 1330 Samples from MS patients were tested by the STRATIFY JCV DxSelect™ assay.
Two groups of prospectively collected and archived clinical samples obtained from the STRATIFY-2 and the AFFIRM clinical studies were used to assess the performance of the STRATIFY JCVg DxSelect™ assay compared to the validated laboratory methodology used in the STRATIFY-2 and AFFIRM clinical studies. One group consisted of patients who were receiving natalizumab, the other group of patients had not received natalizumab therapy (and were considering natalizumab). The samples were blinded and randomly distributed to two external testing sites and one internal testing site.
The STRATIFY JCV% DxSelect™ assay been used to evaluate JCV antibody positivity rate in serum and plasma samples from a geographically diverse cohort of 1330 MS patients. The cohort was comprised from MS patients from clinical trials including a completed Phase 3 clinical study of natalizumab in MS patients (AFFIRM C-1801), an ongoing study to evaluate seroprevalence in the MS population (STRATIFY-2 [101JC402).
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Clinical Performance (Comparative Agreement)
Study Type: Clinical Performance, Comparative Agreement
Sample Size: 31 pre-PML samples, 1330 MS patient samples, 38 PML cases, 13,189 non-PML cases.
Key Results: The 100% JCV antibody positivity demonstrated in the 31 natalizumab-treated PML patients prior to PML diagnosis was significantly different than the 58.7% JCV antibody positivity in the MS population, and represents an approximately 2-fold increased risk of PML compared to the PML incidence in the overall natalizumab-treated population.
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)
Performance with pre-PML samples.
Study Type: Comparative study against cleared Anti-JCV assay
Sample Size: 31 serum samples from confirmed PML patients
Key Results: 100% (31/31) positive agreement (95% CI: 89.0% to 100%) with the validated assay.
Performance with archived clinical specimens
Study Type: Agreement with Cleared Anti-JCV Assay using archived clinical specimens.
Sample Size: Patients receiving natalizumab (N=707), Patients considering natalizumab (N=623)
Key Results (Patients Receiving Natalizumab): Positive Percent Agreement (PPA) = 97.0% (385/397), 95% CI: 94.8 to 98.3%; Negative Percent Agreement (NPA) = 90.6% (281/310), 95% CI: 86.9 to 93.4%.
Key Results (Patients Considering Natalizumab): Positive %Agreement (PPA) = 98.5% (326/331), 95% CI: 96.5 to 99.4%; Negative %Agreement (NPA) = 91.8% (268/292), 95% CI: 88.1 to 94.4%.
Positivity Rate and Expected Values:
Study Type: Seroprevalence evaluation
Sample Size: 1330 MS patients (AFFIRM: N=538, STRATIFY-2: N=792)
Key Results: JCV antibody positivity rate in the MS cohort was 55-59%. Observed seroprevalence: AFFIRM = 55.2% (95% CI: 51.0 to 59.4), STRATIFY-2 = 59.0% (95% CI: 55.5 to 62.3). 100% concordance with 31 pre-PML samples.
Reproducibility
Study Type: Reproducibility based on CLSI guideline
Sample Size: 120 (OD), 90 (Controls), 90 (Plasma and Serum samples)
Key Results (Total %CV): OD = 16.5%, Negative Control = 16.5%, Positive Control = 3.7%, Indeterminate Control = 8.3%, Plasma Low positive = 7.9% (Detection Assay), Plasma Medium positive = 6.9% (Detection Assay), Serum Negative = 12.8% (Detection Assay), Serum Indeterminate = 8.5% (Detection Assay). %Inhibition: Plasma Low positive = 5.4%, Plasma Medium positive = 4.3%, Serum Negative = 30.1%, Serum Indeterminate (6.2% CV=6.2) = 10.5%, Serum Indeterminate (3.9% CV=3.9) = 11.5%.
Reproducibility at the Lower Cut Point
Study Type: Precision near lower cut point
Sample Size: 20 (Plasma), 19 (Serum) for Detection and Confirmation Assay
Key Results: Plasma %CV (Detection Assay) = 2.3%, %CV (Confirmation Assay) = 2.2%. Serum %CV (Detection Assay) = 2.6%, %CV (Confirmation Assay) = 2.0%.
