Human Immunology ELISA Kits 12
Human Flt3L (FMS Like Tyrosine Kinase 3 Ligand) ELISA Kit (HUES01362)
- SKU:
- HUES01362
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P49771
- Sensitivity:
- 9.38pg/mL
- Range:
- 15.63-1000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- FLT3LG, Flt3-L, FL, Fms-Related Tyrosine Kinase 3 ligand
- 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: | 15.63-1000 pg/mL |
| Sensitivity: | 9.38 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human Flt3L in samples. No significant cross-reactivity or interference between Human Flt3L 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 Flt3L. 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 Flt3L 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 Flt3L, 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 Flt3L. The concentration of Human Flt3L in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | FLT3LG: Stimulates the proliferation of early hematopoietic cells by activating FLT3. Synergizes well with a number of other colony stimulating factors and interleukins. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Cytokine; Ligand, receptor tyrosine kinase; Membrane protein, integral Chromosomal Location of Human Ortholog: 19q13. 3 Cellular Component: extracellular space; membrane; integral to membrane Molecular Function:protein homodimerization activity; cytokine activity; receptor tyrosine kinase binding; receptor binding Biological Process: regulation of myeloid dendritic cell activation; positive regulation of natural killer cell differentiation; lymphocyte differentiation; embryonic hemopoiesis; positive regulation of cell proliferation; positive regulation of protein amino acid phosphorylation; signal transduction; positive regulation of myoblast differentiation |
| NCBI Summary: | Dendritic cells (DCs) provide the key link between innate and adaptive immunity by recognizing pathogens and priming pathogen-specific immune responses. FLT3LG controls the development of DCs and is particularly important for plasmacytoid DCs and CD8 (see MIM 186910)-positive classical DCs and their CD103 (ITGAE; MIM 604682)-positive tissue counterparts (summary by Sathaliyawala et al. , 2010 [PubMed 20933441]). [supplied by OMIM, Jan 2011] |
| UniProt Code: | P49771 |
| NCBI GenInfo Identifier: | 1706818 |
| NCBI Gene ID: | 2323 |
| NCBI Accession: | P49771. 1 |
| UniProt Secondary Accession: | P49771,Q05C96, A0AVC2, B9EGH2, |
| UniProt Related Accession: | P49771 |
| Molecular Weight: | approximately 150 kDa |
| NCBI Full Name: | Fms-related tyrosine kinase 3 ligand |
| NCBI Synonym Full Names: | fms-related tyrosine kinase 3 ligand |
| NCBI Official Symbol: | FLT3LG |
| NCBI Official Synonym Symbols: | FL; FLT3L |
| NCBI Protein Information: | fms-related tyrosine kinase 3 ligand; flt3 ligand |
| UniProt Protein Name: | Fms-related tyrosine kinase 3 ligand |
| UniProt Synonym Protein Names: | SL cytokine |
| Protein Family: | Receptor-type tyrosine-protein kinase |
| UniProt Gene Name: | FLT3LG |
| UniProt Entry Name: | FLT3L_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 |
| 1000 | 2.437 2.487 | 2.462 | 2.39 |
| 500 | 1.635 1.639 | 1.637 | 1.565 |
| 250 | 0.967 0.961 | 0.964 | 0.892 |
| 125 | 0.455 0.481 | 0.468 | 0.396 |
| 62.5 | 0.255 0.249 | 0.252 | 0.18 |
| 31.25 | 0.185 0.163 | 0.174 | 0.102 |
| 15.63 | 0.117 0.131 | 0.124 | 0.052 |
| 0 | 0.064 0.08 | 0.072 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human Flt3L were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human Flt3L 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) | 50.50 | 97.06 | 482.64 | 45.71 | 88.32 | 486.28 |
| Standard deviation | 2.77 | 4.68 | 26.30 | 2.46 | 3.86 | 25.82 |
| C V (%) | 5.49 | 4.82 | 5.45 | 5.38 | 4.37 | 5.31 |
Recovery
The recovery of Human Flt3L 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) | 93-107 | 101 |
| EDTA plasma (n=5) | 88-102 | 95 |
| Cell culture media (n=5) | 85-100 | 91 |
Linearity
Samples were spiked with high concentrations of Human Flt3L 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 (%) | 91-103 | 93-106 | 92-106 |
| Average (%) | 97 | 101 | 98 | |
| 1:4 | Range (%) | 93-105 | 82-94 | 82-96 |
| Average (%) | 99 | 88 | 89 | |
| 1:8 | Range (%) | 92-103 | 82-93 | 82-95 |
| Average (%) | 98 | 88 | 87 | |
| 1:16 | Range (%) | 87-100 | 82-95 | 82-92 |
| Average (%) | 92 | 88 | 87 |
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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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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