25OH VITAMIN D TOTAL ELISA TEST
Device Facts
| Record ID | K123364 |
|---|---|
| Device Name | 25OH VITAMIN D TOTAL ELISA TEST |
| Applicant | Diasource Immunoassays, S.A. |
| Product Code | MRG · Clinical Chemistry |
| Decision Date | Jul 15, 2013 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 862.1825 |
| Device Class | Class 2 |
Indications for Use
The DIAsource 25 OH Vitamin D Total ELISA is intended for the quantitative measurement of 25hydroxy vitamin D2 and D3 (25 OH-D2 and 25 OH-D3) in human serum. The results are to be used in conjunction with other clinical and laboratory findings to assess the Vitamin D status of a patient.
Device Story
The DIAsource 25OH Vitamin D Total ELISA is an enzyme-linked immunosorbent assay (ELISA) for human serum. The assay dissociates 25OH Vitamin D from binding proteins; the analyte competes with biotin-labeled 25OH Vitamin D for binding sites on a monoclonal antibody-coated microtiter plate. Horseradish peroxidase (HRP) is used for detection. After incubation and washing, a chromogenic substrate (TMB) is added; the resulting color change is measured colorimetrically. Absorbance is inversely proportional to the total 25OH Vitamin D concentration. The test is performed in a clinical laboratory setting by trained technicians. Results are interpreted by clinicians alongside other patient data to assess Vitamin D status (deficient, insufficient, sufficient, or potential toxicity).
Clinical Evidence
Clinical method comparison study conducted at three sites with 356 serum samples comparing the subject device to the predicate (K021163). Results showed a correlation coefficient of 0.917 (95% CI: 87.6%–93.6%), slope of 0.954, and y-intercept of 3.05. Reference range study performed on 150 healthy individuals (ages 21-90) across three US regions.
Technological Characteristics
Competitive ELISA; 96-well microtiter plate coated with mouse monoclonal anti-25OH Vitamin D2 and D3; HRP-conjugated detection; TMB chromogenic substrate; stop solution (1.5N HCl). Standards: CLSI EP6-A (linearity), CLSI EP17-A (LoD/LoQ). Requires ELISA plate reader at 450nm. Reagents include lyophilized calibrators and controls.
Indications for Use
Indicated for quantitative measurement of 25-hydroxy vitamin D2 and D3 in human serum to assess Vitamin D status in adult populations. Performance not established in pediatric populations.
Regulatory Classification
Identification
A vitamin D test system is a device intended for use in clinical laboratories for the quantitative determination of 25-hydroxyvitamin D (25-OH-D) and other hydroxylated metabolites of vitamin D in serum or plasma to be used in the assessment of vitamin D sufficiency.
Special Controls
*Classification.* Class II (special controls). Vitamin D test systems must comply with the following special controls:(1) Labeling in conformance with 21 CFR 809.10 and (2) Compliance with existing standards of the National Committee on Clinical Laboratory Standards.
Predicate Devices
- Immuno diagnostic systems 25-Hydroxy Vitamin D EIA (K021163)
Related Devices
- K122420 — DIAZYME 25-HYDROXY VITAMIN D EIA KIT DIAZYME 25-HYDROXY VITAMIN D EIA CONTROL SET DIA · Diazyme Laboratories · Nov 28, 2012
- K123660 — 25-OH VITAMIN D ELISA · Euroimmun US · Jul 17, 2013
- K142351 — 25-Hydroxy Vitamin Ds EIA · Immunodiagnostic Systems , Ltd. · Aug 25, 2015
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE
A. 510(k) Number:
k123364
B. Purpose for Submission:
New device
C. Measurand:
25-hydroxy Vitamin D
D. Type of Test:
Quantitative, competitive enzyme immunoassay
E. Applicant:
DIAsource ImmunoAssays
F. Proprietary and Established Names:
25OH Vitamin D Total ELISA Test
G. Regulatory Information:
| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| MRG | Class II | 21 CFR 862.1825 – Vitamin D Test System | 75 |
H. Intended Use:
1. Intended use(s):
See Indications for use below
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2. Indication(s) for use:
The DIA source 25 OH Vitamin D Total ELISA Test is intended for the quantitative measurement of 25-hydroxy vitamin D2 and D3 (25 OH-D2 and 25 OH-D3) in human serum. The results are to be used in conjunction with other clinical and laboratory findings to assess the Vitamin D status of a patient.
