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ELISAGenie/Human IFN- beta(Interferon Beta)ELISA Kit (HUES01371)/Interferon Beta)ELISA Kit (HUES01371/HUES01371

Description
Additional Information

Description

Human IFN- beta(Interferon Beta)ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human IFN- beta . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human IFN- beta and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human IFN- beta, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human IFN- beta. The concentration of Human IFN- beta in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay type	Sandwich
Format	96T
Assay time	4.5h
Reactivity	Human
Detection Method	Colormetric
Detection Range	31.25-2000 pg/mL
Sensitivity	18.75 pg/mL
Sample Volume Required Per Well	100uL
Sample Type	Serum, plasma and other biological fluids

Specificity

This kit recognizes Human IFN- beta in samples. No significant cross-reactivity or interference between Human IFN- beta and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration(pg/mL)	O.D	Average	Corrected
2000	2.512.522	2.516	2.439
1000	1.6711.725	1.698	1.621
500	0.9780.948	0.963	0.886
250	0.4610.473	0.467	0.39
125	0.2810.263	0.272	0.195
62.5	0.1830.173	0.178	0.101
31.25	0.120.138	0.129	0.052
0	0.070.084	0.077	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human IFN- beta were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human IFN- beta 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 (pg/mL)	105.20	289.60	951.90	107.40	261.00	1001.00
Standard deviation	5.90	14.20	40.00	5.40	11.70	54.10
C V (%)	5.61	4.90	4.20	5.03	4.48	5.40

Recovery

The recovery of Human IFN- beta 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=5)	88-102	93
EDTA plasma (n=5)	92-106	98
Cell culture media (n=5)	86-98	93

Linearity

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

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	94-107	91-106	88-99
1:2	Average (%)	101	97	94
1:4	Range (%)	100-117	84-99	96-112
1:4	Average (%)	107	90	103
1:8	Range (%)	98-113	80-96	95-110
1:8	Average (%)	104	87	102
1:16	Range (%)	98-116	82-92	96-110
1:16	Average (%)	106	87	103

Kit Components & Storage

An unopened kit can be stored at 4"C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro ELISA Plate(Dismountable)	8 wells X12 strips	-20"C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100X)	1 vial, 120 uL
Concentrated HRP Conjugate (100X)	1 vial, 120 uL	-20"C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4"C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25X)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	4"C(shading light)
Stop Solution	1 vial, 10 mL	4"C
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Human IFN- beta(Interferon Beta)ELISA Kit (HUES01371) Assay procedure

1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.

2. Aliquot 100µl of standard solutions into the standard wells.

3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.

4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.

5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.

6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.

7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.

8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.

9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.

10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.

11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.

12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human IFN- beta(Interferon Beta)ELISA Kit (HUES01371) Protein Information

UniProt Protein Function:	IFNB1: Has antiviral, antibacterial and anticancer activities. Belongs to the alpha/beta interferon family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 9p21 Cellular Component: extracellular space; extracellular region Molecular Function:interferon-alpha/beta receptor binding; cytokine activity Biological Process: B cell proliferation; adaptive immune response; cytokine and chemokine mediated signaling pathway; response to virus; regulation of MHC class I biosynthetic process; natural killer cell activation; negative regulation of T cell differentiation; humoral immune response; T cell activation during immune response; response to exogenous dsRNA; cell surface receptor linked signal transduction; negative regulation of viral genome replication; B cell differentiation; positive regulation of innate immune response; defense response to bacterium; natural killer cell activation during immune response; innate immune response; positive regulation of transcription from RNA polymerase II promoter; blood coagulation; defense response to virus; B cell activation during immune response; positive regulation of peptidyl-serine phosphorylation of STAT protein
UniProt Code:	P01574
NCBI GenInfo Identifier:	124469
NCBI Gene ID:	3456
NCBI Accession:	P01574.1
UniProt Secondary Accession:	P01574,Q5VWC9,
UniProt Related Accession:	P01574
Molecular Weight:	187
NCBI Full Name:	Interferon beta
NCBI Synonym Full Names:	interferon, beta 1, fibroblast
NCBI Official Symbol:	IFNB1
NCBI Official Synonym Symbols:	IFB; IFF; IFNB
NCBI Protein Information:	interferon beta; IFN-beta; fibroblast interferon
UniProt Protein Name:	Interferon beta
UniProt Synonym Protein Names:	Fibroblast interferon
Protein Family:	Interferon
UniProt Gene Name:	IFNB1
UniProt Entry Name:	IFNB_HUMAN

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Additional Information

Reactivity:	Human
ELISA Type:	Sandwich