|Detection Range:||0.16-10 ng/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Human TLR9 in samples. No significant cross-reactivity or interference between Human TLR9 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 TLR9. 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 TLR9 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 TLR9, 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 TLR9. The concentration of Human TLR9 in samples can be calculated by comparing the OD of the samples to the standard curve.
|UniProt Protein Function:||TLR9: Key component of innate and adaptive immunity. TLRs (Toll-like receptors) control host immune response against pathogens through recognition of molecular patterns specific to microorganisms. TLR9 is a nucleotide-sensing TLR which is activated by unmethylated cytidine-phosphate-guanosine (CpG) dinucleotides. Acts via MYD88 and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. Controls lymphocyte response to Helicobacter infection. Interacts with MYD88 via their respective TIR domains. Interacts (via transmembrane domain) with UNC93B1. Interacts with CD300LH; the interaction may promote full activation of TLR9-triggered innate responses. Interacts with BTK. Interacts with CNPY3 and HSP90B1; this interaction is required for proper folding in the endoplasmic reticulum. Highly expressed in spleen, lymph node, tonsil and peripheral blood leukocytes, especially in plasmacytoid pre- dendritic cells. Levels are much lower in monocytes and CD11c+ immature dendritic cells. Also detected in lung and liver. Belongs to the Toll-like receptor family. 5 isoforms of the human protein are produced by alternative splicing.|
|UniProt Protein Details:|
Protein type:Receptor, misc. ; Membrane protein, integral
Chromosomal Location of Human Ortholog: 3p21. 3
Cellular Component: apical plasma membrane; basolateral plasma membrane; cytoplasm; endoplasmic reticulum; endoplasmic reticulum membrane; endosome; endosome membrane; Golgi membrane; lysosome; plasma membrane
Molecular Function:1-phosphatidylinositol-3-kinase activity; interleukin-1 receptor binding; siRNA binding
Biological Process: activation of NF-kappaB transcription factor; axonogenesis; defense response to Gram-negative bacterium; I-kappaB phosphorylation; inhibition of NF-kappaB transcription factor; innate immune response; maintenance of gastrointestinal epithelium; negative regulation of interleukin-6 production; negative regulation of interleukin-8 production; negative regulation of toll-like receptor signaling pathway; positive regulation of chemokine production; positive regulation of granulocyte macrophage colony-stimulating factor production; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of inflammatory response; positive regulation of interferon-alpha biosynthetic process; positive regulation of interferon-beta biosynthetic process; positive regulation of interferon-beta production; positive regulation of interferon-gamma biosynthetic process; positive regulation of interleukin-10 production; positive regulation of interleukin-12 production; positive regulation of interleukin-18 production; positive regulation of interleukin-6 production; positive regulation of interleukin-8 production; positive regulation of JNK activity; positive regulation of JNK cascade; positive regulation of NF-kappaB import into nucleus; positive regulation of nitric-oxide synthase biosynthetic process; positive regulation of toll-like receptor signaling pathway; positive regulation of transcription from RNA polymerase II promoter; positive regulation of tumor necrosis factor production; response to molecule of bacterial origin; toll-like receptor 9 signaling pathway; toll-like receptor signaling pathway; tumor necrosis factor production
|NCBI Summary:||The protein encoded by this gene is a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity. TLRs are highly conserved from Drosophila to humans and share structural and functional similarities. They recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, and mediate the production of cytokines necessary for the development of effective immunity. The various TLRs exhibit different patterns of expression. This gene is preferentially expressed in immune cell rich tissues, such as spleen, lymph node, bone marrow and peripheral blood leukocytes. Studies in mice and human indicate that this receptor mediates cellular response to unmethylated CpG dinucleotides in bacterial DNA to mount an innate immune response. [provided by RefSeq, Jul 2008]|
|NCBI GenInfo Identifier:||20140872|
|NCBI Gene ID:||54106|
|NCBI Accession:||Q9NR96. 2|
|UniProt Secondary Accession:||Q9NR96,Q6UVZ2, Q9HD68, Q9HD69, Q9HD70, Q9NYC2, Q9NYC3 B3Y661, D1CS56,|
|UniProt Related Accession:||Q9NR96|
|Molecular Weight:||114,237 Da|
|NCBI Full Name:||Toll-like receptor 9|
|NCBI Synonym Full Names:||toll like receptor 9|
|NCBI Official Symbol:||TLR9|
|NCBI Official Synonym Symbols:||CD289|
|NCBI Protein Information:||toll-like receptor 9|
|UniProt Protein Name:||Toll-like receptor 9|
|UniProt Synonym Protein Names:||CD_antigen: CD289|
|Protein Family:||Toll-like receptor|
|UniProt Gene Name:||TLR9|
|UniProt Entry Name:||TLR9_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 TLR9 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TLR9 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||6.12||5.69||4.57||5.88||4.84||4.34|
The recovery of Human TLR9 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)||90-105||97|
|Cell culture media (n=5)||87-101||93|
Samples were spiked with high concentrations of Human TLR9 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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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.