Human Cell Biology ELISA Kits 3
Human HHIP (Hedgehog Interacting Protein) CLIA Kit (HUES00532)
- SKU:
- HUES00532
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 37.5pg/mL
- Range:
- 62.5-4000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Biology
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 HHIP in samples. No significant cross-reactivity or interference between Human HHIP 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 HHIP. 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 HHIP 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 HHIP, 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 HHIP. The concentration of Human HHIP in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | HHIP: Modulates hedgehog signaling in several cell types including brain and lung through direct interaction with members of the hedgehog family. Belongs to the HHIP family. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Cell development/differentiation Chromosomal Location of Human Ortholog: 4q28-q32 Cellular Component: cell surface; integral to plasma membrane; cytoplasm; extracellular region Molecular Function:protein binding; oxidoreductase activity, acting on the CH-OH group of donors, quinone or similar compound as acceptor; zinc ion binding; quinone binding Biological Process: smoothened signaling pathway; dorsal/ventral pattern formation; skeletal morphogenesis; negative regulation of smoothened signaling pathway; carbohydrate metabolic process; neuroblast proliferation; regulation of fibroblast growth factor receptor signaling pathway; negative regulation of signal transduction |
| NCBI Summary: | This gene encodes a member of the hedgehog-interacting protein (HHIP) family. The hedgehog (HH) proteins are evolutionarily conserved protein, which are important morphogens for a wide range of developmental processes, including anteroposterior patterns of limbs and regulation of left-right asymmetry in embryonic development. Multiple cell-surface receptors are responsible for transducing and/or regulating HH signals. The HHIP encoded by this gene is a highly conserved, vertebrate-specific inhibitor of HH signaling. It interacts with all three HH family members, SHH, IHH and DHH. Two single nucleotide polymorphisms (SNPs) near this gene are significantly associated with risk of chronic obstructive pulmonary disease (COPD). A single nucleotide polymorphism in this gene is also strongly associated with human height. [provided by RefSeq, Feb 2011] |
| UniProt Code: | Q96QV1 |
| NCBI GenInfo Identifier: | 118572655 |
| NCBI Gene ID: | 64399 |
| NCBI Accession: | Q96QV1. 3 |
| UniProt Secondary Accession: | Q96QV1,Q6PK09, Q8NCI7, Q9BXK3, Q9H1J4, Q9H7E7, |
| UniProt Related Accession: | Q96QV1 |
| Molecular Weight: | 700 |
| NCBI Full Name: | Hedgehog-interacting protein |
| NCBI Synonym Full Names: | hedgehog interacting protein |
| NCBI Official Symbol: | HHIP |
| NCBI Official Synonym Symbols: | HIP |
| NCBI Protein Information: | hedgehog-interacting protein |
| UniProt Protein Name: | Hedgehog-interacting protein |
| Protein Family: | HHIP-like protein |
| UniProt Gene Name: | HHIP |
| UniProt Entry Name: | HHIP_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 | 58436 59726 | 59081 | 59046 |
| 2000 | 26143 29575 | 27859 | 27824 |
| 1000 | 14269 12699 | 13484 | 13449 |
| 500 | 6033 7177 | 6605 | 6570 |
| 250 | 3486 3000 | 3243 | 3208 |
| 125 | 1588 1574 | 1581 | 1546 |
| 62.50 | 714 796 | 755 | 720 |
| 0 | 34 36 | 35 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human HHIP were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human HHIP 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) | 204.06 | 625.68 | 1486.17 | 197.49 | 637.73 | 1348.08 |
| Standard deviation | 19.86 | 74.96 | 95.26 | 15.98 | 49.42 | 112.70 |
| C V (%) | 9.73 | 11.98 | 6.41 | 8.09 | 7.75 | 8.36 |
Recovery
The recovery of Human HHIP 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) | 100-116 | 106 |
| EDTA plasma (n=5) | 88-99 | 94 |
| Cell culture media (n=5) | 89-102 | 94 |
Linearity
Samples were spiked with high concentrations of Human HHIP 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 (%) | 86-101 | 84-98 | 89-102 |
| Average (%) | 93 | 91 | 95 | |
| 1:4 | Range (%) | 87-97 | 96-110 | 96-106 |
| Average (%) | 92 | 102 | 101 | |
| 1:8 | Range (%) | 85-100 | 93-107 | 86-98 |
| Average (%) | 92 | 100 | 91 | |
| 1:16 | Range (%) | 102-117 | 92-107 | 101-116 |
| Average (%) | 108 | 99 | 109 |
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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