Document 6526408

Diazyme Laboratories
12889 Gregg Court
Poway, CA 92064, USA
Tel: 858-455-4768 / Fax: 858-455-3701
Email: [email protected]
Website: www.diazyme.com
Assay Principle
25-Hydroxy Vitamin D Assay Kit
Configuration
Diazyme’s 25-Hydroxy Vitamin D Assay Kit is provided in the
following microplate (colorimetric) configuration (96 tests):
Instrument
Microplate
reader
Catalog #
Kit size
EX: Extraction solution (1x 30 mL)
R1a: Lyophilized Reagent (1 vial)
R1: Reconstitution Buffer (1x 8 mL)
R2a: Lyophilized Reagent (1 vial)
R2: Reconstitution Buffer (1x 6 mL)
R3a: Lyophilized Reagent (1 vial)
DZ688A-K R3: Reconstitution Buffer (1x 12.5 mL)
STOP: STOP Reagent (1x 8 mL)
8-well Microtube Strips (12)
Microtube Rack (1)
96-well Microplate (1)
Microplate Sealing Film (3)
*Cal: DZ688A-CAL (6x 1mL)
*Note: Diazyme 25-Hydroxy Vitamin D Assay Calibrator Set is sold separately.
Intended Use
The Diazyme 25-hydroxy Vitamin D Assay is designed for the
quantification of total 25-hydroxy Vitamin D in human serum and
plasma. The assay results are to be used in parallel with other clinical data to assess the Vitamin D status of a patient. For investigational use only in the USA.
Clinical Significance
The assay is based on the principle of α-complementation of the
enzyme β-galactosidase and the competition between an enzyme
donor-25-hydroxy Vitamin D conjugate, Vitamin D binding protein and the 25-hydroxy Vitamin D content of a serum sample.
Samples with higher 25-hydroxy Vitamin D concentrations produce higher β-galactosidase activities and vice versa. Chlorophenol red-β-D- galactopyranoside (CPRG) is used as the enzyme
substrate and the accumulation of the reaction's product (chlorophenol red) is monitored at 560 nm. The 25-hydroxy Vitamin D
concentration of a patient sample is proportional to the measured
β-galactosidase activity. The assay consists of a sample extraction
step during which Vitamin D is irreversibly dissociated from its
serum transporter. Extracted samples are then analyzed on a microplate reader after the sequential addition of three reagents. Unlike most microplate-based assays, the Diazyme assay does not
require tedious washing steps between reagents. The whole assay
procedure takes about 2 hours.
Materials Required but not Provided
1. Six level calibrators (DZ688A-CAL): Human serum containing
specific amounts of 25-hydroxy Vitamin D. Calibrators are
supplied in aliquots of 1.0 mL. The exact concentration (ng/mL)
of each calibrator is indicated on the included certificate of
analysis. 6 x 1 mL vials are provided per kit.
2. Two Level controls (DZ688A-CON): Human serum containing
specific amounts of 25-hydroxy Vitamin D. Controls are supplied in aliquots of 1.0 mL. Control level 1 has a concentration
of 25-OH Vitamin D that corresponds to a deficient patient.
Control level 2 has a concentration of Vitamin D that corresponds to a normal patient. 2 x 1 mL vials are provided per kit.
3. Flat-head Vortex mixer, micro pipettes.
Vitamin D is a steroid hormone involved in the active intestinal
absorption of calcium and in the regulation of its homeostasis.
Vitamin D has two isomers: Vitamin D2 and Vitamin D3. Vitamin
D2 is obtained from dairy products whereas Vitamin D3 is produced in the skin after exposure to ultraviolet light. In the liver,
Vitamin D is hydroxylated at its carbon 25 to form 25-hydroxy
Vitamin D. This metabolite is the predominant circulating form of
Vitamin D and is considered to be an accurate indicator of the
general Vitamin D status of an individual. Vitamin D deficiency
has been linked to many diseases including osteoporosis, rickets,
osteomalacia, cancers, and cardiovascular diseases. Both dietary
supplements of Vitamin D that are currently available in the market (Vitamin D2 and Vitamin D3) are converted to 25-hydroxy
Vitamin D in the liver. The sum of the concentrations of 25hydroxy Vitamin D2 and 25-hydroxy Vitamin D3, in serum or
plasma, is referred to as “Total 25-hydroxy Vitamin D”. Accurate
monitoring of total 25-hydroxy Vitamin D (25-OH) level is critical
in clinical settings. Vitamin D deficient patients who are prescribed a daily Vitamin D supplement should regularly monitor
their serum or plasma Vitamin D levels in order to reach an optimal level and prevent their 25-hydroxy Vitamin D concentrations
from reaching excessive levels that are considered toxic1-5.
