Description
Human MFGE8 (Milk Fat Globule EGF Factor 8) ELISA Kit
The Human MFGE8 (Milk Fat Globule EGF Factor 8) ELISA Kit is a highly sensitive and specific assay designed for the accurate detection of MFGE8 levels in human samples including serum, plasma, and cell culture supernatants. This kit provides reliable and reproducible results, making it an ideal tool for a variety of research applications.MFGE8, also known as lactadherin, is a multifunctional protein involved in various biological processes, including phagocytosis, inflammation, and tissue repair. It plays a crucial role in maintaining tissue homeostasis and immune regulation.
Dysregulation of MFGE8 has been implicated in conditions such as cancer, inflammatory diseases, and autoimmune disorders, making it a valuable biomarker for studying these pathologies and potential therapeutic interventions.With its high sensitivity and specificity, the Human MFGE8 ELISA Kit offers researchers a powerful tool for quantitative analysis of MFGE8 levels in human samples, further advancing our understanding of its roles in health and disease.
| 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 MFGE8 in samples. No significant cross-reactivity or interference between Human MFGE8 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 MFGE8. 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 MFGE8 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 MFGE8, 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 MFGE8. The concentration of Human MFGE8 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | MFGE8: Plays an important role in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing. Promotes VEGF-dependent neovascularization. Contributes to phagocytic removal of apoptotic cells in many tissues. Specific ligand for the alpha-v/beta-3 and alpha-v/beta-5 receptors. Also binds to phosphatidylserine-enriched cell surfaces in a receptor-independent manner. Zona pellucida-binding protein which may play a role in gamete interaction. Binds specifically to rotavirus and inhibits its replication. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 15q25 Cellular Component: extrinsic to plasma membrane; extracellular matrix; extracellular space; membrane; extracellular region; vesicle; external side of plasma membrane Molecular Function:integrin binding; phosphatidylethanolamine binding; phosphatidylserine binding Biological Process: viral reproduction; single fertilization; phagocytosis, recognition; angiogenesis; phagocytosis, engulfment; cell adhesion |
| UniProt Code: | Q08431 |
| NCBI GenInfo Identifier: | 2506380 |
| NCBI Gene ID: | |
| NCBI Accession: | Q08431. 2 |
| Molecular Weight: | |
| NCBI Full Name: | Lactadherin |
| UniProt Protein Name: | Lactadherin |
| UniProt Synonym Protein Names: | Breast epithelial antigen BA46; HMFG; MFGM; Milk fat globule-EGF factor 8; MFG-E8; SED1Cleaved into the following 2 chains:Lactadherin short form; Medin |
| UniProt Gene Name: | MFGE8 |
| UniProt Entry Name: | MFGM_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 | 2.228 2.276 | 2.252 | 2.178 |
| 2000 | 1.617 1.647 | 1.632 | 1.558 |
| 1000 | 0.899 0.873 | 0.886 | 0.812 |
| 500 | 0.435 0.475 | 0.455 | 0.381 |
| 250 | 0.287 0.259 | 0.273 | 0.199 |
| 125 | 0.183 0.155 | 0.169 | 0.095 |
| 62.50 | 0.122 0.124 | 0.123 | 0.049 |
| 0 | 0.066 0.082 | 0.074 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human MFGE8 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human MFGE8 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) | 220.86 | 478.83 | 1900.42 | 201.64 | 525.61 | 2026.24 |
| Standard deviation | 12.99 | 25.43 | 90.46 | 13.79 | 29.17 | 97.46 |
| C V (%) | 5.88 | 5.31 | 4.76 | 6.84 | 5.55 | 4.81 |
Recovery
The recovery of Human MFGE8 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) | 90-107 | 98 |
| EDTA plasma (n=5) | 86-99 | 92 |
| Cell culture media (n=5) | 93-108 | 99 |
Linearity
Samples were spiked with high concentrations of Human MFGE8 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 (%) | 97-112 | 98-112 | 90-102 |
| Average (%) | 105 | 105 | 96 | |
| 1:4 | Range (%) | 90-101 | 83-94 | 86-101 |
| Average (%) | 96 | 89 | 93 | |
| 1:8 | Range (%) | 94-106 | 85-96 | 83-95 |
| Average (%) | 99 | 91 | 89 | |
| 1:16 | Range (%) | 88-100 | 84-98 | 88-100 |
| Average (%) | 93 | 90 | 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.
