Human Cell Death ELISA Kits
Human FAS/CD95 (Factor Related Apoptosis) CLIA Kit (HUES00065)
- SKU:
- HUES00065
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 9.38pg/mL
- Range:
- 15.63-1000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- FASR, TNFRSF6, APO1, APT1, FAS1, FASTM, ALPS1A
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Death
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 15.63-1000 pg/mL |
| Sensitivity: | 9.38 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human FAS/CD95 in samples. No significant cross-reactivity or interference between Human FAS/CD95 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 FAS/CD95. 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 FAS/CD95 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 FAS/CD95, 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 FAS/CD95. The concentration of Human FAS/CD95 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | FAS: Receptor for TNFSF6/FASLG. The adapter molecule FADD recruits caspase-8 to the activated receptor. The resulting death- inducing signaling complex (DISC) performs caspase-8 proteolytic activation which initiates the subsequent cascade of caspases (aspartate-specific cysteine proteases) mediating apoptosis. FAS- mediated apoptosis may have a role in the induction of peripheral tolerance, in the antigen-stimulated suicide of mature T-cells, or both. The secreted isoforms 2 to 6 block apoptosis (in vitro). Binds DAXX. Interacts with HIPK3. Part of a complex containing HIPK3 and FADD. Binds RIPK1 and FAIM2. Interacts with BRE and FEM1B. Interacts with FADD. Isoform 1 and isoform 6 are expressed at equal levels in resting peripheral blood mononuclear cells. After activation there is an increase in isoform 1 and decrease in the levels of isoform 6. 6 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Apoptosis; Membrane protein, integral; Receptor, cytokine; Cell surface Chromosomal Location of Human Ortholog: 10q24. 1 Cellular Component: cell surface; cytoplasm; plasma membrane; integral to membrane; CD95 death-inducing signaling complex; nucleus; cytosol; lipid raft; external side of plasma membrane Molecular Function:identical protein binding; protein binding; signal transducer activity; transmembrane receptor activity; receptor activity; kinase binding Biological Process: circadian rhythm; caspase activation; spleen development; regulation of myeloid cell differentiation; renal system process; transformed cell apoptosis; apoptosis; positive regulation of apoptosis; positive regulation of protein homooligomerization; regulation of lymphocyte differentiation; response to toxin; response to glucocorticoid stimulus; negative regulation of B cell activation; signal transduction; negative thymic T cell selection; regulation of apoptosis; inflammatory cell apoptosis; B cell mediated immunity; induction of apoptosis via death domain receptors; gene expression; protein complex assembly; immunoglobulin production; activated T cell apoptosis; protein homooligomerization; negative regulation of apoptosis Disease: Autoimmune Lymphoproliferative Syndrome |
| NCBI Summary: | The protein encoded by this gene is a member of the TNF-receptor superfamily. This receptor contains a death domain. It has been shown to play a central role in the physiological regulation of programmed cell death, and has been implicated in the pathogenesis of various malignancies and diseases of the immune system. The interaction of this receptor with its ligand allows the formation of a death-inducing signaling complex that includes Fas-associated death domain protein (FADD), caspase 8, and caspase 10. The autoproteolytic processing of the caspases in the complex triggers a downstream caspase cascade, and leads to apoptosis. This receptor has been also shown to activate NF-kappaB, MAPK3/ERK1, and MAPK8/JNK, and is found to be involved in transducing the proliferating signals in normal diploid fibroblast and T cells. Several alternatively spliced transcript variants have been described, some of which are candidates for nonsense-mediated mRNA decay (NMD). The isoforms lacking the transmembrane domain may negatively regulate the apoptosis mediated by the full length isoform. [provided by RefSeq, Mar 2011] |
| UniProt Code: | P25445 |
| NCBI GenInfo Identifier: | 119833 |
| NCBI Gene ID: | 355 |
| NCBI Accession: | P25445. 1 |
| UniProt Secondary Accession: | P25445,Q14292, Q14293, Q14294, Q14295, Q16652, Q5T9P1 Q5T9P2, Q5T9P3, Q6SSE9, A9UJX4, B6VNV4, |
| UniProt Related Accession: | P25445 |
| Molecular Weight: | 37,732 Da |
| NCBI Full Name: | Tumor necrosis factor receptor superfamily member 6 |
| NCBI Synonym Full Names: | Fas cell surface death receptor |
| NCBI Official Symbol: | FAS |
| NCBI Official Synonym Symbols: | APT1; CD95; FAS1; APO-1; FASTM; ALPS1A; TNFRSF6 |
| NCBI Protein Information: | tumor necrosis factor receptor superfamily member 6; Fas AMA; FAS 827dupA; CD95 antigen; FASLG receptor; apoptosis antigen 1; Delta Fas/APO-1/CD95; FAS receptor variant 9; APO-1 cell surface antigen; TNF receptor superfamily member 6; apoptosis-mediating surface antigen FAS; Fas (TNF receptor superfamily, member 6); tumor necrosis factor receptor superfamily, member 6 |
| UniProt Protein Name: | Tumor necrosis factor receptor superfamily member 6 |
| UniProt Synonym Protein Names: | Apo-1 antigen; Apoptosis-mediating surface antigen FAS; FASLG receptor |
| Protein Family: | Development-specific protein |
| UniProt Gene Name: | FAS |
| UniProt Entry Name: | TNR6_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 |
| 1000 | 49923 57427 | 53675 | 53650 |
| 500 | 21166 23812 | 22489 | 22464 |
| 250 | 11033 9389 | 10211 | 10186 |
| 125 | 4620 5182 | 4901 | 4876 |
| 62.5 | 2539 2367 | 2453 | 2428 |
| 31.25 | 1378 1184 | 1281 | 1256 |
| 15.63 | 664 752 | 708 | 683 |
| 0 | 24 26 | 25 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human FAS/CD95 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human FAS/CD95 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) | 49.71 | 144.43 | 466.80 | 52.94 | 149.22 | 443.94 |
| Standard deviation | 4.31 | 14.76 | 49.11 | 4.46 | 10.71 | 27.35 |
| C V (%) | 8.67 | 10.22 | 10.52 | 8.42 | 7.18 | 6.16 |
Recovery
The recovery of Human FAS/CD95 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) | 85-97 | 90 |
| EDTA plasma (n=5) | 98-109 | 104 |
| Cell culture media (n=5) | 98-110 | 105 |
Linearity
Samples were spiked with high concentrations of Human FAS/CD95 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 (%) | 95-111 | 92-107 | 96-109 |
| Average (%) | 102 | 99 | 103 | |
| 1:4 | Range (%) | 97-113 | 95-109 | 97-108 |
| Average (%) | 103 | 101 | 103 | |
| 1:8 | Range (%) | 85-99 | 93-110 | 92-108 |
| Average (%) | 92 | 101 | 98 | |
| 1:16 | Range (%) | 102-116 | 102-120 | 91-105 |
| Average (%) | 109 | 109 | 98 |
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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