Reagent Composition
Extraction solution EX:
Acetonitrile-based extraction solution.
Reagent R1:
(R1a): Lyophilized enzyme acceptor, Vitamin D binding protein and
phosphate salts.
(R1): Reconstitution buffer containing phosphate salts and
preservatives.
Reagent R2:
(R2a): Lyophilized enzyme donor.
(R2): Reconstitution buffer containing phosphate salts and
preservatives.
Reagent R3:
(R3a): Lyophilized substrate (CPRG).
(R3): Reconstitution buffer containing phosphate salts and
preservatives.
STOP reagent:
Sodium carbonate solution.
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Preparation and Storage
Calibrators and Controls
Calibrators and controls are serum-based solutions and stable
when stored at -20°C until the expiration date on the label. If calibrators and controls are stored at 2-8°C, the material should be
used within 11 days. Equilibrate the needed volume of calibrators
and controls to room temperature for 30 minutes before use. Avoid
repeated freeze-thaw cycles.
absence of infectious agents, this material and all patient samples
should be handled as though capable of transmitting infectious
disease and such biohazardous material should be disposed of
according to relevant local, state or federal regulations.
6. Additional safety information concerning storage and handling
of this product is provided within the Material Safety Data Sheet
for this product. To obtain an MSDS, please contact our customer
service department at 858-455-4768 or email [email protected].
Extraction solution (EX)
This reagent is ready to use. It is stable when stored at room temperature until the expiration date on the label. EX buffer contains
an organic solvent, Acetonitrile, and should be handled per MSDS
instructions. Keep bottle tightly closed after use.
Reagents 1, 2, and 3
Reagents are supplied as lyophilized powders, and need to be reconstituted with the provided reconstitution buffers before use.
The lyophilized reagents and their reconstitution buffers are stable
when stored at 2-8°C until the expiration date on the label. The
reconstituted reagents are stable for 11 days at 2-8°C.
Reconstitute R1a, R2a and R3a with the entire contents of the
provided R1, R2 and R3 buffer, respectively. Invert 4-5 times and
incubate at room temperature for 30 minutes. Use immediately or
store in a tightly closed bottle at 2-8°C. Allow cold reagents (28°C) to equilibrate to room temperature before use.
Assay Procedure
Reconstitute and prepare reagents as described in the “Preparation
and Storage” section.
1. Pipette 90 µL of samples, calibrators and controls into the
wells of the provided microtube strips (use the provided microtube
rack).
2. Using a multi-channel pipette, carefully add 180 µL of Extraction solution (EX) to each well containing samples. Make sure
that reagent EX is fully equilibrated to room temperature before
use.
STOP reagent
This reagent is ready to use and should be stored at room temperature upon receipt of the kit. It is stable until the expiration date.
Specimen Collection and Handling
Serum, heparinized plasma or EDTA plasma samples can be used
for the assay. For serum, collect whole blood by venipuncture and
allow clotting. For plasma, mix the sample by gentle inversion
prior to centrifugation. Centrifuge and separate serum or plasma as
soon as possible after collection.
The specimens may be refrigerated at 2-8°C for two weeks. For
long term storage, they can be stored at -20°C. Avoid repeated
freeze-thaw cycles.
Allow the refrigerated or frozen-thawed samples to equilibrate to
room temperature for 30 minutes before use; samples must be
mixed well before analysis.
Precautions
1. DO NOT INGEST. Avoid contact with skin and eyes.
3. Cap each microtube tightly and vortex each strip for 30
seconds. Hold the strip with both hands to make sure that all 8
microtube of the strip are uniformly mixed. Let stand for 10 minutes at room temperature to ensure clear top phase separation.
2. EX buffer contains an organic solvent, Acetonitrile, and should
be handled per MSDS instructions.
3. Reagents contain sodium azide, which may react with lead or
copper plumbing to form explosive compounds. Flush drains with
copious amounts of water when disposing of reagents.
4. Specimens containing human sourced materials should be handled as if potentially infectious, using safe laboratory procedures
such as those outlined in Biosafety in Microbiological and Biomedical Laboratories, 5th Ed (HHS Publication Number [CDC] 211112).
