Human Cell Biology ELISA Kits 4
Human POMC (Pro-Opiomelanocortin) CLIA Kit (HUES00599)
- SKU:
- HUES00599
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 18.75pg/mL
- Range:
- 31.25-2000pg/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: | 31.25-2000 pg/mL |
| Sensitivity: | 18.75 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human POMC in samples. No significant cross-reactivity or interference between Human POMC 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 POMC. 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 POMC 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 POMC, 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 POMC. The concentration of Human POMC in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | POMC: ACTH stimulates the adrenal glands to release cortisol. Defects in POMC may be associated with susceptibility to obesity (OBESITY). It is a condition characterized by an increase of body weight beyond the limitation of skeletal and physical requirements, as the result of excessive accumulation of body fat. Defects in POMC are the cause of pro-opiomelanocortinin deficiency (POMCD). Affected individuals present early-onset obesity, adrenal insufficiency and red hair. Belongs to the POMC family. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 2p23. 3 Cellular Component: peroxisomal matrix; extracellular space; cytoplasm; extracellular region; peroxisome; secretory granule Molecular Function:type 3 melanocortin receptor binding; G-protein-coupled receptor binding; type 4 melanocortin receptor binding; hormone activity; receptor binding Biological Process: generation of precursor metabolites and energy; cellular protein metabolic process; cell-cell signaling; neuropeptide signaling pathway; regulation of blood pressure; peptide hormone processing; regulation of appetite; positive regulation of transcription from RNA polymerase II promoter; negative regulation of tumor necrosis factor production; signal transduction; glucose homeostasis; cellular pigmentation Disease: Obesity; Proopiomelanocortin Deficiency |
| NCBI Summary: | This gene encodes a polypeptide hormone precursor that undergoes extensive, tissue-specific, post-translational processing via cleavage by subtilisin-like enzymes known as prohormone convertases. There are eight potential cleavage sites within the polypeptide precursor and, depending on tissue type and the available convertases, processing may yield as many as ten biologically active peptides involved in diverse cellular functions. The encoded protein is synthesized mainly in corticotroph cells of the anterior pituitary where four cleavage sites are used; adrenocorticotrophin, essential for normal steroidogenesis and the maintenance of normal adrenal weight, and lipotropin beta are the major end products. In other tissues, including the hypothalamus, placenta, and epithelium, all cleavage sites may be used, giving rise to peptides with roles in pain and energy homeostasis, melanocyte stimulation, and immune modulation. These include several distinct melanotropins, lipotropins, and endorphins that are contained within the adrenocorticotrophin and beta-lipotropin peptides. The antimicrobial melanotropin alpha peptide exhibits antibacterial and antifungal activity. Mutations in this gene have been associated with early onset obesity, adrenal insufficiency, and red hair pigmentation. Alternatively spliced transcript variants encoding the same protein have been described. [provided by RefSeq, Nov 2014] |
| UniProt Code: | P01189 |
| NCBI GenInfo Identifier: | 116880 |
| NCBI Gene ID: | 5443 |
| NCBI Accession: | P01189. 2 |
| UniProt Secondary Accession: | P01189,P78442, Q53T23, Q9UD39, Q9UD40, |
| UniProt Related Accession: | P01189 |
| Molecular Weight: | 267 |
| NCBI Full Name: | Pro-opiomelanocortin |
| NCBI Synonym Full Names: | proopiomelanocortin |
| NCBI Official Symbol: | POMC |
| NCBI Official Synonym Symbols: | LPH; MSH; NPP; POC; ACTH; CLIP |
| NCBI Protein Information: | pro-opiomelanocortin; beta-LPH; beta-MSH; alpha-MSH; gamma-LPH; gamma-MSH; beta-endorphin; met-enkephalin; lipotropin beta; lipotropin gamma; melanotropin beta; melanotropin alpha; melanotropin gamma; pro-ACTH-endorphin; adrenocorticotropin; corticotropin-lipotropin; adrenocorticotropic hormone; opiomelanocortin prepropeptide; proopiomelanocortin preproprotein; beta-melanocyte-stimulating hormone; alpha-melanocyte-stimulating hormone; corticotropin-like intermediary peptide |
| UniProt Protein Name: | Pro-opiomelanocortin |
| UniProt Synonym Protein Names: | Corticotropin-lipotropinCleaved into the following 11 chains:NPP; Melanotropin gammaAlternative name(s):Gamma-MSH |
| Protein Family: | Pro-opiomelanocortin |
| UniProt Gene Name: | POMC |
| UniProt Entry Name: | COLI_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 |
| 2000 | 54564 56878 | 55721 | 55693 |
| 1000 | 21686 26262 | 23974 | 23946 |
| 500 | 11928 10136 | 11032 | 11004 |
| 250 | 5003 5585 | 5294 | 5266 |
| 125 | 2661 2555 | 2608 | 2580 |
| 62.5 | 1324 1298 | 1311 | 1283 |
| 31.25 | 645 703 | 674 | 646 |
| 0 | 27 29 | 28 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human POMC were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human POMC 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) | 111.10 | 236.43 | 880.16 | 117.42 | 226.88 | 807.21 |
| Standard deviation | 10.65 | 20.83 | 68.21 | 13.94 | 19.49 | 81.85 |
| C V (%) | 9.59 | 8.81 | 7.75 | 11.87 | 8.59 | 10.14 |
Recovery
The recovery of Human POMC 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) | 92-105 | 97 |
| EDTA plasma (n=5) | 94-107 | 101 |
| Cell culture media (n=5) | 90-105 | 96 |
Linearity
Samples were spiked with high concentrations of Human POMC 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 (%) | 93-111 | 85-99 | 89-102 |
| Average (%) | 101 | 91 | 94 | |
| 1:4 | Range (%) | 100-112 | 94-110 | 86-97 |
| Average (%) | 106 | 101 | 91 | |
| 1:8 | Range (%) | 99-110 | 88-99 | 98-115 |
| Average (%) | 105 | 93 | 105 | |
| 1:16 | Range (%) | 93-108 | 101-115 | 89-102 |
| Average (%) | 99 | 108 | 96 |
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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