Human AChE (Acetylcholinesterase) CLIA Kit (HUES00187)
Human AChE (Acetylcholinesterase) CLIA Kit
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 AChE . 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 AChE 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 AChE, 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 AChE. The concentration of Human AChE in the samples can be calculated by comparing the RLU of the samples to the standard curve.
|Detection range||0.31-20 ng/mL|
|Sample type||Serum, plasma and other biological fluids|
|Repeatability||CV < 15%|
This kit recognizes Human AChE in samples. No significant cross-reactivity or interference between Human AChE and analogues was observed.
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human AChE were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human AChE were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||11.01||10.73||9.50||9.57||7.05||11.45|
The recovery of Human AChE 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)||94-107||101|
|Cell culture media (n=5)||85-96||91|
Samples were spiked with high concentrations of Human AChE 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 CLIA 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 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|
Human AChE (Acetylcholinesterase) CLIA Kit (HUES00187) 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 100µl of standard solutions into the standard wells.
- 3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- 4. Add 100µl 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 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.
- 7. 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.
- 8. Add 100µL 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 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.
- 11. Determine the RLU value of each well immediately.
Human AChE (Acetylcholinesterase) CLIA Kit (HUES00187) Protein Information
|UniProt Protein Function:||ACHE: Terminates signal transduction at the neuromuscular junction by rapid hydrolysis of the acetylcholine released into the synaptic cleft. Role in neuronal apoptosis. Belongs to the type-B carboxylesterase/lipase family. 4 isoforms of the human protein are produced by alternative splicing.|
|UniProt Protein Details:|
Protein type:EC 126.96.36.199; Hydrolase; Membrane protein, GPI anchor; Lipid Metabolism - glycerophospholipid
Chromosomal Location of Human Ortholog: 7q22
Cellular Component: Golgi apparatus; extracellular space; cell surface; membrane; perinuclear region of cytoplasm; plasma membrane; extracellular region; synapse; basal lamina; nucleus; neuromuscular junction; cell junction
Molecular Function:collagen binding; serine hydrolase activity; protein binding; protein homodimerization activity; protein self-association; cholinesterase activity; hydrolase activity; beta-amyloid binding; laminin binding; acetylcholinesterase activity; acetylcholine binding
Biological Process: negative regulation of synaptic transmission, cholinergic; nervous system development; muscle development; acetylcholine catabolic process in synaptic cleft; acetylcholine catabolic process; glycerophospholipid biosynthetic process; osteoblast development; cell proliferation; synaptic transmission; synaptogenesis; positive regulation of protein secretion; amyloid precursor protein metabolic process; retina development in camera-type eye; receptor internalization; phospholipid metabolic process; neurotransmitter receptor biosynthetic process; response to wounding; phosphatidylcholine biosynthetic process; regulation of receptor recycling; DNA replication; cell adhesion; protein tetramerization; neurotransmitter biosynthetic process
Disease: Yt Blood Group Antigen
|NCBI Summary:||Acetylcholinesterase hydrolyzes the neurotransmitter, acetylcholine at neuromuscular junctions and brain cholinergic synapses, and thus terminates signal transmission. It is also found on the red blood cell membranes, where it constitutes the Yt blood group antigen. Acetylcholinesterase exists in multiple molecular forms which possess similar catalytic properties, but differ in their oligomeric assembly and mode of cell attachment to the cell surface. It is encoded by the single ACHE gene, and the structural diversity in the gene products arises from alternative mRNA splicing, and post-translational associations of catalytic and structural subunits. The major form of acetylcholinesterase found in brain, muscle and other tissues is the hydrophilic species, which forms disulfide-linked oligomers with collagenous, or lipid-containing structural subunits. The other, alternatively spliced form, expressed primarily in the erythroid tissues, differs at the C-terminal end, and contains a cleavable hydrophobic peptide with a GPI-anchor site. It associates with the membranes through the phosphoinositide (PI) moieties added post-translationally. [provided by RefSeq, Jul 2008]|
|NCBI GenInfo Identifier:||113037|
|NCBI Gene ID:||43|
|UniProt Secondary Accession:||P22303,Q16169, Q29S23, Q2M324, Q504V3, Q53F46, Q86TM9 Q86YX9, Q9BXP7, A4D2E2, B7ZKZ0, D6W5X7,|
|UniProt Related Accession:||P22303|
|Molecular Weight:||58,352 Da|
|NCBI Full Name:||Acetylcholinesterase|
|NCBI Synonym Full Names:||acetylcholinesterase (Yt blood group)|
|NCBI Official Symbol:||ACHE|
|NCBI Official Synonym Symbols:||YT; ACEE; ARACHE; N-ACHE|
|NCBI Protein Information:||acetylcholinesterase; Yt blood group; apoptosis-related acetylcholinesterase|
|UniProt Protein Name:||Acetylcholinesterase|
|UniProt Gene Name:||ACHE|
|UniProt Entry Name:||ACES_HUMAN|