3. Special conditions for use statement(s):
- For prescription use only
4. Special instrument requirements:
ELISA plate reader at 450nm.
I. Device Description:
The kit contains the following:
Microtiter-Plate consisting of 96 wells with Monoclonal anti-25OH Vitamin D₂ and D₃
Calibrator 0, 1 vial lyophilized, biological matrix (human plasma) with gentamycin and proclin
Calibrators 1-5, 1 vial each lyophilized with horse serum with gentamycin and proclin
Controls 1-2, 1 vial each lyophilized with human serum and proclin
Incubation Buffer, 20 ml, ready to use
Conjugate Concentrate 100x, 0.4ml. 25OH Vitamin D Concentrated Conjugate
HRP Conjugate 200x, 0.2ml. Concentrated HRP
Conjugate Buffer, 30ml. ready to use.
Wash Solution Concentrate 200x, 10ml
Chromogenic solution TMB, 12ml
Stop Solution, 12 ml, contains 1.5N HCl.
The human blood components in the kit have been tested by FDA-approved methods
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and found negative for the presence of HBsAg, anti-HIV-1/2 and anti-HCV.
# J. Substantial Equivalence Information:
1. Predicate device name(s):
IDS 25-Hydroxy Vitamin D EIA
2. Predicate K number(s):
k021163
3. Comparison with predicate
| IDS 25OH Vitamin D ELISA ( Predicate) | The DIAsource 25OH Vitamin D Total ELISA ( Candidate) |
| --- | --- |
| Intended Use - For the quantitative determination of 25-hydroxyvitamin D (25-OH D) in human serum. The results are to be used in conjunction with other clinical and laboratory data to assist the clinician in the assessment of Vitamin D sufficiency in adult populations. | Same |
| Platform - ELISA plate read on a plate reader | Same |
| Microtiter Plate - 96 wells coated with sheep polyclonal anti 25OH D | Microtiter Plate - 96 wells coated with mouse monoclonal anti 25OH D2 and D3 |
| Wash Solution Concentrate - 20x | Wash Solution Concentrate - 200x |
| Incubation Buffer - Proprietary buffer for dissociating vitamin D | Same |
| Conjugate Concentrate - None | 100x |
| HRP Concentrate -Ready to Use | HRP Conjugate - 200x |
| Conjugate Buffer - None | Use to dilute Conjugate and HRP Concentrate |
| Substrate - Tetramethylbenzidine (TMB) | Same |
| Stop Solution - Acid mixture | Same |
| Calibrators 0-6 Ready to Use | Calibrators 0-5 - Lyophilized |
| Controls 1-2 - Lyophilized | Same |
| Interpretation of Results - Standard Curve | Same |
| Expected Values: Deficient <10 ng/ml; Insufficient 10-29 ng/ml; Sufficient 20-100 ng/ml; Potential Toxicity >100ng/ml | Same |
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K. Standard/Guidance Document Referenced (if applicable):
- CLSI EP6-A, Evaluation of the Linearity of Quantitative Measurement Procedures
- CLSI EP17-A, Protocols for Determination of Limits of Detection and Limits of Quantitation.
