|Detection Range:||0.31-20 ng/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, 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.|
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.
|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]|
|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|
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.
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|
|C V (%)||5.10||5.23||4.32||6.54||4.42||3.82|
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 (%)|
|EDTA plasma (n=5)||95-109||101|
|Cell culture media (n=5)||89-101||95|
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)|
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.
|Micro ELISA Plate(Dismountable)||8 wells ×12 strips||-20°C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100×)||1 vial, 120 µL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 µL||-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 (25×)||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|
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.