Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 7.81-500 pg/mL |
| Sensitivity: | 4.69 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human Flt3L in samples. No significant cross-reactivity or interference between Human Flt3L 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 Flt3L. 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 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 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 Flt3L. The concentration of Human Flt3L in the samples can be calculated by comparing the RLU 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 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 |
| 500 | 50657 52417 | 51537 | 51508 |
| 250 | 18936 23060 | 20998 | 20969 |
| 125 | 10133 8659 | 9396 | 9367 |
| 62.5 | 4203 4821 | 4512 | 4483 |
| 31.25 | 2483 2115 | 2299 | 2270 |
| 15.63 | 1320 1180 | 1250 | 1221 |
| 7.81 | 708 772 | 740 | 711 |
| 0 | 29 29 | 29 | -- |
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) | 27.22 | 42.60 | 226.45 | 26.78 | 46.32 | 221.91 |
| Standard deviation | 2.21 | 4.17 | 25.29 | 3.21 | 5.49 | 13.36 |
| C V (%) | 8.12 | 9.79 | 11.17 | 11.99 | 11.85 | 6.02 |
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) | 92-107 | 98 |
| EDTA plasma (n=5) | 97-110 | 105 |
| Cell culture media (n=5) | 99-115 | 105 |
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 (%) | 87-99 | 95-110 | 83-98 |
| Average (%) | 94 | 101 | 90 | |
| 1:4 | Range (%) | 93-106 | 94-107 | 84-96 |
| Average (%) | 100 | 102 | 91 | |
| 1:8 | Range (%) | 99-115 | 89-104 | 91-106 |
| Average (%) | 107 | 97 | 98 | |
| 1:16 | Range (%) | 97-115 | 96-114 | 99-116 |
| Average (%) | 105 | 104 | 108 |
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.
