Human Immunology ELISA Kits 13
Human MUC5B (Mucin 5 Subtype B) ELISA Kit (HUES03163)
- SKU:
- HUES03163
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q9HC84
- Sensitivity:
- 0.38ng/mL
- Range:
- 0.63-40ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- MUC5B, MG1, MUC-5B, MUC9
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.63-40 ng/mL |
| Sensitivity: | 0.38 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human MUC5B in samples. No significant cross-reactivity or interference between Human MUC5B 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 MUC5B. 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 MUC5B 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 MUC5B, 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 MUC5B. The concentration of Human MUC5B in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | MUC5B: Gel-forming mucin that is thought to contribute to the lubricating and viscoelastic properties of whole saliva and cervical mucus. Defects in MUC5B are a cause of susceptibility to pulmonary fibrosis idiopathic (IPF). Pulmonary fibrosis is a lung disease characterized by shortness of breath, radiographically evident diffuse pulmonary infiltrates, and varying degrees of inflammation and fibrosis on biopsy. It results in acute lung injury with subsequent scarring and endstage lung disease. A common polymorphism in the promoter of MUC5B is associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis, suggesting that dysregulated MUC5B expression in the lung may be involved in the pathogenesis of pulmonary fibrosis (PubMed:21506741). |
| UniProt Protein Details: | Protein type:Cell adhesion; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 11p15. 5 Cellular Component: extracellular space; Golgi lumen Molecular Function:protein binding Biological Process: protein amino acid O-linked glycosylation; cellular protein metabolic process; O-glycan processing; regulation of macrophage activation; defense response to bacterium; post-translational protein modification Disease: Pulmonary Fibrosis, Idiopathic |
| NCBI Summary: | This gene encodes a member of the mucin family of proteins, which are highly glycosylated macromolecular components of mucus secretions. This family member is the major gel-forming mucin in mucus. It is a major contributor to the lubricating and viscoelastic properties of whole saliva, normal lung mucus and cervical mucus. This gene has been found to be up-regulated in some human diseases, including sinus mucosa of chronic rhinosinusitis (CRS), CRS with nasal polyposis, chronic obstructive pulmonary disease (COPD) and H. pylori-associated gastric disease, and it may be involved in the pathogenesis of these diseases. [provided by RefSeq, Jul 2010] |
| UniProt Code: | Q9HC84 |
| NCBI GenInfo Identifier: | 308153579 |
| NCBI Gene ID: | 727897 |
| NCBI Accession: | Q9HC84. 3 |
| UniProt Secondary Accession: | Q9HC84,O00447, O00573, O14985, O15494, O95291, O95451 Q14881, Q7M4S5, Q99552, Q9UE28, |
| UniProt Related Accession: | Q9HC84 |
| Molecular Weight: | 596,340 Da |
| NCBI Full Name: | Mucin-5B |
| NCBI Synonym Full Names: | mucin 5B, oligomeric mucus/gel-forming |
| NCBI Official Symbol: | MUC5B |
| NCBI Official Synonym Symbols: | MG1; MUC5; MUC9; MUC-5B |
| NCBI Protein Information: | mucin-5B; cervical mucin MUC5B; sublingual gland mucin; mucin 5, subtype B, tracheobronchial; high molecular weight salivary mucin MG1 |
| UniProt Protein Name: | Mucin-5B |
| UniProt Synonym Protein Names: | Cervical mucin; High molecular weight salivary mucin MG1; Mucin-5 subtype B, tracheobronchial; Sublingual gland mucin |
| Protein Family: | Mucin |
| UniProt Gene Name: | MUC5B |
| UniProt Entry Name: | MUC5B_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 (ng/mL) | O.D | Average | Corrected |
| 40 | 2.488 2.524 | 2.506 | 2.418 |
| 20 | 1.625 1.637 | 1.631 | 1.543 |
| 10 | 0.97 0.946 | 0.958 | 0.87 |
| 5 | 0.455 0.473 | 0.464 | 0.376 |
| 2.5 | 0.287 0.263 | 0.275 | 0.187 |
| 1.25 | 0.189 0.185 | 0.187 | 0.099 |
| 0.63 | 0.133 0.145 | 0.139 | 0.051 |
| 0 | 0.083 0.093 | 0.088 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human MUC5B were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human MUC5B 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 (ng/mL) | 1.92 | 6.00 | 14.33 | 1.79 | 5.85 | 14.62 |
| Standard deviation | 0.11 | 0.34 | 0.58 | 0.11 | 0.32 | 0.57 |
| C V (%) | 5.73 | 5.67 | 4.05 | 6.15 | 5.47 | 3.90 |
Recovery
The recovery of Human MUC5B 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) | 86-97 | 92 |
| EDTA plasma (n=5) | 95-108 | 101 |
| Cell culture media (n=5) | 86-100 | 91 |
Linearity
Samples were spiked with high concentrations of Human MUC5B 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 (%) | 90-101 | 89-105 | 90-106 |
| Average (%) | 95 | 96 | 97 | |
| 1:4 | Range (%) | 88-102 | 80-93 | 87-102 |
| Average (%) | 93 | 85 | 93 | |
| 1:8 | Range (%) | 90-104 | 88-98 | 85-95 |
| Average (%) | 96 | 93 | 90 | |
| 1:16 | Range (%) | 93-105 | 84-99 | 84-96 |
| Average (%) | 98 | 91 | 90 |
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.
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