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Universal 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312)

Standard Curve - 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312) - Antibodies.com Linearity - Universal 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312) - Antibodies.com
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$40
Dispatched from St. Louis, MO.
Lead Time: 7-11 business days.
Name
Universal 1,25-Dihydroxy Vitamin D3 ELISA Kit
Description
Universal 1,25-Dihydroxy Vitamin D3 ELISA Kit is a 120 minute competitive Enzyme-Linked Immunosorbent Assay (cELISA) designed for the in vitro quantitative determination of universal 1,25-Dihydroxy Vitamin D3 in serum, plasma, cell culture supernatant, cell or tissue lysate, and other biological fluids.
Assay Type
Competitive (quantitative)
Principle of Assay
Universal 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312) is a 2 hour competitive Enzyme-Linked Immunosorbent Assay (cELISA) designed for the in vitro quantitative determination of universal 1,25-Dihydroxy Vitamin D3 in serum, plasma, cell culture supernatant, cell or tissue lysate, and other biological fluids. A 96-well microtiter plate has been pre-coated with 1,25-Dihydroxy Vitamin D3. During the assay, 1,25-Dihydroxy Vitamin D3 present in the standards or samples competes with immobilized 1,25-Dihydroxy Vitamin D3 for binding to an assay-specific antibody. Following incubation, unbound components are removed by washing and HRP-Streptavidin conjugate is added to the wells. After further incubation and washing, TMB substrate solution is added to visualize the HRP enzymatic reaction. This produces a blue-coloured product that changes to yellow following addition of acidic stop solution. The intensity of the colour is measured at 450 nm using a microplate reader. The concentration of 1,25-Dihydroxy Vitamin D3 in the samples is inversely proportional to the OD450 value and is determined by comparison with the standard curve.
Platform
Pre-coated Microplate (12 x 8 well strips)
Detection Type
Colorimetric Detection (OD450)
Instrument
Colorimetric Microplate Reader
Sample Type
Serum, plasma, cell culture supernatant, cell or tissue lysate, and other biological fluids.
Sensitivity
3.75 pg/mL
Product Range
6.25-400 pg/mL
Assay Time
2 hours
Reactivity
Universal
Cross Reactivity
Specific for 1,25-Dihydroxy Vitamin D3 with no reported cross-reactivity with other analogs or variants.
Recovery
Sample Type n Range Average
Serum 10 88% - 98% 92%
EDTA Plasma 10 89% - 99% 92%
Heparin Plasma 10 88% - 100% 95%
Linearity
Sample Type n 1:2 1:4 1:8
Serum 10 86-100% 92-101% 85-100%
EDTA Plasma 10 85-102% 88-96% 87-100%
Heparin Plasma 10 86-103% 85-100% 86-90%
Storage
Shipped at +4°C. Store kit components at +4°C or -20°C as instructed in the Protocol Manual. Do not use past expiration date! Avoid freeze/thaw cycles.
Product Note
Information online is representative. Always refer to the Product Manual inside the kit.
Disclaimer
This product is for research use only. It is not intended for diagnostic or therapeutic use.
Kit Components
Item Quantity Storage
Pre-Coated 96 Well Microplate 12 x 8 Well Strips +4°C
Lyopholized Standard 2 Vials +4°C
Sample Dilution Buffer 20ml +4°C
Biotinylated Detection Antibody 60µl +4°C
Antibody Dilution Buffer 10ml +4°C
HRP-Streptavidin Conjugate 120µl +4°C
SABC Dilution Buffer 10ml +4°C
TMB Substrate 10ml +4°C
Stop Solution 10ml +4°C
Wash Buffer (25X) 30ml +4°C
Plate Sealers 5 Adhesive Strips -
Foil Pouch 1 Zip-Sealed Pouch -
Required Materials
The following materials are not included in the kit, but are required to run this assay:
  • Microplate reader capable of measuring absorbance at 450nm ± 10nm.
  • Squirt bottle, multichannel pipette reservoir, or automated microplate washer.
  • Graph paper or computer software capable of generating logarithmic functions.
  • Test tubes and Eppendorf tubes to prepare standards and samples.
  • Multi- and single-channel pipettes and sterile pipette tips.
  • Deionized or distilled water.
  • Absorbent paper towels.
  • 37°C incubator.
Important Notes
  • Sample concentrations should be estimated before being used in the assay and a proper dilution factor should be selected to make the diluted target protein concentration fall in the optimal detection range of this kit.
  • Sample matrix components will interfere with the test results. All samples must be diluted at least 1:2 with Sample Dilution Buffer.
  • Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.
  • This ELISA kit is intended for the analysis of biological samples and has not been validated for purified/recombinant proteins, industrial, or commercial materials. Performance with such samples cannot be guaranteed, as differences in expression, sequence, tags, folding, post-translational modifications, purity, activity, or buffer composition may affect assay results. For further guidance, please contact our technical support team.
Bring all reagents and samples to room temperature before use.
We recommend that all standards and samples be assayed in duplicate.
Step 1
Prepare all reagents, samples, and working standards as directed in the previous sections.
Step 2
Determine the number of microplate strips required for the assay and insert them into the plate frame. Unused strips should be stored in the foil pouch at +4°C.
Step 3
Wash the plate two times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time. Note: Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Blot the plate onto paper towels or other absorbent material. Do not let the wells dry completely at any time!
Step 4
Add 50μl of standard solutions to the standard wells. Note: A plate layout is provided to record standards and samples assayed.
Step 5
Add 50μl of samples to the sample wells. Note: All samples must be properly diluted in order to produce sample values within the dynamic range of the assay.
Step 6
Immediately add 50μl of Biotinylated Detection Antibody working solution to each well. Gently tap the plate to ensure thorough mixing for 1 minute.
Step 7
Cover with an adhesive plate sealer provided and incubate at 37°C for 45 minutes.
Step 8
Remove the plate sealer, aspirate the liquid from the plate, and wash the plate three times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time.
Step 9
Add 100μl of HRP-Streptavidin Conjugate (SABC) working solution to each well.
Step 10
Cover with a new adhesive plate sealer and incubate at 37°C for 30 minutes.
Step 11
Remove the plate sealer, aspirate the liquid from the plate, and wash the plate five times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time.
Step 12
Add 90μl of TMB Substrate to each well. Cover with a new adhesive plate sealer and incubate at 37°C in the dark for 10-20 minutes. Note: This incubation time is for reference only, the optimal reaction time should be determined by the end-user and can be shortened or extended according to the actual color change. The reaction may be terminated when a gradient is apparent in the standard wells. Do not exceed 30 minutes!
Step 13
Add 50μl of Stop Solution to each well. The color in the wells should change from blue to yellow immediately. Note: If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
Step 14
Determine the optical density (O.D. value) of each well immediately using a microplate reader set to 450 nm.

  • Scientific Validation DataValidation Data (2)

Standard Curve - 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312) - Antibodies.comEnlarge Image

Standard Curve - Universal 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312)

Representative standard curve for 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312).
Linearity - Universal 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312) - Antibodies.comEnlarge Image

Universal 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312)

Linearity assessment of the Universal 1,25-Dihydroxy Vitamin D3 ELISA Kit (A326312) across serial sample dilutions. Linearity was evaluated using Serum, EDTA Plasma, and Heparin Plasma samples serially diluted from 1:2 to 1:8. Recovery (%) was calculated relative to the expected Universal 1,25-Dihydroxy Vitamin D3 concentration at each dilution. Data are presented as mean recovery values from 10 replicates (n = 10).
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