Human Immunology ELISA Kits 1
Human CFD (Complement Factor D) CLIA Kit (HUES00040)
- SKU:
- HUES00040
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 46.88pg/mL
- Range:
- 78.13-5000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- ADN, Adipsin, DF, PFD, Factor D, Properdin Factor D Esterase
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Immunology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 78.13-5000 pg/mL |
| Sensitivity: | 46.88 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human CFD in samples. No significant cross-reactivity or interference between Human CFD 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 CFD. 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 CFD 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 CFD, 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 CFD. The concentration of Human CFD in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | CFD: Factor D cleaves factor B when the latter is complexed with factor C3b, activating the C3bbb complex, which then becomes the C3 convertase of the alternate pathway. Its function is homologous to that of C1s in the classical pathway. Defects in CFD are the cause of complement factor D deficiency (CFDD). CFDD is an immunologic disorder characterized by increased susceptibility to bacterial infections, particularly Neisseria infections, due to a defect in the alternative complement pathway. Belongs to the peptidase S1 family. |
| UniProt Protein Details: | Protein type:Secreted; EC 3. 4. 21. 46; Secreted, signal peptide; Protease Chromosomal Location of Human Ortholog: 19p13. 3 Cellular Component: extracellular region Molecular Function:serine-type endopeptidase activity; serine-type peptidase activity Biological Process: complement activation; complement activation, alternative pathway; platelet degranulation Disease: Complement Factor D Deficiency |
| NCBI Summary: | This gene encodes a member of the S1, or chymotrypsin, family of serine peptidases. This protease catalyzes the cleavage of factor B, the rate-limiting step of the alternative pathway of complement activation. This protein also functions as an adipokine, a cell signaling protein secreted by adipocytes, which regulates insulin secretion in mice. Mutations in this gene underlie complement factor D deficiency, which is associated with recurrent bacterial meningitis infections in human patients. Alternative splicing of this gene results in multiple transcript variants. At least one of these variants encodes a preproprotein that is proteolytically processed to generate the mature protease. [provided by RefSeq, Nov 2015] |
| UniProt Code: | P00746 |
| NCBI GenInfo Identifier: | 158515408 |
| NCBI Gene ID: | 1675 |
| NCBI Accession: | P00746. 5 |
| UniProt Secondary Accession: | P00746,Q5U5S1, Q86VJ5, Q8N4E0, Q8WZB4, B4DV76, |
| UniProt Related Accession: | P00746 |
| Molecular Weight: | 27,033 Da |
| NCBI Full Name: | Complement factor D |
| NCBI Synonym Full Names: | complement factor D |
| NCBI Official Symbol: | CFD |
| NCBI Official Synonym Symbols: | DF; ADN; PFD; ADIPSIN |
| NCBI Protein Information: | complement factor D |
| UniProt Protein Name: | Complement factor D |
| UniProt Synonym Protein Names: | Adipsin; C3 convertase activator; Properdin factor D |
| Protein Family: | Complement factor |
| UniProt Gene Name: | CFD |
| UniProt Entry Name: | CFAD_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 |
| 5000 | 57784 64728 | 61256 | 61233 |
| 2500 | 33813 34895 | 34354 | 34331 |
| 1250 | 19789 16549 | 18169 | 18146 |
| 625 | 8818 9966 | 9392 | 9369 |
| 312.5 | 5212 4454 | 4833 | 4810 |
| 156.25 | 2611 2411 | 2511 | 2488 |
| 78.13 | 1316 1362 | 1339 | 1316 |
| 0 | 22 24 | 23 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CFD were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CFD 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) | 233.24 | 687.31 | 2486.95 | 242.57 | 721.32 | 2632.72 |
| Standard deviation | 22.39 | 49.56 | 250.68 | 31.39 | 85.19 | 235.89 |
| C V (%) | 9.60 | 7.21 | 10.08 | 12.94 | 11.81 | 8.96 |
Recovery
The recovery of Human CFD 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) | 96-111 | 103 |
| EDTA plasma (n=5) | 84-95 | 90 |
| Cell culture media (n=5) | 100-116 | 107 |
Linearity
Samples were spiked with high concentrations of Human CFD 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 (%) | 88-101 | 96-111 | 95-112 |
| Average (%) | 95 | 101 | 103 | |
| 1:4 | Range (%) | 86-98 | 101-117 | 92-109 |
| Average (%) | 91 | 109 | 99 | |
| 1:8 | Range (%) | 99-112 | 86-97 | 100-113 |
| Average (%) | 105 | 92 | 107 | |
| 1:16 | Range (%) | 85-95 | 89-103 | 100-115 |
| Average (%) | 90 | 94 | 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.
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