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Porcine Cortisol ELISA Kit

Cat.No.:E-EL-0159
Reactivity: Porcine

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Product Details

Properties

Assay type	Competitive-ELISA
Format	96T/48T/24
Assay time	2.0h
Detection range	0.78-50 ng/mL
Sensitivity	0.48 ng/mL
Sample type &Sample volume	serum and plasma; 50μL
Specificity	This kit recognizes Cortisol in samples. No significant cross-reactivity or interference between Cortisol 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 Cortisol concentrations in serum and plasma.

Test Principle

This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with Cortisol. During the reaction, Cortisol in the sample or standard competes with a fixed amount of Cortisol on the solid phase supporter for sites on the Biotinylated Detection Ab specific to Cortisol. 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 Cortisol 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℃, 12 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), 12 months
Reference Standard & Sample Diluent	1 vial, 20 mL	2-8°C, 12 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 Cortisol were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Cortisol 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)	50.32	109.34	275.80	51.24	107.22	279.31
Standard deviation	4.14	8.16	20.74	4.59	8.61	21.17
CV (%)	8.23	7.46	7.52	8.95	8.03	7.58

Recovery

The recovery of Cortisol 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)	81-90	85
EDTA plasma(n=8)	86-97	89
Urine(n=8)	85-100	94

Linearity

Samples were spiked with high concentrations of Porcine Cortisol 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

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