Description
Human CYPD (Cyclophilin D) ELISA Kit
The Human CypD (Cyclophilin D) ELISA Kit is a powerful tool for detecting and measuring levels of CypD in human samples such as serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit provides accurate and reliable results for a variety of research applications.Cyclophilin D is a key regulator of mitochondrial function, playing a critical role in cell death pathways and diseases such as Alzheimer's, Parkinson's, and ischemia-reperfusion injury.
By measuring CypD levels, researchers can gain valuable insights into the mechanisms underlying these conditions and potentially identify new therapeutic targets.Overall, the Human CypD (Cyclophilin D) ELISA Kit is a valuable tool for studying mitochondrial function, cell death pathways, and disease processes, making it a must-have for any laboratory conducting research in these areas.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 62.50-4000 pg/mL |
| Sensitivity: | 37.50 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human CYPD in samples. No significant cross-reactivity or interference between Human CYPD 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 CYPD. 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 CYPD 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 CYPD, 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 CYPD. The concentration of Human CYPD in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | PPID: PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides. Belongs to the cyclophilin-type PPIase family. PPIase D subfamily. |
| UniProt Protein Details: | Protein type:Nucleolus; Isomerase; EC 5. 2. 1. 8 Chromosomal Location of Human Ortholog: 4q31. 3 Cellular Component: cytoplasm; nucleolus; nucleus Molecular Function:cyclosporin A binding; estrogen receptor binding; heat shock protein binding; Hsp70 protein binding; Hsp90 protein binding; peptidyl-prolyl cis-trans isomerase activity; protein binding; transcription factor binding Biological Process: negative regulation of transcription from RNA polymerase II promoter; positive regulation of apoptosis; positive regulation of protein secretion; positive regulation of viral genome replication; protein complex assembly; protein folding; release of virus from host |
| NCBI Summary: | The protein encoded by this gene is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family. PPIases catalyze the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. This protein has been shown to possess PPIase activity and, similar to other family members, can bind to the immunosuppressant cyclosporin A. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q08752 |
| NCBI GenInfo Identifier: | 729274 |
| NCBI Gene ID: | 5481 |
| NCBI Accession: | Q08752. 3 |
| UniProt Secondary Accession: | Q08752,B2R9V2, |
| UniProt Related Accession: | Q08752 |
| Molecular Weight: | 40,764 Da |
| NCBI Full Name: | Peptidyl-prolyl cis-trans isomerase D |
| NCBI Synonym Full Names: | peptidylprolyl isomerase D |
| NCBI Official Symbol: | PPID |
| NCBI Official Synonym Symbols: | CYPD; CYP-40 |
| NCBI Protein Information: | peptidyl-prolyl cis-trans isomerase D |
| UniProt Protein Name: | Peptidyl-prolyl cis-trans isomerase D |
| UniProt Synonym Protein Names: | 40 kDa peptidyl-prolyl cis-trans isomerase; Cyclophilin-40; CYP-40; Cyclophilin-related protein; Rotamase D |
| Protein Family: | Peptidyl-prolyl cis-trans isomerase |
| UniProt Gene Name: | PPID |
| UniProt Entry Name: | PPID_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 |
| 4000 | 2.444 2.47 | 2.457 | 2.39 |
| 2000 | 1.615 1.635 | 1.625 | 1.558 |
| 1000 | 0.897 0.863 | 0.88 | 0.813 |
| 500 | 0.47 0.494 | 0.482 | 0.415 |
| 250 | 0.243 0.225 | 0.234 | 0.167 |
| 125 | 0.168 0.158 | 0.163 | 0.096 |
| 62.50 | 0.11 0.124 | 0.117 | 0.05 |
| 0 | 0.061 0.073 | 0.067 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CYPD were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CYPD 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) | 198.69 | 469.78 | 1867.98 | 187.83 | 435.71 | 1685.54 |
| Standard deviation | 10.05 | 25.70 | 102.55 | 11.44 | 24.57 | 71.47 |
| C V (%) | 5.06 | 5.47 | 5.49 | 6.09 | 5.64 | 4.24 |
Recovery
The recovery of Human CYPD 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) | 91-102 | 96 |
| EDTA plasma (n=5) | 85-97 | 92 |
| Cell culture media (n=5) | 89-100 | 94 |
Linearity
Samples were spiked with high concentrations of Human CYPD 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 (%) | 88-102 | 86-98 | 92-106 |
| Average (%) | 94 | 93 | 97 | |
| 1:4 | Range (%) | 88-102 | 85-97 | 86-99 |
| Average (%) | 94 | 91 | 91 | |
| 1:8 | Range (%) | 94-107 | 83-96 | 84-94 |
| Average (%) | 99 | 90 | 89 | |
| 1:16 | Range (%) | 90-103 | 85-96 | 85-97 |
| Average (%) | 95 | 90 | 91 |
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 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 90µL 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.