Cross Reactivity
Study Type: Cross-reactivity in three parts
Sample Size: 9 samples for Part One, 334 samples in total for Part Two, 40 clinical specimens for Part Three.
Key Results (Part One): No observed reactivity with spiked antibodies (Escherichia coli, Mycobacterium tuberculosis, Pneumocystis jiroveci).
Key Results (Part Two): Four groups (C. pneumoniae, HIV, CMV, HSV 1) exhibited a positivity rate slightly above that observed in previously reported studies, suggesting potential cross reactivity. Overall Positive: 66.8%, Overall Negative: 33.2%.
Key Results (Part Three): No samples exhibited >45% change in the OD signal when spiked with BKV VLP, indicating that the assay does not cross react with BKV.
Interferences
Study Type: Evaluation of potential interference due to endogenous substances
Key Results: For all potential interferents except gamma globulin, observed differences in signal did not cause changes in interpretation. Gamma globulin caused a significant change in index value (Plasma: 831.6%, Serum: 758.5%).
HOOK EFFECT
Study Type: Hook effect investigation
Key Results: No Hook Effect was observed at the optimal coating VLP concentration and conjugate dilution for this assay using the panel tested.
SAMPLE MATRIX COMPARISON
Study Type: Comparison of sera and plasma samples
Sample Size: 109 paired sera and plasma samples
Key Results: Passing-Bablok regression: slope of 1 (95% CI: 0.9928 to 1.0167) and intercept of 0 (95% CI: -0.0068 to 0.0016). Qualitative results: Positive Percent Agreement = 96.7% (58/60), 95% CI: 88.6 to 99.1%; Negative Percent Agreement = 97.9% (47/48), 95% CI: 89.1 to 99.6%.
Sample Comparison -Fresh vs. Frozen
Study Type: Comparison of fresh and frozen plasma/sera specimens
Sample Size: 53 pairs of fresh and frozen plasma, 53 pairs of fresh and frozen sera. Total 106 pairs.
Key Results: Passing-Bablok regression: slope of 1 (95% CI: 0.9900 to 1.0221) and intercept of 0 (95% CI: -0.0090 to 0.0028). Qualitative results: Positive Percent Agreement = 96% (72/75), 95% CI: 88.9 to 98.6%; Negative Percent Agreement = 96.7% (29/30), 95%CI: 83.3 to 99.4%.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Not directly reported as Sensitivity/Specificity/PPV/NPV, but agreements:
Positive Percent Agreement (PPA):
- Clinical Samples - Patients Receiving Natalizumab: 97.0% (385/397) (95% CI: 94.8 to 98.3%)
- Clinical Samples - Patients Considering Natalizumab: 98.5% (326/331) (95% CI: 96.5 to 99.4%)
- Sample Matrix Comparison: 96.7% (58/60) (95% Cl: 88.6 to 99.1%)
- Fresh vs. Frozen: 96% (72/75) (95% C1: 88.9 to 98.6%)
Negative Percent Agreement (NPA):
- Clinical Samples - Patients Receiving Natalizumab: 90.6% (281/310) (95% CI: 86.9 to 93.4%)
- Clinical Samples - Patients Considering Natalizumab: 91.8% (268/292) (95% CI: 88.1 to 94.4%)
- Sample Matrix Comparison: 97.9% (47/48) (95%Cl: 89.1 to 99.6%)
- Fresh vs. Frozen: 96.7% (29/30) (95%CI: 83.3 to 99.4%)
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.
STRATIFY JCV™ Antibody ELISA
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).
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§ 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).
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STRATIFY JCV @ DxSelect™ (EL1950) Page 1 of 10
| Applicant | Focus Diagnostics, Inc.
11331 Valley View Street
Cypress, California 90630
USA | | AUG 16 2012 |
|--------------------------------|----------------------------------------------------------------------------------------------------------|--|-------------|
| Establishment Registration No. | 2023365 | | |
| Contact Person | Tara Viviani
tel 562-240-6115
fax 562.240.6530
tviviani@focusdx.com | | |
| Summary Date | July 24, 2012 | | |
| Proprietary Name | STRATIFY JCV ® DxSelect™ | | |
| Generic Name | JCV ELISA | | |
| Classification | Class II, Special Controls
§ 21 CFR 866.3336, Anti-JCV antibody detection assay.