L. Test Principle:
The DIAsource 25 OH Vitamin D Total ELISA Test is an enzyme linked immunosorbent assay to detect total 25OH Vitamin D (D2 and D3) present in human serum. During the first incubation at room temperature, 25OH Vitamin D is dissociated from binding serum proteins to fix on binding sites of a specific monoclonal antibody. After washing, a fixed amount of 25OH Vitamin D-labeled with biotin in presence of horseradish peroxidase (HRP) compete with unlabeled 25OH Vitamin D2 and 25OH vitamin D3 present on the binding sites of the specific monoclonal antibody. After another incubation at room temperature, the microtiterplate is washed to stop the competition reaction. A chromogenic solution (TMB) is added and then stopped with a Stop Solution after the last incubation period. The amount of substrate turnover is determined colorimetrically by measuring the absorbance which is inversely proportional to the total 25OH Vitamin D concentration.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
The inter-assay and intra-assay precision were performed in two separate studies to cover the entire claimed range. Precision was calculated by running four serum samples in singlicate at the given n value for 20 days using 3 different lots. The results are summarized in the table below:
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| Intra-Assay | | | | | Inter-Assay | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | N | <X> ± SD (ng/ml) | CV (%) | | Sample | N | <X> ± SD (ng/ml) | CV (%) |
| A | 24 | 5.6 ± 0.4 | 7.8 | | A | 42 | 17.7 ± 1.3 | 7.4 |
| B | 35 | 27.4 ± 1.5 | 5.5 | | B | 10 | 26.3 ± 1.3 | 4.9 |
| C | 35 | 43.0 ± 1.2 | 2.7 | | C | 10 | 42.0 ± 1.9 | 4.5 |
| D | 24 | 81.2 ± 2.0 | 2.5 | | D | 42 | 85.4 ± 4.0 | 9.4 |
The reproducibility of the assay was done by testing 60 serum samples each at 3 concentrations in duplicate for five days, twice a day, at three sites with two technicians per site. The mean results are summarized in the table below:
| Sample | n | ng/ml | | Within-Run | Between-Run | Between-Day | Between-Tech | Between-Site | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 60 | 25.5 | SD | 0.217 | 0.611 | 0.975 | 1.537 | 2.206 | 2.593 |
| | | | CV | 0.3% | 0.9% | 3.8% | 6.0% | 8.7% | 10.2% |
| 2 | 60 | 52.9 | SD | 0.638 | 1.571 | 1.108 | 2.285 | 4.310 | 5.192 |
| | | | CV | 0.9% | 2.3% | 2.1% | 4.3% | 8.2% | 9.8% |
| 3 | 60 | 124.8 | SD | 1.00 | 1.735 | 1.834 | 3.391 | 4.906 | 6.190 |
| | | | CV | 1.4% | 2.5% | 1.5% | 2.7% | 3.9% | 5.0% |
b. Linearity/assay reportable range:
Two samples with known concentrations (96.7 and 122.9 ng/mL) were tested at equidistant dilutions to determine the linear range of the assay. A linear regression analysis was performed. The data showed the following linear regression: Y = 1.005X + 0.435, r = 0.99.
The sponsor claimed that the assay range is 7.7 to 122.9 ng/dL.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Traceability and value assignment of calibrator:
The master calibrator stock is an ethanolic solution prepared in house by weighing 25OH Vitamin D3. The value assignment for the calibrator stock is made by UV absorbance at 254 nm using a molar extinction coefficient of 18000 L mol⁻¹ cm⁻¹. The stock solution of 25OH Vitamin D is then used to make calibrators by diluting into horse serum. Calibrator values for all six levels have been assigned by using native serum samples that have been
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assayed by LC/MS-MS. The LC/MS-MS method has been validated against a reference method.
Value assignment for 2 levels of controls:
The mean values of multiple replicates obtained from 10 independent runs were assigned as the control target values. The controls target ranges were assigned using the mean with specified precision values.
Stability:
Open Vial Stability – Calibrators and Controls are provided in lyophilized form. Once reconstituted with DI water, they were stored at 4°C for 7 days, and frozen at -20°C for one month, two months and three months with one thawing. The study protocol and acceptance criteria have been reviewed and found to be acceptable. The sponsor claims that the calibrators and controls can be stored for 7 days at 4°C or up to three months at -20°C.
