ELISA/assay

25-OH Vitamin D ELISA Kit

DEIA4458

96T

25-OH Vitamin D ELISA Kit

25-OH Vitamin D

25-OH Vitamin D ELISA Kit

cELISA

25-OH Vitamin D

Human

Quantitative

Serum, Plasma

96T

On arrival, store the 25-OH Vitamin D ELISA assay kit at 2-8 °C. Once opened the kit is stable until its expiry date. Do not use components beyond the expiration date shown on the kit labels.

This 25-OH Vitamin D ELISA assay kit detects 25-OH Vitamin D2 and D3 specifically. Cross reactions with other metabolites are given in the following table.

25-OH Vitamin D

Human

calcifediol; calcidiol; 25-OH Vitamin D; 25-hydroxyvitamin D; 25OHD; 25-OH VitD; 25-hydroxycholecalciferol; 25-hydroxycholecalciferol monohydrate; 25-OH Vitamin D3; 25-OHD3; 25-OH VitD3; 25-hydroxyvitamin D3; 25-OH Vitamin D2

The 25-OH Vitamin D ELISA assay test kit is designed for the determination of 25-OH Vitamin D in human serum or plasma samples.

The 25-OH Vitamin D ELISA assay kit is designed for the serological determination of the vitamin D concentration in the human organism types of vitamin D that are differentiated are vitamin D2 (ergocalciferol) that is contained in plant food (mushrooms, avocado) and vitamin D3 (cholecalciferol) that is produced from 7-dehydrocholesterol in the skin under ultra-violet irradiation or found in animal food or products (sea fish, egg yolk, butter). These two forms of vitamin D, which are not yet biologically active, are bound by a protein called VDBP (vitamin D binding protein) in the bloodstream, then metabolised in the liver and converted into 25-OH vitamin D2 (calcidiol) and 25-OH vitamin D3 (calcitriol), respectively, which are storage forms of the vitamin with little activity. In contrast to other commercially available tests, the 25-OH Vitamin D ELISA assay kit uses a newly designed monoclonal antibody which is specific for both vitamin D2 and vitamin D3 at 100% specificity. This is necessary because sometimes vitamin D2 instead of D3 is used in therapy.

The 25-OH Vitamin D ELISA assay test kit is designed for the determination of 25-OH Vitamin D in human serum or plasma samples. In the first analysis step, the calibrators and patient samples are diluted with biotin-labelled 25-OH vitamin D and added to microplate wells coated with monoclonal anti-25-OH Vitamin D antibodies. During the incubation an unknown amount of 25-OH Vitamin D in the patient sample and a known amount of biotin-labelled 25-OH vitamin D compete for the antibody binding sites in the microplate wells plate. Unbound 25-OH vitamin D is removed by washing. For the detection of bound biotin-labelled 25-OH vitamin D, a second incubation is performed using peroxidase-labelled streptavidin. In a third incubation using the peroxidase substrate tetramethylbenzidine (TMB) the bound peroxidase promotes a colour reaction. The colour intensity is inversely proportional to the 25-OH vitamin D concentration.

1. MT-Strips, 12 strips 8 wells per strip, single break apart, coated with monoclonal antibody (vitamin D2 and D3) 2. Standards 1-6, 6 vials 1 ml each, colored red-brown; To be diluted 1:26 in working strength biotin. 3. Control 1+2, 2 vials 1 ml each, colored red-brown, To be diluted 1:26 in working strength biotin. values for the Control are given on the vial label 4. Biotin Biotin, 1.2 ml, 100 × concentrated; colored blue 1 vial 5. Sample Buffer, 1 vial 100 ml, colored yellow 6. Enzyme Conjugate, 1 vial 12 ml, colored blue, ready for use 7. Substrate, 1 vial 12 ml tetramethyl benzidine (TMB)/ H 2 O 2 , ready for use 8. Wash Buffer, 1 vial 100 ml, 10 × concentrated 9. Stop Solution, 1 vial 12 ml, ready for use, 0.5M sulphuric acid

1. Pipettes for 10 μl, 100 μl, 200 μl, 1 ml, and 4.5 ml 2. Pure water 3. Microtiter plate reader (450 nm/ 620 nm)

Sample material: Human serum or EDTA, heparin or citrate plasma. Stability: Patient samples to be investigated can generally be stored at 2°C to 8°C for up to 14 days. Severely haemolytic or lipemic serum samples should not be used. Please note: Diluted samples should only be used for one test run and subsequently discarded. Always use fresh samples and calibrator dilutions for every test run. Always pipette samples and calibrators first, then add the working strength biotin within 5 minutes to the dilution tubes, particularly if the test is performed manually or if large sample series (> 20 samples) are analysed in order to avoid any drift effects. Performance: The calibrators/controls and patient samples for analysis are diluted 1:26 in working strength biotin. Example: a. Add 20 μl of sample (calibrators, controls, samples) to suitable dilution tubes b. Add 0.5 ml diluted biotin to all tubes within 5 minutes and mix thoroughly (vortex). c. Incubate the mixture for at least 10 minutes at room temperature (+18°C to +25°C). d. The samples can subsequently be pipetted into the reagent wells according to the pipetting scheme.

