Human AChE (Acetylcholinesterase) CLIA Kit (HUES00187)

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HUES00187
€599

Description

ELISA Kit Technical ManualMSDS

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.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human AChE in samples. No significant cross-reactivity or interference between Human AChE and analogues was observed.

Typical Data

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
(ng/mL)
RLU Average Corrected
2032754
35210
3398233950
1012253
12687
1247012438
55512
4988
52505218
2.52373
2675
25242492
1.251406
1360
13831351
0.63892
842
867835
0.31601
645
623591
032
32
32--

Precision

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
Sample123123
n202020202020
Mean (ng/mL)1.092.339.791.152.279.26
Standard deviation0.120.250.930.110.161.06
C V (%)11.0110.739.509.577.0511.45

Recovery

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 (%)
Serum (n=5)84-9791
EDTA plasma (n=5)94-107101
Cell culture media (n=5)85-9691

Linearity

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)
1:2Range (%)103-11986-9695-108
Average (%)11091101
1:4Range (%)96-10893-10593-108
Average (%)10198100
1:8Range (%)99-115100-115102-118
Average (%)108108108
1:16Range (%)89-9998-11499-111
Average (%)94105105

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.

Item Specifications Storage
Micro CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 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 Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 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 3.1.1.7; 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]
UniProt Code:P22303
NCBI GenInfo Identifier:113037
NCBI Gene ID:43
NCBI Accession:P22303.1
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
Protein Family:Achelase
UniProt Gene Name:ACHE  
UniProt Entry Name:ACES_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
CLIA
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