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VDVitamin D ELISA Kit - E-EL-0012

VDVitamin D ELISA Kit - E-EL-0012

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Product Details

Properties

Assay type Competitive-ELISA
Format 96T/48T
Assay time 2.0h
Detection range 6.25-400 ng/mL
Sensitivity 3.75 ng/mL
Sample type &Sample volume serum, plasma and other biological fluids; 50μL
Specificity This kit recognizes VD in samples. No significant cross-reactivity or interference between VD and analogues was observed.
Reproducibility Both intra-CV and inter-CV are < 10%.
Application This ELISA kit applies to the in vitro quantitative determination of VD concentrations in serum, plasma and other biological fluids.

Test Principle

This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with VD. During the reaction, VD in the sample or standard competes with a fixed amount of VD on the solid phase supporter for sites on the Biotinylated Detection Ab specific to VD. Excess conjugate and unbound sample or standard are washed away, and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns from blue to yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of VD in tested samples can be calculated by comparing the OD of the samples to the standard curve.

Kit components & Storage

An unopened kit can be stored at 2-8℃ for 1 month. If the kit is not supposed to be used within 1 month, store the components separately according to the following conditions once the kit is received.
Item Specifications Storage
Micro ELISA Plate(Dismountable) 96T: 8 wells ×12 strips
48T: 8 wells ×6 strips		 -20℃, 6 months
Reference Standard 96T: 2 vials
48T: 1 vial
Concentrated Biotinylated Detection Ab (100×) 96T: 1 vial, 120 μL
48T: 1 vial, 60 μL
Concentrated HRP Conjugate (100×) 96T: 1 vial, 120 μL
48T: 1 vial, 60 μL		 -20℃(Protect from light), 6 months
Reference Standard & Sample Diluent 1 vial, 20 mL 2-8°C, 6 months
Biotinylated Detection Ab Diluent 1 vial, 14 mL
HRP Conjugate Diluent 1 vial, 14 mL
Concentrated Wash Buffer (25×) 1 vial, 30 mL
Substrate Reagent 1 vial, 10 mL 2-8℃(Protect from light)
Stop Solution 1 vial, 10 mL 2-8℃
Plate Sealer 5 pieces
Manual 1 copy
Certificate of Analysis 1 copy

Technical Data

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level VD were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level VD were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample 1 2 3 1 2 3
n 20 20 20 20 20 20
Mean (ng/mL) 21.62 53.66 157.91 21.41 57.30 152.81
Standard deviation 1.28 2.70 5.98 1.47 2.35 8.34
CV (%) 5.92 5.03 3.79 6.87 4.10 5.46

Recovery

The recovery of VD spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum(n=8) 96-112 102
EDTA plasma(n=8) 88-99 93
Cell culture media(n=8) 85-97 91

Target Information

Research Area Metabolism

Assay Procedures

elisa assay procedure 1

1. Add 50μL standard or sample to the wells, immediately add 50μL Biotinylated Detection Ab working solution to each well. Incubate for 45 min at 37°C
elisa assay procedure 2

2. Aspirate and wash the plate for 3 times
elisa assay procedure 3

3. Add 100μL HRP conjugate working solution. Incubate for 30 min at 37°C. Aspirate and wash the plate for 5 times
elisa assay procedure 4

4. Add 90μL Substrate Reagent. Incubate for 15 min at 37°C
elisa assay procedure 5

5. Add 50μL Stop Solution
elisa assay procedure 6

6. Read the plate at 450nm immediately. Calculation of the results