Product Code: OYP | | |
| Predicate Devices | STRATIFY JCV™ Antibody ELISA | | |
INTENDED USE
The Focus Diagnostics' STRATIFY JCV® DxSelect™ assay is intended for the qualitative detection of antibodies to JC virus in human serum or plasma. The assay is intended for use in conjunction with other clinical data, in multiple sclerosis patients receiving or considering natalizumab therapy, as an aid in risk stratification for progressive multifocal leukoencephalopathy development. The assay is for professional use only.
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 patients with different disease conditions or undergoing other treatments or in neonates and pediatric patient populations.
SUMMARY AND EXPLANATION OF THE TEST
Progressive multifocal leukoencephalopathy (PML) is an opportunistic infection of the central nervous system (CNS) caused by JC virus (JCV), a polyoma virus, that is pathogenic only in humans. It is thought that exposure to the JC virus occurs early in life (pre-adolescence) and recently published studies have reported that approximately 50% to 60% of adults have been infected with JCV, as evidenced by the presence of antibody to JCV in the serum. In rare instances, the virus reactivates and progresses to PML such as in individuals who are immune compromised. Treatment with immunomodulatory therapies increase the risk of developing PML in JCV infected individuals. While JCV exposure is necessary for the development of PML, the development of the disease is also dependent on both host and viral factors, as well as immune status. Since JCV infection is a necessary step for PML development, an assay to detect JCV exposure in patients may be a potentially useful tool to stratify patients for PML risk (i.e., identifying patients who may be at a lower or higher risk of developing PML).
There is an increased risk of PML in natalizumab treated multiple sclerosis (MS) and Crohn's disease (CD) patients. Three risk factors for PML have been identified in MS and CD patient populations: natalizumab treatment duration, prior immunosuppressant use, and the presence of antibodies to JCV. Utilizing these three risk factors, sub-groups of patients can be identified with both higher and lower risk for PML. Please consult the current, locally available, prescribing or supplemental physician information for natalizumab (Tysabri®) for detailed information on the known risks associated with JCV serological status and the development of PML in natalizumab-treated patient's JCV antibody status should be considered in combination with other known PML risk factors when evaluating the benefit and risk of initiating or continuing therapy with natalizumab. Patients with all three known risk factors have the highest risk for the development of PML.
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120986 510(k) Summarv STRATIFY JCVg DxSelect™ (EL1950) Page 2 of 10
TEST PRINCIPLE
In the Focus Diagnostics' STRATIFY JCVg DxSelect™ test, JC virus-like particles (VLP) are pre-coated onto 96-well microiter plates. Diluted serum or plasma specimens and controls are incubated in the wells to allow JCV-specific antibodies present in the specimens to react with the JC VLP antigen. Nonspecific reactants are removed by washing. Peroxidase-conjugated anti-human antibodies are added to react with JCV-specific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromogen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of optical density (OD). Specimen OD readings are compared with Cut-Off Calibrator OD readings to determine results. Each specimen result is reported as an index value. A specimen with an index value that is greater than a specified upper cut-off is reported as positive for detectable JCV-specific antibodies, whereas a specimen with an index value less than the specified lower cut-off is reported as negative for detectable JCV-specific antibodies. A specimen with an index value that is equal to or between the upper and lower cut-off values is reported as indeterminate. An indeterminate result requires further evaluation in the confirmation (inhibition) assay.
In the confirmation assay, soluble JC VLP antigen will compete with plate bound JC VLP antigen for the JCV-specific antibodies present in the serum or plasma specimens. After washing away the unbound antibodies, peroxidase-conjugated anti-human antibodies are added and bind to any captured JCVspecific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromagen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of OD. The percent inhibition is calculated to confirm presence of JCV-specific antibodies in the specimens with a percent inhibition value that is greater than the specified cut-off are reported as positive for detectable JCV-specific antibodies, whereas specimens with percent inhibition values less than or equal to the cut-off are reported as negative for detectable JCV-specific antibodies.