Note: All reagents of the 25-OH Vitamin D ELISA assay kit must be brought to room temperature (+18°C to +25°C) approx. 30 minutes before use. After first use, the reagents are stable until the indicated expiry date if stored at +2°C to +8°C and protected from contamination, unless stated otherwise below. Coated wells: Ready for use. Tear open the resealable protective wrapping of the microplate at the recesses above the grip seam. Do not open until the microplate has reached room temperature to prevent the individual strips from moistening. Immediately replace the remaining wells of a partly used microplate in the protective wrapping and tightly seal with the integrated grip seam (Do not remove the desiccant bag). Once the protective wrapping has been opened for the first time, the wells coated with antigens can be stored in a dry place and at a temperature between +2°C and +8°C for 4 months. Calibrators and controls: The reagents must be mixed thoroughly before use. Calibrators and controls are to be diluted 1:26 in working strength biotin prior to use, see point 5 on ASSAY PROCEDURE. Biotin: The biotin is a 100× concentrate. Mix thoroughly before diluting. The required volume should be removed with a clean pipette tip and diluted in sample buffer (1 part biotin plus 99 parts sample buffer). Example: 1 ml biotin concentrate plus 99 ml sample buffer. The workingstrength biotin is stable for 2 weeks when stored at +2°C to +8°C. For longer storage freeze at -20 °C. Sample buffer: It can be used for sample dilution after adding the biotin concentrate. Enzyme conjugate: Ready for use. The enzyme conjugate must be mixed thoroughly before use. Wash buffer: The wash buffer is a 10× concentrate. If crystallization occurs in the concentrated buffer, warm it to 37°C and mix well before diluting. The quantity required should be removed from the bottle using a clean pipette and diluted with deionized or distilled water (1 part reagent plus 9 parts distilled water). The working-strength wash buffer is stable until the expiry date when stored at +2 °C to +8 °C and handled properly. Chromogen/substrate solution: Ready for use. Close the bottle immediately after use, as the contents are sensitive to light. The chromogen/substrate solution must be clear on use. Do not use the solution if it is blue coloured. Stop solution: Ready for use. Storage and stability: The 25-OH Vitamin D ELISA assay kit has to be stored at a temperature between +2°C to +8°C. Do not freeze. Unopened, all test kit components are stable until the indicated expiry date. Waste disposal: Patient samples, calibrators, controls and incubated microplate strips should be handled as infectious waste. All reagents must be disposed of in accordance with local disposal regulations. Warning: The controls and calibrators contain serum of animal origin. Therefore, all materials should be treated as being a potential infection hazard and should be handled with care. Some of the reagents contain the toxic agent sodium azide. Avoid skin contact!

Calculate the number of individual ELISA plate wells needed for the assay. Allow all the reagents supplied, including the appropriate number of packets of strips to reach room temperature (at least 30 min), remove the number of strip wells required and fit them firmly into the frame provided. Controls must always be included in each assay run. 1. Pipette each 200 μl of sample diluted in biotin/sample buffer into each well to be used in the assay. 2. Pipette each 200 μl of Standards 1 - 6, Control1 and Control2 into the appropriate wells. 3. Incubate at room temperature ((+18°C to +25°C) for 2 hours. 4. After the 2-hour incubation, aspirate or discard the samples from the wells, add 300 μl of Wash Buffer and aspirate or discard again. Repeat washing with each 300 μl Wash Buffer two more times for a total of three washings. Tap the inverted wells gently on a clean dry absorbent surface to remove any droplets of Wash Buffer. 5. Pipette 100 μl of Enzyme Conjugate into each well and incubate for 30 min at room temperature (+18°C to +25°C). 6. After the 30 minute incubation, aspirate or discard the reagent from the wells, add 300 μl of Wash Buffer and aspirate or discard again. Repeat washing with each 300 μl Wash Buffer two more times for a total of three washings. Tap the inverted wells gently on a clean dry absorbent surface to remove any droplets of Wash Buffer. 7. Pipette 100 μl of diluted chromogen/ substrate solution into each well and incubate for 15 minutes at room temperature without shaking (protect from direct sunlight!). 8. Stop the substrate reaction by addition of 100 μl of Stop Solution to each well (this will cause the blue colour to turn yellow). 9. Photometric measurement of the colour intensity should be made at a wavelength of 450 nm and a reference wavelength between 620 nm and 650 nm within 30 minutes of adding the stop solution. Prior to measuring, slightly shake the microplate to ensure a homogeneous distribution of the solution.

