Description
Human RIPK2 (Receptor TNFRSF Interacting Serine Threonine Kinase 2) ELISA Kit
The Human RIPK2 (Receptor Interacting Serine/Threonine Kinase 2) ELISA Kit is a valuable tool for the quantitative detection of RIPK2 levels in human serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and reproducible results, making it an ideal choice for a variety of research applications.RIPK2 is a critical regulator of immune responses and inflammatory signaling pathways. Dysregulation of RIPK2 has been implicated in various diseases, including inflammatory disorders and autoimmune conditions.
The ability to accurately measure RIPK2 levels is essential for understanding its role in disease pathogenesis and potentially identifying novel therapeutic targets.Overall, the Human RIPK2 ELISA Kit provides researchers with a reliable method for studying the role of RIPK2 in health and disease, offering valuable insights into potential diagnostic and therapeutic strategies.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 62.50-4000 pg/mL |
| Sensitivity: | 37.50 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human RIPK2 in samples. No significant cross-reactivity or interference between Human RIPK2 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 RIPK2. 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 RIPK2 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 RIPK2, 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 RIPK2. The concentration of Human RIPK2 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | RIPK2: a tyrosine kinase-like kinase of the RIPK family. Activates pro-caspase-1 and pro-caspase-8. Potentiates casp-8-mediated apoptosis. May activate NF-kappaB. |
| UniProt Protein Details: | Protein type:EC 2. 7. 11. 1; Kinase, protein; EC 2. 7. 10. 2; Protein kinase, Ser/Thr (non-receptor); Protein kinase, TKL; TKL group; RIPK family Chromosomal Location of Human Ortholog: 8q21 Cellular Component: cytoplasm; cytoskeleton; cytosol; protein complex; vesicle Molecular Function:ATP binding; CARD domain binding; LIM domain binding; non-membrane spanning protein tyrosine kinase activity; protein binding; protein homodimerization activity; protein serine/threonine kinase activity; receptor binding; signal transducer activity Biological Process: activation of MAPK activity; activation of NF-kappaB transcription factor; adaptive immune response; apoptosis; defense response to Gram-positive bacterium; I-kappaB kinase/NF-kappaB cascade; inflammatory response; innate immune response; JNK cascade; lipopolysaccharide-mediated signaling pathway; MyD88-dependent toll-like receptor signaling pathway; MyD88-independent toll-like receptor signaling pathway; negative regulation of apoptosis; nerve growth factor receptor signaling pathway; peptidyl-tyrosine phosphorylation; positive regulation of alpha-beta T cell proliferation; positive regulation of apoptosis; positive regulation of chemokine production; positive regulation of cytokine and chemokine mediated signaling pathway; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of immature T cell proliferation; positive regulation of interferon-alpha production; positive regulation of interferon-beta production; positive regulation of interferon-gamma production; positive regulation of interleukin-12 production; positive regulation of interleukin-2 production; positive regulation of interleukin-6 production; positive regulation of JNK cascade; positive regulation of peptidyl-serine phosphorylation; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of protein ubiquitination; positive regulation of T-helper 1 cell differentiation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of tumor necrosis factor production; response to exogenous dsRNA; signal transduction; stress-activated MAPK cascade; T cell proliferation; T cell receptor signaling pathway; toll-like receptor 10 signaling pathway; toll-like receptor 2 signaling pathway; toll-like receptor 3 signaling pathway; toll-like receptor 4 signaling pathway; toll-like receptor 5 signaling pathway; toll-like receptor 9 signaling pathway; toll-like receptor signaling pathway |
| NCBI Summary: | This gene encodes a member of the receptor-interacting protein (RIP) family of serine/threonine protein kinases. The encoded protein contains a C-terminal caspase activation and recruitment domain (CARD), and is a component of signaling complexes in both the innate and adaptive immune pathways. It is a potent activator of NF-kappaB and inducer of apoptosis in response to various stimuli. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O43353 |
| NCBI GenInfo Identifier: | 20455217 |
| NCBI Gene ID: | 8767 |
| NCBI Accession: | O43353. 2 |
| UniProt Secondary Accession: | O43353,Q6UWF0, B7Z748, |
| UniProt Related Accession: | O43353 |
| Molecular Weight: | 45,582 Da |
| NCBI Full Name: | Receptor-interacting serine/threonine-protein kinase 2 |
| NCBI Synonym Full Names: | receptor interacting serine/threonine kinase 2 |
| NCBI Official Symbol: | RIPK2 |
| NCBI Official Synonym Symbols: | CCK; RICK; RIP2; CARD3; GIG30; CARDIAK |
| NCBI Protein Information: | receptor-interacting serine/threonine-protein kinase 2 |
| UniProt Protein Name: | Receptor-interacting serine/threonine-protein kinase 2 |
| UniProt Synonym Protein Names: | CARD-containing interleukin-1 beta-converting enzyme-associated kinase; CARD-containing IL-1 beta ICE-kinase; RIP-like-interacting CLARP kinase; Receptor-interacting protein 2; RIP-2; Tyrosine-protein kinase RIPK2 (EC:2. 7. 10. 2) |
| Protein Family: | Putative zinc metalloprotease |
| UniProt Gene Name: | RIPK2 |
| UniProt Entry Name: | RIPK2_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.
| Concentration (pg/mL) | O.D | Average | Corrected |
| 4000 | 2.21 2.268 | 2.239 | 2.183 |
| 2000 | 1.441 1.495 | 1.468 | 1.412 |
| 1000 | 0.882 0.864 | 0.873 | 0.817 |
| 500 | 0.397 0.417 | 0.407 | 0.351 |
| 250 | 0.226 0.204 | 0.215 | 0.159 |
| 125 | 0.162 0.14 | 0.151 | 0.095 |
| 62.50 | 0.1 0.11 | 0.105 | 0.049 |
| 0 | 0.051 0.061 | 0.056 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human RIPK2 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human RIPK2 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) | 192.96 | 526.29 | 1921.41 | 198.64 | 498.34 | 1892.55 |
| Standard deviation | 12.41 | 23.21 | 63.79 | 13.55 | 29.80 | 56.78 |
| C V (%) | 6.43 | 4.41 | 3.32 | 6.82 | 5.98 | 3.00 |
Recovery
The recovery of Human RIPK2 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) | 93-105 | 100 |
| EDTA plasma (n=5) | 87-101 | 93 |
| Cell culture media (n=5) | 84-98 | 91 |
Linearity
Samples were spiked with high concentrations of Human RIPK2 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 (%) | 94-109 | 98-109 | 91-108 |
| Average (%) | 100 | 104 | 99 | |
| 1:4 | Range (%) | 90-105 | 84-95 | 86-101 |
| Average (%) | 97 | 90 | 93 | |
| 1:8 | Range (%) | 91-106 | 87-97 | 88-99 |
| Average (%) | 97 | 92 | 94 | |
| 1:16 | Range (%) | 90-101 | 81-92 | 87-101 |
| Average (%) | 95 | 87 | 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 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.