| Item | Device | Predicate |
|---|---|---|
| Name | STRATIFY JCV® DxSelect™ | STRATIFY JCV™ Antibody ELISA |
| Intended Use | The Focus Diagnostics' STRATIFY | |
| JCV® DxSelect™ assay is intended for | ||
| the qualitative detection of antibodies to | ||
| JC virus in human serum or plasma. | ||
| The assay is intended for use in | ||
| conjunction with other clinical data, in | ||
| multiple sclerosis patients receiving or | ||
| considering natalizumab therapy, as an | ||
| aid in risk stratification for progressive | ||
| multifocal leukoencephalopathy | ||
| development. The assay is for | ||
| professional use only. | ||
| 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 patients with different | The STRATIFY JCV™ 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 | ||
| disease conditions or undergoing other | ||
| treatments or in neonates and pediatric | ||
| patient populations. | in other immunocompromised patient | |
| populations or in neonates and | ||
| pediatrics patient populations. | ||
| Assay Methodology | Detection ELISA with a secondary | |
| Confirmation ELISA for specimens that | ||
| have results between the two cut-off | ||
| levels. | Detection ELISA with a secondary | |
| Confirmation ELISA for specimens that | ||
| have results between the two cut-off | ||
| levels. | ||
| Assay Target | Antibodies to John Cunningham Virus | |
| (JCV) | Antibodies to John Cunningham Virus | |
| (JCV) | ||
| Sample Type(s) | Serum and plasma | Serum and plasma |
Comparison to Predicate
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STRATIFY JCVg DxSelect™ (EL1950) Page 3 of 10
| Item | Device | Predicate |
|---|---|---|
| Name | STRATIFY JCV® DxSelect™ | STRATIFY JCV™ Antibody ELISA |
| Sample Preparation | 1:101 dilution in sample diluent | 1:200 dilution in sample diluent |
| Detection Assay |
- Confirmation Assay | 1:101 dilution in confirmation diluent | 1:200 dilution in assay diluent spiked with JCV antigen. |
| Assay Cut-Offs | Index 70%) it may be an indicator of potential cross reactivity. Four groups of patients (C. pneumoniae, HIV, CMV, HSV 1) exhibited a positivity rate that was slightly above that observed in previously reported studies. The results suggest that these groups demonstrate potential cross reactivity.
| Sample
Matrix | Cross Reactant | Total No.
of
Replicates
or
Samples | Screening
Result Count
(Based on
Index) | | | Confirmation
Result Count
(Based on
%Inhibition) | | | | Final Interpretation | | % | |
|------------------|--------------------|------------------------------------------------|--------------------------------------------------|-----|----|-----------------------------------------------------------|----|----|-----|----------------------|------|------|--|
| | | | D | IND | ND | Not
Tested | D | ND | D | ND | D | ND | |
| Unknown | HIV | 22 | 16 | 2 | 4 | 20 | 1 | 1 | 17 | 5 | 77.3 | 22.7 | |
| | C. pneumoniae | 48 | 35 | 5 | 8 | 43 | 2 | 3 | 37 | 11 | 77.1 | 22.9 | |
| Serum | C. trachomatis | 20 | 11 | 2 | 7 | 18 | 1 | 1 | 12 | 8 | 60.0 | 40.0 | |
| | CMV | 40 | 27 | 7 | 6 | 33 | 5 | 2 | 32 | 8 | 80.0 | 20.0 | |
| | Candida | 40 | 26 | 6 | 8 | 34 | 1 | 5 | 27 | 13 | 67.5 | 32.5 | |
| | EBV | 40 | 23 | 9 | 8 | 31 | 2 | 7 | 25 | 15 | 62.5 | 37.5 | |
| | HSV 1 | 48 | 32 | 6 | 10 | 42 | 2 | 4 | 34 | 14 | 70.8 | 29.2 | |
| | HSV 2 | 20 | 8 | 5 | 7 | 15 | 1 | 4 | 9 | 11 | 45.0 | 55.0 | |
| | HHV 6 | 20 | 11 | 2 | 7 | 18 | | 2 | 11 | 9 | 55.0 | 45.0 | |
| | Listeria | 17 | 8 | 3 | 6 | 14 | | 3 | 8 | 9 | 47.1 | 52.9 | |
| | Mycoplasma | 20 | 10 | 4 | 6 | 16 | | 4 | 10 | 10 | 50.0 | 50.0 | |
| | Treponema pallidum | 19 | 12 | 2 | 5 | 17 | 1 | 1 | 13 | 6 | 68.4 | 31.6 | |
| | VZV | 20 | 12 | 4 | 4 | 16 | 1 | 3 | 13 | 7 | 65.0 | 35.0 | |
| | All | 334 | 208 | 48 | 78 | 286 | 15 | 33 | 223 | 111 | 66.8 | 33.2 | |
Table 8: Cross Reactivity - Part Two -- Sero-prevalence Comparison
D= Detected, ND = Not Detected, IND = Indeterminate
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STRATIFY JCVg DxSelect™ (EL1950) Page 8 of 10