Quantitative: The standard curve from which the 25-OH vitamin D concentrations in the serum samples can be taken is obtained by point-to-point plotting of the extinction values measured for the 6 calibration sera against the corresponding units (linear/log). Use "cubic spline" or "4-PL" plotting for calculation of the standard curve by computer. For correct logarithmic representation it might be necessary to set the concentration of calibrator 1 from 0 to e.g. 0.1 ng/ml. The following plot is an example of a typical calibration curve. Please do not use this curve for the determination of concentrations in samples. 25-OH Vitamin D3 (ng/mL) x 2.5 = 25-OH Vitamin D3 (nmol/L)

If the extinction of a sample lies below the value of calibrator 6 (120 ng/ml), the result should be given as ">120 ng/ml". It is recommended that the sample be re-tested at a dilution of 1:2 with calibrator 1 before following the test instructions. The result in ng/ml read from the calibration curve for this sample must then be multiplied by a factor of 2. For duplicate determinations the mean of the two values should be taken. If the two values deviate substantially from one another the sample should be retested.

359 plasma samples from apparently healthy blood donors in the age range of 13 to 99 years old were investigated using the ELISA. The mean 25-OH vitamin D concentration was 20.9 ng/ml with a 5-95% percentile range of 8.2 to 37.4 ng/ml.

Calibration As there is no international standard for 25-OH Vitamin D, the standards and controls are calibrated gravimetrically using UV-Vis (264nm) verified stock standards and compared with NIST standards (National Institute of Standards and Technology, USA), DEQAS (Vitamin D External Quality Assessment Scheme, UK) quality assessment data and in-house quality control sera. For every group of tests performed, the values of the concentrations must lie within the limits stated for the relevant test kit lot. A quality control certificate containing these reference values is included. If the values specified for the controls are not achieved, the test results may be inaccurate and the test should be repeated. Antibodies The reagent wells of the 25-OH Vitamin D ELISA assay kit are coated with monoclonal antibodies which identify specifically 25-OH vitamin D3 and 25-OH vitamin D2.

The reproducibility of the 25-OH Vitamin D ELISA assay kit was investigated by determining the intra- and interassay coefficients of variation using 3 sera from different areas of the calibration curve. The intra-assay CVs are based on 40 measurements for each serum and the inter-assay CVs on four measurements performed in six different test runs.

The lower detection limit of the 25-OH Vitamin D ELISA assay kit is defined as the mean value of an analyte-free sample minus three times the standard deviation and is the smallest detectable 25-OH Vitamin D concentration. The detection limit of 25-OH Vitamin D ELISA is 1.6 ng/ml.

The linearity of the 25-OH Vitamin D ELISA assay kit was investigated by diluting three samples with calibrator 1 and determining the concordance. The average concordance amounted to 98% (85-117%).

Haemolytic, lipaemic and icteric samples showed no influence at the result up to a concentration of 5 mg/ml for hemoglobin, 5 mg/ml for triglycerides and 0.2 mg/ml for bilirubin in this ELISA.

The 25-OH Vitamin D ELISA assay test kit is designed for the determination of 25-OH Vitamin D in human serum or plasma samples.

1. MT-Strips, 12 strips 8 wells per strip, single break apart, coated with monoclonal antibody (vitamin D2 and D3) 2. Standards 1-6, 6 vials 1 ml each, colored red-brown; To be diluted 1:26 in working strength biotin. 3. Control 1+2, 2 vials 1 ml each, colored red-brown, To be diluted 1:26 in working strength biotin. values for the Control are given on the vial label 4. Biotin Biotin, 1.2 ml, 100 × concentrated; colored blue 1 vial 5. Sample Buffer, 1 vial 100 ml, colored yellow 6. Enzyme Conjugate, 1 vial 12 ml, colored blue, ready for use 7. Substrate, 1 vial 12 ml tetramethyl benzidine (TMB)/ H 2 O 2 , ready for use 8. Wash Buffer, 1 vial 100 ml, 10 × concentrated 9. Stop Solution, 1 vial 12 ml, ready for use, 0.5M sulphuric acid

If the extinction of a sample lies below the value of calibrator 6 (120 ng/ml), the result should be given as ">120 ng/ml". It is recommended that the sample be re-tested at a dilution of 1:2 with calibrator 1 before following the test instructions. The result in ng/ml read from the calibration curve for this sample must then be multiplied by a factor of 2. For duplicate determinations the mean of the two values should be taken. If the two values deviate substantially from one another the sample should be retested.

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet