General 1, 25-Dihydroxyvitamin D3 (DHVD3) ELISA Kit

SKU: orb1806547

Description

General 1, 25-Dihydroxyvitamin D3 (DHVD3) ELISA Kit

Research Area

Metabolism Research, Musculoskeletal & Connective Tissue Research

Images & Validation

• Application Notes:
  This assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with 1,25-Dihydroxyvitamin D3(DHVD3) protein. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to 1,25-Dihydroxyvitamin D3(DHVD3). Next,Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of 1,25-Dihydroxyvitamin D3(DHVD3) in the samples is then determined by comparing the OD of the samples to the standard curve.

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Key Properties

• Reactivity: All
• Sample Types: serum, plasma and other biological fluids
• Assay Type: Competitive
• Assay Time: 1.5H
• Range: 156.25-10000pg/mL
• Sensitivity: 49.5 pg/mL

Procedure & Performance

• Assay Principle:
  This kit is based on a competitive ELISA detection method. The microtiter plate is pre-coated with target antigen. During the assay, the target antigen in the sample or standard competes with the immobilized target for binding sites on the biotinylated detection antibody specific to the target antigen. After incubation, excess conjugate and unbound sample or standard are removed by washing. HRP–streptavidin (SABC) is then added to each well and incubated, followed by the addition of TMB substrate solution. The enzymatic reaction is terminated by adding an acidic stop solution, and the color change is measured at 450 nm using a microplate reader. The concentration of the target antigen in the samples is determined by comparison with the standard curve and is inversely proportional to the OD450 value.
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• Kit Components:
  1. ELISA Microplate; 2. Standards; 3. Detection Antibody ; 4. HRP-Streptavidin Conjugate; 5. TMB Substrate; 6. Dilution buffers; 7. Stop Solution; 8. Wash Buffer; 9. Plate Sealers; 10. Manual
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• Reagent Preparation:
  1. Wash Buffer: Prepare the 1X Wash Buffer using distilled water according to the manual.; 2. Standard: Perform gradient dilution according to the instructions in the manual. ; 3. Other Concentrated Reagents: Dilute the concentrated reagents using the Dilution Buffers provided in the kit to 1 X working solutions as instructed in the manual. Always use a clean pipette tip for each different solution.
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• Assay Procedure:
  This procedure is for reference only.; ; After the kit equilibrates to room temperature, add standard or sample to the wells and incubate.; Aspirate and wash the plate for three times.; Immediately add Biotinylated Detection Antibody Working Solution to each well and incubate.; Aspirate and wash the plate for three times ; Add Streptavidin-HRP Working Solution to each well and incubate.; Add TMB Substrate Reagent to each well and incubate.; Add Stop Reagent to each well, read the plate at OD 450 nm immediately, and calculate the results.
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• Materials Required:
  1. Microplate readers; 2. Centrifuge; 3. Incubator; 4. Automated plate washer; 5. Single-channel or multi-channel high-precision pipettes; 6. Disposable pipette tips; 7. Sterile tubes; 8. Eppendorf tubes; 9. Absorbent paper; 10. Loading slots
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• Precision:
  Intra-assay Precision (Precision within an assay): CV% ≤ 10% ; Intra-assay precision was evaluated by testing multiple replicates of samples within the same plate.; ; Inter-assay Precision (precision between assays): CV% ≤ 10% ; Inter-assay precision was evaluated by testing samples across different plates.
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• Calculation of Results:
  1. Average the duplicate readings for each Standard, Control, and Sample, and subtract the mean optical density of the zero Standard. ; 2. Construct a standard curve by plotting the target concentration on the y-axis against absorbance on the x-axis and draw a curve through the data points. ; 3. Determine the sample concentration by substituting the OD450 value into the standard curve. For diluted samples, multiply the calculated value by the corresponding dilution factor.
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• Curve Fitting Softwares:
  1. Curve Expert; 2. Thermo SkanIt RE; 3. SciDAVis; 4. LabPlot; 5. ……
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Storage & Handling

• Storage: Refer to the Storage Guidelines in the Manual
• Expiration Date: Please enquire.
• Disclaimer: For research use only

Alternative Names

DH-VD3; Calcitriol; Rocaltrol; Calcijex; 1,25-(OH)2D3; 1,25-Dihydroxycholecalciferol; 1,25-Dihydroxy Vitamin D3

Quick Database Links

Gene Symbol

DHVD3