Human Immunology ELISA Kits 1
Human TLR4 (Toll-Like Receptor 4) CLIA Kit (HUES00872)
- SKU:
- HUES00872
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 37.5pg/mL
- Range:
- 62.5-4000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- TLR-4, ARMD10, CD284, TOLL, hToll
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Immunology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 62.50-4000 pg/mL |
| Sensitivity: | 37.50 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human TLR4 in samples. No significant cross-reactivity or interference between Human TLR4 and analogues was observed. |
This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human TLR4. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human TLR4 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 TLR4, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human TLR4. The concentration of Human TLR4 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | TLR4: Cooperates with LY96 and CD14 to mediate the innate immune response to bacterial lipopolysaccharide (LPS). Acts via MYD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. Also involved in LPS- independent inflammatory responses triggered by Ni(2+). These responses require non-conserved histidines and are, therefore, species-specific. Belongs to the lipopolysaccharide (LPS) receptor, a multi-protein complex containing at least CD14, LY96 and TLR4. Binding to bacterial LPS leads to homodimerization. Interacts with LY96 via the extracellular domain. Interacts with MYD88 and TIRAP via their respective TIR domains. Interacts with NOX4. Interacts with CNPY3. Interacts with HSP90B1. The interaction with both CNPY3 and HSP90B1 is required for proper folding in the endoplasmic reticulum. Highly expressed in placenta, spleen and peripheral blood leukocytes. Detected in monocytes, macrophages, dendritic cells and several types of T-cells. Belongs to the Toll-like receptor family. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Receptor, misc. Chromosomal Location of Human Ortholog: 9q33. 1 Cellular Component: integral to plasma membrane; perinuclear region of cytoplasm; cytoplasm; plasma membrane; lipopolysaccharide receptor complex; endosome membrane; external side of plasma membrane Molecular Function:protein binding; transmembrane receptor activity; lipopolysaccharide binding; lipopolysaccharide receptor activity; receptor activity Biological Process: I-kappaB kinase/NF-kappaB cascade; positive regulation of nitric oxide biosynthetic process; activation of MAPK activity; response to lipopolysaccharide; positive regulation of NF-kappaB import into nucleus; toll-like receptor 3 signaling pathway; positive regulation of interleukin-10 production; activation of NF-kappaB transcription factor; positive regulation of interferon-beta production; negative regulation of interleukin-6 production; positive regulation of B cell proliferation; toll-like receptor 4 signaling pathway; positive regulation of interferon-alpha production; T-helper 1 type immune response; production of nitric oxide during acute inflammatory response; positive regulation of interleukin-6 production; positive regulation of interleukin-8 biosynthetic process; positive regulation of tumor necrosis factor production; positive regulation of chemokine production; toll-like receptor 2 signaling pathway; negative regulation of interleukin-23 production; macrophage activation; detection of lipopolysaccharide; defense response to bacterium; positive regulation of transcription from RNA polymerase II promoter; I-kappaB phosphorylation; positive regulation of nitric-oxide synthase biosynthetic process; negative regulation of osteoclast differentiation; regulation of cytokine secretion; positive regulation of interleukin-12 production; positive regulation of JNK cascade; defense response to Gram-negative bacterium; positive regulation of interleukin-8 production; negative regulation of interferon-gamma production; positive regulation of MHC class II biosynthetic process; interferon-gamma production; lipopolysaccharide-mediated signaling pathway; B cell proliferation during immune response; positive regulation of tumor necrosis factor biosynthetic process; detection of fungus; MyD88-independent toll-like receptor signaling pathway; negative regulation of tumor necrosis factor production; MyD88-dependent toll-like receptor signaling pathway; positive regulation of interferon-gamma production; positive regulation of interleukin-12 biosynthetic process; negative regulation of interleukin-17 production; positive regulation of interferon-beta biosynthetic process; toll-like receptor signaling pathway; innate immune response; immune response; positive regulation of interleukin-1 production; positive regulation of inflammatory response Disease: Macular Degeneration, Age-related, 10 |
| 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 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 receptor has been implicated in signal transduction events induced by lipopolysaccharide (LPS) found in most gram-negative bacteria. Mutations in this gene have been associated with differences in LPS responsiveness. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jan 2012] |
| UniProt Code: | O00206 |
| NCBI GenInfo Identifier: | 20140413 |
| NCBI Gene ID: | 7099 |
| NCBI Accession: | O00206. 2 |
| UniProt Secondary Accession: | O00206,Q5VZI8, Q5VZI9, Q9UK78, Q9UM57, A8K1Y8, A9XLP9 A9XLQ0, A9XLQ1, B4E194, D1CS52, D1CS53, |
| UniProt Related Accession: | O00206 |
| Molecular Weight: | 73,301 Da |
| NCBI Full Name: | Toll-like receptor 4 |
| NCBI Synonym Full Names: | toll-like receptor 4 |
| NCBI Official Symbol: | TLR4 |
| NCBI Official Synonym Symbols: | TOLL; CD284; TLR-4; ARMD10 |
| NCBI Protein Information: | toll-like receptor 4; hToll; homolog of Drosophila toll |
| UniProt Protein Name: | Toll-like receptor 4 |
| UniProt Synonym Protein Names: | hToll; CD_antigen: CD284 |
| Protein Family: | Toll-like receptor |
| UniProt Gene Name: | TLR4 |
| UniProt Entry Name: | TLR4_HUMAN |
As the RLU 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) | RLU | Average | Corrected |
| 4000 | 58749 59413 | 59081 | 59046 |
| 2000 | 26972 28746 | 27859 | 27824 |
| 1000 | 14115 12853 | 13484 | 13449 |
| 500 | 6234 6976 | 6605 | 6570 |
| 250 | 3467 3019 | 3243 | 3208 |
| 125 | 1596 1566 | 1581 | 1546 |
| 62.50 | 718 792 | 755 | 720 |
| 0 | 35 35 | 35 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TLR4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TLR4 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) | 193.73 | 335.07 | 1584.16 | 207.30 | 367.47 | 1733.16 |
| Standard deviation | 20.79 | 30.79 | 127.52 | 21.25 | 40.16 | 191.86 |
| C V (%) | 10.73 | 9.19 | 8.05 | 10.25 | 10.93 | 11.07 |
Recovery
The recovery of Human TLR4 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) | 85-97 | 91 |
| EDTA plasma (n=5) | 101-116 | 107 |
| Cell culture media (n=5) | 99-112 | 105 |
Linearity
Samples were spiked with high concentrations of Human TLR4 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 (%) | 98-115 | 93-107 | 93-109 |
| Average (%) | 105 | 99 | 99 | |
| 1:4 | Range (%) | 91-101 | 96-109 | 88-102 |
| Average (%) | 96 | 104 | 94 | |
| 1:8 | Range (%) | 88-102 | 98-115 | 98-112 |
| Average (%) | 94 | 105 | 106 | |
| 1:16 | Range (%) | 92-108 | 87-99 | 87-102 |
| Average (%) | 99 | 93 | 94 |
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 CLIA 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 A | 1 vial, 5 mL | 4°C (shading light) |
| Substrate Reagent B | 1 vial, 5 mL | 4°C (shading light) |
| 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
- Determine the RLU value of each well immediately.
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