Part three of the cross reactivity evaluation consisted of an evaluation of the potential cross reactivity with other polyoma viruses. This included a cross absorption study using BKV virus like particles (VLP). A total of 40 clinical specimens that have previously tested positive for JCV antibodies in the STRATIFY JCV% DxSelect™ assay were tested in a confirmation style assay using JCV VLP and BKV VLP. A control set consisted of samples spiked with JCV VLP at the same concentration of VLP that is used in the confirmation assay. The test samples were with BKV VLP at the same concentration. The tests samples would be considered to be cross reactive if the % change in signal between the unspiked sample and the sample spiked with BKV VLP is > 45%. The control set demonstrated % change in OD that was consistent with expectations. The test samples demonstrated % change in OD values due to BKV VLP that ranged from -15 to 27%. No samples exhibited >45% change in the OD signal when spiked with BKV VLP, indicating that the assay does not cross react with BKV.
Additional analysis of the structure of the VP1 protein of other polyoma viruses indicates that BKV and JCV are more closely related (79.4% similarity) than other polyoma virus such as Merkel Cell virus and WU virus and KI virus (30.8 to 50.8 % similarity). Due to the differences in viral structure a similar experiment with other polyoma virus VLP was not conducted.
Interferences
Potential interference due to endogenous substances were evaluated using a protocol based on a CLSI guideline for estimating the effect of interfering substances. The testing panel consisted of sera and plasma samples that contain JCV antibody with an index value that is close to the assay cut off. The samples are spiked with the potential interferent at the highest possible endogenous level and compared to baseline testing of the same serum and plasma samples that did not contain the interferent. 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. A potentially interference is suspected if the % Change from the baseline sample is > 20%. 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 it is expected to react with this assay.
| Substance
Name | Substance
Concentration | Plasma | | | Serum | | |
|-------------------|----------------------------|-------------------------------------|-----------------------------------------|-----------------------------|-------------------------------------|-----------------------------------------|-----------------------------|
| | | Average Index
Baseline
Sample | Average Index
Interference
Sample | %Change
from
Baseline | Average Index
Baseline
Sample | Average Index
Interference
Sample | %Change
from
Baseline |
| Albumin | 120 mg/mL | 0.35 | 0.38 | 8.6 | 0.43 | 0.37 | -14.0 |
| Ascorbic Acid | 0.03 mg/mL | 0.40 | 0.42 | 5.0 | 0.42 | 0.44 | 4.8 |
| Bilirubin | 0.2 mg/mL | 0.33 | 0.33 | 0.0 | 0.42 | 0.38 | -9.5 |
| Cholesterol | 5 mg/mL | 0.48 | 0.40 | -16.7 | 0.46 | 0.44 | -4.3 |
| Gamma Globulin | 60 mg/mL | 0.38 | 3.54 | 831.6 | 0.41 | 3.52 | 758.5 |
| Hemoglobin | 110 mg/mL | | 0.41 | 13.5 | | 0.46 | 5.0 |
| Hemoglobin | 165 mg/mL | 0.36 | 0.38 | 6.1 | 0.44 | 0.39 | -10.1 |
| Hemoglobin | 220 mg/mL | | 0.35 | -2.4 | | 0.37 | -16.0 |
| Triglycerides | 10 mg/mL | 0.36 | 0.35 | -2.8 | 0.40 | 0.37 | -7.5 |
Table 9: Interference Summary - Signal Comparison to Baseline
Note: Three dilution of Gamma Globulin stock solution exhibited Index values - 3.22, 3.57 and 3.40.
HOOK EFFECT
The Hook Effect was investigated during the optimization of the JCV VLP coating concentration and conjugate dilution using a panel of serum samples ranging from negative reactivity to strong positive reactivity (beyond the limit of the plate reader at OD450 - 4.000) to JCV. No Hook Effect was observed at the optimal coating VLP concentration and conjugate dilution for this assay using the panel tested.
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