Closed vial stability and kit stability:
The shelf life studies were based on accelerated studies that supported the sponsor’s claim of is 18 months at 4°C. Real time studies are ongoing.
d. Detection limit:
Limit of Detection:
The Limit of Blank (LoB), Limit of Detection (LoD), and the Limit of Quantitation (LoQ), were determined in accordance with the CLSI guideline EP17-A.
The LoB was calculated by measuring the blank several times and calculating the 95th percentile of the distribution of the test values. The LoB was calculated to be 1.69 ng/ml.
The LoD was calculated by using the formula LoD = LoB + 1.65 x SDs (from the CLSI EP17-A) where SDs is the standard deviation of a low value serum tested 50 times. The LoD was calculated to be 2.81 ng/ml.
For LoQ determination, 5 samples with concentrations above the LoD were tested 10 times. The LoQ was calculated to be 4.32 ng/mL based on an inter-assay precision of 20% CV.
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e. Analytical specificity:
Interfering Substances
The effect of potential interfering substances on samples using the DIAsource 25 OH Vitamin D Total ELISA test was evaluated. Different levels of Hemoglobin, Bilirubin, Triglyceride, Vitamin C, Bilirubin Conjugate and Unconjugated and Zemplar in serum samples were tested on samples with different 25OH Vitamin D Concentration. The sponsor defined the acceptance criteria of < 10% difference to be non significant. The labeling states that hemolyzed samples should not be used.
Results are summarized in the table below:
| Substance | 25OH Vitamin D Concentration (ng/ml) | Concentration of Interferent (mg/dL) | Mean Percent Variation |
| --- | --- | --- | --- |
| Hemoglobin | 7.6 | 250 | -0.5% |
| | | 500 | |
| | 29.3 | 250 | |
| | | 500 | |
| | 42.5 | 250 | |
| | | 500 | |
| Bilirubin Conjugated | 6.0 | 50 | -3.5% |
| | | 100 | |
| | 21.5 | 50 | |
| | | 100 | |
| | 38.6 | 50 | |
| | | 100 | |
| Bilirubin Unconjugated | 7.6 | 50 | 2.5% |
| | | 100 | |
| | 29.3 | 50 | |
| | | 100 | |
| | 42.5 | 50 | |
| | | 100 | |
| Triglyceride | 7.6 | 7.5 | -4.3% |
| | | 125 | |
| | | 250 | |
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| | | 500 | |
| --- | --- | --- | --- |
| | 29.3 | 7.5 | |
| | | 125 | |
| | | 250 | |
| | | 500 | |
| | 42.5 | 7.5 | |
| | | 125 | |
| | | 250 | |
| | | 500 | |
| Vitamin C | 6.0 | 1 | 4.5% |
| | | 10 | |
| | | 100 | |
| | 21.5 | 1 | |
| | | 10 | |
| | | 100 | |
| | 38.6 | 1 | |
| | | 10 | |
| | | 100 | |
| Biotin | 8.7 | 0.2 | 4.6% |
| | | 2 | |
| | | 4 | |
| | 19.8 | 0.2 | |
| | | 2 | |
| | | 4 | |
| | 36.1 | 0.2 | |
| | | 2 | |
| | | 4 | |
| Zemplar | 17.6 | 1.25x10-3 | -4.3% |
| | | 2.5x10-3 | |
| | | 5.0x10-3 | |
| | 33.5 | 1.25x10-3 | |
| | | 2.5x10-3 | |
| | | 5.0x10-3 | |
Cross Reactivity
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Cross reactivity of the 25OH Vitamin D Total ELISA assay was determined by testing sera with spiked and unspiked cross reactants. The results are summarized in the following table:
| Compound and Concentration | Spiked Vitamin D (ng/ml) | Unspiked Vitamin D (ng/ml) | % Cross Reaction |
| --- | --- | --- | --- |
| 1,25(OH)_{2}-Vitamin D_{3} at 200 ng/mL | 57.3 | 16.7 | 20.3 |
| 1,25(OH)_{2}-Vitamin D_{2} at 690 ng/mL | 29.9 | 16.7 | 1.9 |
| Vitamin D_{3} at 200 ng/mL | 22.5 | 16.7 | 2.9 |
| Vitamin D_{2} at 200 ng/mL | 19.3 | 16.7 | 1.3 |
