Human AChE (Acetylcholinesterase) ELISA Kit (HUES01488)

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HUES01488
€539

Description

ELISA Kit Technical ManualMSDS

Human AChE (Acetylcholinesterase) 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 AChE . 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 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 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 AChE. The concentration of Human AChE in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range1.56-100 ng/mL
Sensitivity0.94 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

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 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
1002.497
2.517
2.5072.437
501.661
1.689
1.6751.605
250.934
0.904
0.9190.849
12.50.507
0.529
0.5180.448
6.250.25
0.238
0.2440.174
3.130.181
0.161
0.1710.101
1.560.116
0.128
0.1220.052
00.064
0.076
0.07--

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)5.0012.5439.125.0312.8638.83
Standard deviation0.250.721.880.290.631.88
C V (%)5.005.744.815.774.904.84

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)94-106101
EDTA plasma (n=5)86-9891
Cell culture media (n=5)86-9991

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 (%)85-9796-11489-104
Average (%)9210496
1:4Range (%)87-10380-9481-96
Average (%)948788
1:8Range (%)91-10783-9587-100
Average (%)998794
1:16Range (%)87-9985-9786-97
Average (%)939091

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 ELISA 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 Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human AChE (Acetylcholinesterase) ELISA Kit (HUES01488) 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 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.
    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 AChE (Acetylcholinesterase) ELISA Kit (HUES01488) 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:
Sandwich
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