Human TLR9/Toll-like receptor 9 ELISA Kit- High Sensitivity

Product Name:	Human TLR9/Toll-like receptor 9 ELISA Kit
Product Code:	HUFI00774
Size:	96 Assays
Alias:	TLR9, CD289, Toll-like receptor 9, CD289 antigen
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TLR9 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human TLR9 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TLR9 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-104	96
EDTA plasma(n=5)	91-102	98
UFH plasma(n=5)	91-103	98

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TLR9 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-104%	93-99%	88-97%
EDTA plasma(n=5)	82-90%	86-101%	90-101%
UFH plasma(n=5)	81-93%	87-96%	81-96%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

Q9NR96

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]
UniProt Code:	Q9NR96
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

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.