Human CAD (Caspase Activated Deoxyribonuclease) ELISA Kit
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 CAD . 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 CAD 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 CAD, 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 CAD. The concentration of Human CAD in samples can be calculated by comparing the OD of the samples to the standard curve.
|Detection Range||1.56-100 ng/mL|
|Sample Volume Required Per Well||100uL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes Human CAD in samples. No significant cross-reactivity or interference between Human CAD and analogues was observed.
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CAD were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CAD were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||6.31||4.77||5.31||6.71||5.39||4.20|
The recovery of Human CAD spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||90-105||97|
|Cell culture media (n=5)||89-102||96|
Samples were spiked with high concentrations of Human CAD 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)|
Kit Components & Storage
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.
|Micro ELISA Plate(Dismountable)||8 wells X12 strips||-20'C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100X)||1 vial, 120 uL|
|Concentrated HRP Conjugate (100X)||1 vial, 120 uL||-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 (25X)||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|
Human CAD (Caspase Activated Deoxyribonuclease) ELISA Kit (HUES01821) Assay procedure
- 1. 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.
- 2. Aliquot 100 µL of standard solutions into the standard wells.
- 3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- 4. Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- 5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
- 6. 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.
- 7. 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.
- 8. Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
- 9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 10. 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.
- 11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- 12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
Human CAD (Caspase Activated Deoxyribonuclease) ELISA Kit (HUES01821) Protein Information
|UniProt Protein Function:||DFFB: Nuclease that induces DNA fragmentation and chromatin condensation during apoptosis. Degrades naked DNA and induces apoptotic morphology. 4 isoforms of the human protein are produced by alternative splicing.|
|UniProt Protein Details:|
Protein type:EC 3.-.-.-; Apoptosis; Deoxyribonuclease
Chromosomal Location of Human Ortholog: 1p36.3
Cellular Component: cytosol; nuclear chromatin; nucleolus; nucleoplasm; nucleus
Molecular Function:deoxyribonuclease activity; enzyme binding
Biological Process: apoptotic chromosome condensation; DNA fragmentation during apoptosis
|NCBI Summary:||Apoptosis is a cell death process that removes toxic and/or useless cells during mammalian development. The apoptotic process is accompanied by shrinkage and fragmentation of the cells and nuclei and degradation of the chromosomal DNA into nucleosomal units. DNA fragmentation factor (DFF) is a heterodimeric protein of 40-kD (DFFB) and 45-kD (DFFA) subunits. DFFA is the substrate for caspase-3 and triggers DNA fragmentation during apoptosis. DFF becomes activated when DFFA is cleaved by caspase-3. The cleaved fragments of DFFA dissociate from DFFB, the active component of DFF. DFFB has been found to trigger both DNA fragmentation and chromatin condensation during apoptosis. Alternatively spliced transcript variants encoding distinct isoforms have been found for this gene but the biological validity of some of these variants has not been determined. [provided by RefSeq, Sep 2013]|
|NCBI GenInfo Identifier:||9087148|
|NCBI Gene ID:||1677|
|UniProt Secondary Accession:||O76075,O60521, Q5SR22, Q9BYI4, Q9BYI5, Q9BYI6,|
|UniProt Related Accession:||O76075|
|Molecular Weight:||11,482 Da|
|NCBI Full Name:||DNA fragmentation factor subunit beta|
|NCBI Synonym Full Names:||DNA fragmentation factor subunit beta|
|NCBI Official Symbol:||DFFB|
|NCBI Official Synonym Symbols:||CAD; CPAN; DFF2; DFF40; DFF-40|
|NCBI Protein Information:||DNA fragmentation factor subunit beta|
|UniProt Protein Name:||DNA fragmentation factor subunit beta|
|UniProt Synonym Protein Names:||Caspase-activated deoxyribonuclease; CAD; Caspase-activated DNase; Caspase-activated nuclease; CPAN; DNA fragmentation factor 40 kDa subunit; DFF-40|
|Protein Family:||DNA fragmentation factor|
|UniProt Gene Name:||DFFB|
|UniProt Entry Name:||DFFB_HUMAN|