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VD3(Vitamin D3) ELISA Kit

Cat.No.:E-EL-0014
Reactivity: Universal

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96T
48T
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96T*5
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Product Details

Properties

Assay type	Competitive-ELISA
Format	96T/48T
Assay time	2.0h
Detection range	1.56-100 ng/mL
Sensitivity	0.94 ng/mL
Sample type &Sample volume	serum, plasma and other biological fluids; 50μL
Specificity	This kit recognizes VD3 in samples. No significant cross-reactivity or interference between VD3 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 VD3 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 VD3. During the reaction, VD3 in the sample or standard competes with a fixed amount of VD3 on the solid phase supporter for sites on the Biotinylated Detection Ab specific to VD3. 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 VD3 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 VD3 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level VD3 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)	4.75	12.74	45.67	4.34	12.36	44.41
Standard deviation	0.29	0.63	2.42	0.27	0.67	2.15
CV (%)	6.11	4.95	5.30	6.22	5.42	4.83

Recovery

The recovery of VD3 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)	94-106	101
EDTA plasma(n=8)	85-97	92
Cell culture media(n=8)	89-100	95

Linearity

Samples were spiked with high concentrations of VD3 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Target Information

Research Area

Signal transduction, Metabolism

Assay Procedures

	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
	2. Aspirate and wash the plate for 3 times
	3. Add 100μL HRP conjugate working solution. Incubate for 30 min at 37°C. Aspirate and wash the plate for 5 times
	4. Add 90μL Substrate Reagent. Incubate for 15 min at 37°C
	5. Add 50μL Stop Solution
	6. Read the plate at 450nm immediately. Calculation of the results

Citations

CALCIFIED TISSUE INTERNATIONAL (2022) IF: 4.333
Lactobacillus rhamnosus GG Promotes Intestinal Vitamin D Absorption by Upregulating Vitamin D Transporters in Senile Osteoporosis

DOI: 10.1007/s00223-022-00975-z

PMID: 35616697
Sample: plasma
JOURNAL OF BONE AND MINERAL METABOLISM (2022) IF: 2.626
Alterations in the composition of the gut microbiota affect absorption of cholecalciferol in severe osteoporosis

DOI: 10.1007/s00774-021-01303-5

PMID: 35103840
Sample: plasma
Archives of Endocrinology Metabolism (2019) IF: 1.571
Is there any association between vitamin D levels and polycystic ovary syndrome (PCOS) phenotypes?

DOI: 10.20945/2359-3997000000177

Sample: serum
Reports of Biochemistry and Molecular Biology (2022)
Correlation of Vitamin D3, PAI-1, and HCG Hormone in Pre- and Post-Menopausal in Babylon Province

DOI: 10.52547/rbmb.11.1.36

Sample: serum

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