Description
Human CHAc (Choline acetylase) ELISA Kit
The Human CHAC (Choline Acetylase) ELISA Kit is specifically designed for the precise measurement of CHAC levels in human samples, including serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for a variety of research purposes.CHAC is an important enzyme involved in choline metabolism and acetylcholine synthesis, playing a key role in various physiological processes. Dysregulation of CHAC has been linked to diseases such as Alzheimer's, Parkinson's, and cardiovascular disorders, making it a valuable biomarker for studying these conditions and potential therapeutic interventions.
With its reliability and reproducibility, the Human CHAC ELISA Kit is an indispensable tool for researchers seeking to investigate the role of CHAC in health and disease, providing valuable insights for the development of novel treatments and therapies.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.31-20 ng/mL |
| Sensitivity: | 0.19 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human CHAc in samples. No significant cross-reactivity or interference between Human CHAc 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 CHAc. 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 CHAc 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 CHAc, 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 CHAc. The concentration of Human CHAc in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | CHAT: an enzyme that catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Four alternative splice variants have been described. |
| UniProt Protein Details: | Protein type:EC 2. 3. 1. 6; Lipid Metabolism - glycerophospholipid; Acetyltransferase Chromosomal Location of Human Ortholog: 10q11. 2 Cellular Component: cell soma; mitochondrion; axon; cytoplasm; nucleus; cytosol Molecular Function:choline O-acetyltransferase activity Biological Process: rhythmic excitation; muscle development; neurotransmitter secretion; glycerophospholipid biosynthetic process; adult walking behavior; synaptic transmission; rhythmic behavior; phospholipid metabolic process; phosphatidylcholine biosynthetic process; dendrite development; neuromuscular synaptic transmission; establishment of synaptic specificity at neuromuscular junction; neurotransmitter biosynthetic process Disease: Myasthenic Syndrome, Congenital, Associated With Episodic Apnea |
| NCBI Summary: | This gene encodes an enzyme which catalyzes the biosynthesis of the neurotransmitter acetylcholine. This gene product is a characteristic feature of cholinergic neurons, and changes in these neurons may explain some of the symptoms of Alzheimer's disease. Polymorphisms in this gene have been associated with Alzheimer's disease and mild cognitive impairment. Mutations in this gene are associated with congenital myasthenic syndrome associated with episodic apnea. Multiple transcript variants encoding different isoforms have been found for this gene, and some of these variants have been shown to encode more than one isoform. [provided by RefSeq, May 2010] |
| UniProt Code: | P28329 |
| NCBI GenInfo Identifier: | 281185509 |
| NCBI Gene ID: | 1103 |
| NCBI Accession: | P28329. 4 |
| UniProt Secondary Accession: | P28329,Q16488, Q9BQ23, Q9BQ35, Q9BQE1, A2BDF4, A2BDF5 |
| UniProt Related Accession: | P28329 |
| Molecular Weight: | 748 |
| NCBI Full Name: | Choline O-acetyltransferase |
| NCBI Synonym Full Names: | choline O-acetyltransferase |
| NCBI Official Symbol: | CHAT |
| NCBI Official Synonym Symbols: | CMS1A; CMS1A2; CHOACTASE |
| NCBI Protein Information: | choline O-acetyltransferase; choline acetylase; acetyl CoA:choline O-acetyltransferase |
| UniProt Protein Name: | Choline O-acetyltransferase |
| Protein Family: | (Z)-3-hexen-1-ol acetyltransferase |
| UniProt Gene Name: | CHAT |
| UniProt Entry Name: | CLAT_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 (ng/mL) | O.D | Average | Corrected |
| 20 | 2.343 2.359 | 2.351 | 2.283 |
| 10 | 1.478 1.48 | 1.479 | 1.411 |
| 5 | 0.852 0.852 | 0.852 | 0.784 |
| 2.5 | 0.374 0.384 | 0.379 | 0.311 |
| 1.25 | 0.235 0.217 | 0.226 | 0.158 |
| 0.63 | 0.16 0.16 | 0.16 | 0.092 |
| 0.31 | 0.108 0.122 | 0.115 | 0.047 |
| 0 | 0.061 0.075 | 0.068 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CHAc were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CHAc 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 (ng/mL) | 0.93 | 2.85 | 8.10 | 0.86 | 3.03 | 8.45 |
| Standard deviation | 0.05 | 0.15 | 0.44 | 0.05 | 0.17 | 0.46 |
| C V (%) | 5.38 | 5.26 | 5.43 | 5.81 | 5.61 | 5.44 |
Recovery
The recovery of Human CHAc 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) | 90-104 | 96 |
| EDTA plasma (n=5) | 88-99 | 93 |
| Cell culture media (n=5) | 88-101 | 95 |
Linearity
Samples were spiked with high concentrations of Human CHAc 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 (%) | 92-107 | 98-109 | 86-100 |
| Average (%) | 100 | 103 | 93 | |
| 1:4 | Range (%) | 89-104 | 82-96 | 85-97 |
| Average (%) | 96 | 87 | 91 | |
| 1:8 | Range (%) | 88-101 | 78-90 | 83-98 |
| Average (%) | 93 | 85 | 89 | |
| 1:16 | Range (%) | 94-108 | 85-96 | 83-96 |
| Average (%) | 99 | 90 | 89 |
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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 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.
- 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.
- Add 100 µL 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 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.
- 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.