5. Calibrators and controls contain human source material. Each
donor unit of serum in the preparation of these materials were
tested by FDA-approved methods and found negative for the Human Immunodeficiency Virus Antibody (HIV I/II Ab), Hepatitis B
Surface Antigen (HBsAg), and Hepatitis C Virus Antibody
(HCV). Because no method can offer complete assurance as to the
4. Pipette 20 µL of the top phase into the provided 96-well microplate. Make sure that the precipitated proteins are not carried
over.
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deficient: 15.0 to 31.9 ng/mL; optimal: 32.0 to 100.0 ng/mL and
possible toxicity over 100.0 ng/mL1. It is recommended, however,
that each laboratory establishes the range of normal values that
corresponds to the population of their region.
Limitations
1.
2.
For investigational use only in the USA.
The assay is designed for use with human serum and plasma
samples only.
3.
5. Incubate the 96-well microplate on a lab bench, uncovered, for
45 minutes to evaporate the organic solvent (see MSDS).
Samples suspected of containing analyte values greater than
120 ng/mL should be reported as >120 ng/mL.
4.
6. Add 50 µL of Reagent R1 into each well. Pipette-mix 3-4
times. Seal the microplate with the provided film and incubate for
30 minutes at room temperature.
As with any diagnostic test procedure, results should be interpreted considering all other test results and the clinical status
of the patient.
5.
As with any diagnostic test it is possible that technical, procedural errors as well as substances and factors not listed may
interfere with the proper functioning of the test kit.
7. Add 50 µL of Reagent R2 into each well. Pipette-mix 3-4
times. Seal the microplate with the provided film and incubate for
30 minutes at room temperature.
8. Add 100 µL of Reagent R3 into each well. Pipette-mix 3-4
times. Seal the microplate with the provided film and incubate for
20 minutes at room temperature.
9. Add 50 µL of STOP reagent into each well. Pipette-mix 3-4
times and read at 560 nm using a microplate reader. The developed
color is stable for 90 minutes.
Calibration
Six calibration levels are needed for each run. Calibrators and
controls should be treated exactly the same as patient samples. We
recommend fitting the calibration data to the 4-parameter model. A
typical calibration curve is shown below.
Performance Characteristics
Sensitivity
The analytical sensitivity of the assay was found to be 4.2 ng/mL.
Accuracy
The performance of this assay was compared to the performance
of a legally marketed 25-OH Vitamin D enzyme immunoassay.
Using 76 serum samples (with 25-OH Vitamin D concentrations
ranging from 7.4 ng/mL to 90.3 ng/mL), the correlation coefficient
between the two methods was 0.92, the slope was 1.097 and the y
intercept was - 3.89 ng/mL.
Precision
Using seven precision levels covering the dynamic range of the
assay, intra-assay precisions were under 6.5% and total precisions
were under 9.7%.
(OD)
Linearity
The assay was found to be linear between 4.2 and 120.2 ng/mL.
Interference
The assay’s results were not significantly affected by the presence
of bilirubin (up to 40 mg/dL), hemoglobin (up to 250 mg/dL),
ascorbic acid (up to 10 mM) and triglycerides (up to 500 mg/dL).
(ng/mL)
References
Quality control
We recommend that each laboratory uses 25-OH Vitamin D controls to validate the performance of reagents. A bi-level set of controls is available from Diazyme Laboratories (Catalog # DZ688ACON).
Results
Results are expressed in ng/mL. Note: Samples with values greater
than 120 ng/mL should be reported as >120 ng/mL.
1. Baudeoin D. 25-OH Vitamin D and Calcium Reference Ranges
Update. January 2008. The Legacy Health System.
2. Morris H. A. Vitamin D: A Hormone for All Seasons-How Much is
enough? Clin. Biochem. Rev., 2005, 26, 21-32.
3. Bikle D. D. Vitamin D and the skin. J. Bone Miner. Metab., 2010,
28, 117-30.
4. Zerwekh J. E. Blood biomarkers of vitamin D status. Am. J. Clin.
Nutr., 2008, 87, 1087S-91S.
5. Moyad M. A. Vitamin D: a rapid review. Dermatol Nurs., 2009, 21,
25-30.
Reference Range
Although the normal range of 25-hydroxy vitamin D is populationdependent, it has been established through a large scale analysis
(3200 patients) that the following reference ranges should be considered by physicians: severely deficient: 0 to 14.9 ng/mL; mildly
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