Human IRF3(Interferon Regulatory Factor 3)ELISA Kit  (HUES03351)

SKU:
HUES03351
€539

Description

ELISA Kit Technical ManualMSDS

Human IRF3(Interferon Regulatory Factor 3)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 IRF3 . 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 IRF3 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 IRF3, 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 IRF3. The concentration of Human IRF3 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human IRF3 in samples. No significant cross-reactivity or interference between Human IRF3 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
(ng/mL)
O.D Average Corrected
202.453
2.495
2.4742.403
101.677
1.691
1.6841.613
50.98
0.946
0.9630.892
2.50.425
0.451
0.4380.367
1.250.262
0.236
0.2490.178
0.630.186
0.16
0.1730.102
0.310.114
0.132
0.1230.052
00.069
0.073
0.071--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human IRF3 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human IRF3 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.981.726.951.071.817.07
Standard deviation0.050.090.300.070.080.27
C V (%)5.105.234.326.544.423.82

Recovery

The recovery of Human IRF3 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)89-10294
EDTA plasma (n=5)95-109101
Cell culture media (n=5)89-10195

Linearity

Samples were spiked with high concentrations of Human IRF3 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)
1:2Range (%)86-10189-10285-99
Average (%)929592
1:4Range (%)90-10383-9683-97
Average (%)958889
1:8Range (%)94-11085-10180-92
Average (%)1009287
1:16Range (%)91-10687-10086-101
Average (%)979392

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 Standard2 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 Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human IRF3(Interferon Regulatory Factor 3)ELISA Kit (HUES03351) 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 IRF3(Interferon Regulatory Factor 3)ELISA Kit (HUES03351) Protein Information

UniProt Protein Function:IRF3: interferon regulatory factor 3, a member of the interferon regulatory transcription factor (IRF) family. IRF3 is found in an inactive cytoplasmic form that upon serine/threonine phosphorylation forms a complex with CREBBP. This complex translocates to the nucleus and activates the transcription of interferons alpha and beta, as well as other interferon-induced genes.
UniProt Protein Details:

Protein type:DNA-binding; Transcription factor

Chromosomal Location of Human Ortholog: 19q13.3-q13.4

Cellular Component: nucleoplasm; cytoplasm; nucleus; cytosol

Molecular Function:identical protein binding; protein binding; protein homodimerization activity; DNA binding; transcription cofactor activity; transcription factor activity

Biological Process: transcription from RNA polymerase II promoter; negative regulation of interferon-beta biosynthetic process; positive regulation of I-kappaB kinase/NF-kappaB cascade; viral reproduction; apoptosis; MyD88-independent toll-like receptor signaling pathway; cytokine and chemokine mediated signaling pathway; toll-like receptor 3 signaling pathway; negative regulation of transcription from RNA polymerase II promoter; negative regulation of defense response to virus by host; response to exogenous dsRNA; positive regulation of interferon type I production; lipopolysaccharide-mediated signaling pathway; toll-like receptor signaling pathway; positive regulation of interferon-beta production; innate immune response; interferon type I biosynthetic process; defense response to virus; toll-like receptor 4 signaling pathway; response to DNA damage stimulus; negative regulation of interferon type I production; positive regulation of interferon-alpha production; positive regulation of cytokine secretion

Disease: Herpes Simplex Encephalitis, Susceptibility To, 7

NCBI Summary:This gene encodes a member of the interferon regulatory transcription factor (IRF) family. The encoded protein is found in an inactive cytoplasmic form that upon serine/threonine phosphorylation forms a complex with CREBBP. This complex translocates to the nucleus and activates the transcription of interferons alpha and beta, as well as other interferon-induced genes. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Nov 2011]
UniProt Code:Q14653
NCBI GenInfo Identifier:2497442
NCBI Gene ID:3661
NCBI Accession:Q14653.1
UniProt Secondary Accession:Q14653,Q5FBY1, Q5FBY2, Q5FBY4, Q7Z5G6, A8K7L2, B2RAZ3
UniProt Related Accession:Q14653
Molecular Weight:12,330 Da
NCBI Full Name:Interferon regulatory factor 3
NCBI Synonym Full Names:interferon regulatory factor 3
NCBI Official Symbol:IRF3
NCBI Protein Information:interferon regulatory factor 3
UniProt Protein Name:Interferon regulatory factor 3
Protein Family:Interferon regulatory factor
UniProt Gene Name:IRF3
UniProt Entry Name:IRF3_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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