| 24,25(OH)_{2}-Vitamin D_{3} at 20 ng/mL | 87.9 | 16.7 | >100 |
| 25,26(OH)_{2}-Vitamin D_{3} at 4 ng/mL | 31.1 | 16.7 | >100 |
| 3-epi-25 hydroxy Vitamin D_{3} at20 μg/mL | 31.58 | 16.7 | 0.07 |
| 25 OH Vitamin D_{3} at 10 ng/mL | 26.7 | 16.7 | 100 |
| 25 OH Vitamin D_{2} at 10 ng/mL | 25.0 | 16.7 | 83 |
f. Assay cut-off:
Not applicable
2. Comparison studies:
a. Method comparison with predicate device:
The performance of the DIAsource 25OH Vitamin D Total ELISA test was determined by conducting a correlation study tested at three different sites using a total of 359 serum patient samples. The samples were tested on both the DIAsource 25OH Vitamin D Total ELISA test and a commercially available 25OH Vitamin D ELISA test manufactured by ImmunoDiagnostics System (IDS). The results ranged from 8.0 ng/mL to 123.0 ng/mL,
The correlation coefficient between the two methods was 0.917, with the 95% confidence interval of 87.3% to 93.6%, slope of 0.954 and y-intercept of 3.05.
b. Matrix comparison:
Not applicable, serum is the only sample type indicated
3. Clinical studies:
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a. Clinical Sensitivity:
Not applicable
b. Clinical specificity:
Not applicable
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable
4. Clinical cut-off:
Not applicable
5. Expected values/Reference range:
Reference Range:
To determine the reference range, the total 25OH Vitamin D of 150 apparently healthy individuals were measured. The individual patient serum samples used were obtained from a certified commercial source (Dx Biosamples, San Diego, CA.) and were collected from an FDA Licensed Donor Center with informed consent. 50 samples were from Northern US (Pennsylvania), 50 samples were from Central US (Tennessee), and 50 samples were from Southern US (Florida).
All samples met the following criteria:
Age between 21-90 years old
Subjects from three different geographical locations
Samples collected in the Winter season (January, February, and March)
Subjects were not taking any vitamin D supplements
Subjects were of different skin tones
Subjects had no family history of parathyroid, or calcium regulatory disease
Subjects had no history or Kidney, Liver, Parathyroid, Calcium related disease or bariatric surgery.
Subjects were not taking any medications known to affect absorption or catabolism of Vitamin D.
The following results were obtained:
Ages between 20-62 years old.
Population consisted of 75 light skin (50%) and 75 dark skin (50%).
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No subjects were taking vitamin D supplements.
No subjects had a family history of parathyroid, or calcium regulatory disease.
No subjects had a history of Kidney, Liver, Parathyroid, Calcium related disease or bariatric surgery.
No subjects were taking any medications known to affect absorption or catabolism of Vitamin D.
| | Florida | Tennessee | Pennsylvania | Overall |
| --- | --- | --- | --- | --- |
| Highest Conc. (ng/mL) | 88.6 | 71.7 | 54.6 | 88.6 |
| Lowest Conc. (ng/mL) | 6.1 | 4.9 | 5.9 | 4.9 |
| Median Conc. (ng/mL) | 20.8 | 15.9 | 14.3 | 17.2 |
Central 95% (2.5% - 97.5%) of the results observed were used. The labeling states that the performance of this assay has not been established in a pediatric population.
N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
O. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
