Human ACHE ELISA Kit

Sample

cell culture supernates, tissue lysates, serum, plasma

Species Reactivity

Human

Intended Use

For the quantitative determination of human Acetylcholinesterase (ACHE) concentrations in cell culture supernates, tissue lysates, serum, and plasma.

Contents of Kit

1. Human ACHE Microplate: 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody specific for human ACHE.
Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 1 month at 2-8 °C.*

2. Human ACHE Standard: 2 vials of recombinant human ACHE in a buffered protein base with preservatives; lyophilized. Refer to the vial label for reconstitution volume.
Discard after use. Use a new standard for each assay.

3. Human ACHE Conjugate: 21 mL of a polyclonal antibody specific for human ACHE conjugated to horseradish peroxidase with preservatives.
4. Assay Diluent CD2-74: 12 mL of a buffered protein base with and preservatives.
5. Calibrator Diluent CD6-37 Concentrate: 21 mL of a concentrated buffered protein base with preservatives. Use diluted 1:4 in this assay.
6. Wash Buffer Concentrate: 21 mL of a 25-fold concentrated solution of buffered surfactant with preservative. May turn yellow over time.
7. Color Reagent A: 12 mL of stabilized hydrogen peroxide.
8. Color Reagent B: 12 mL of stabilized chromogen (tetramethylbenzidine).
9. Stop Solution: 6 mL of 2 N sulfuric acid.
May be stored for up to 1 month at 2-8 °C.*

10. Plate Sealers: 4 adhesive strips.

* Provided this is within the expiration date of the kit.

Precision

Intra-assay Precision (Precision within an assay)
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay Precision (Precision between assays)
Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.

Sensitivity

Twenty-three assays were evaluated and the minimum detectable dose (MDD) of human ACHE ranged from 11.5-43.7 pg/mL. The mean MDD was 24.8 pg/mL.
The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

General Description

Acetylcholinesterase (ACHE), a member of the carboxylesterase family of enzymes, is a serine hydrolase that plays an indispensable role in cholinergic neurotransmission. It rapidly and selectively hydrolyzes the neurotransmitter acetylcholine (ACH) at cholinergic synapses and neuromuscular junctions (1). ACHE is expressed throughout the nervous system and is found in both cholinergic and noncholinergic fibers (2). ACHE is also expressed in many non-neural cells and tissues including muscle, haematopoietic cells, leukocytes, vascular endothelial cells, and osteoblasts, (3, 4).
In the ACHE gene, alternate promoters and alternative splicing produces multiple ACHE isoforms with different N- and C-termini, respectively (1, 4-6). ACHE-Synaptic (ACHE-S) contains a 40 amino acid (aa) C-terminal peptide and can exist in both monomeric and multimeric forms (5-7). The human ACHE-S monomer is synthesized as a 614 aa precursor protein with a predicted molecule weight of approximately 68 kDa. It shares 89% aa sequence identity with the mouse and rat orthologs. ACHE-Erythrocyte (ACHE-E) is a GPI-anchored dimeric protein found in the membranes of erythrocytes. It constitutes the Yt blood group antigen (7-9). ACHERead-Through (ACHE-R) is a soluble monomer that is produced by the inclusion of the normally spliced-out intron 4 (6, 7). Its expression has been reported to be elevated during stress (1, 9). In addition to its classical function of ACH hydrolysis, ACHE has been shown to be involved in many non-cholinergic functions including playing a role in the pathology of Alzheimer's disease (AD). ACHE is found in excess in the aged brain and has been found to co-localize with Aβ deposits (10-12). Considered a chaperone molecule, ACHE has been shown to accelerate the assembly of Aβ peptides into fibrils by complexing with Aβ via its peripheral anionic site (12-16). It also increases the neurotoxicity of aggregated Aβ, augmenting the effects induced by Aβ oligomers and fibrils, such as calcium homeostasis dysregulation and mitochondria dysfunction (12, 14). Additionally, ACHE has been shown to promote neurodegeneration of cholinergic neurons by inducing tau hyperphosphorylation and apoptosis (12, 17). ACHE inhibitors have been used to delay symptoms of AD patients by virtue of their ability to enhance ACH availability, as well as reduce amyloidogenesis and subsequent neurotoxicity (13, 16). ACHE has also been shown to be involved in cell growth, stem cell differentiation, neurite outgrowth, cell adhesion, apoptosis, synaptogenesis, activation of dopaminergic neurons, haematopoiesis, and thrombopoiesis (1, 3, 4, 7, 9, 13, 18-20). It is believed to play a role in tumorigenesis, and low ACHE levels are associated with pesticide toxicity (9, 21). Furthermore, ACHE is involved in the cholinergic anti-inflammatory pathway and is associated with lowgrade systemic inflammation in obesity, hypertension, type II diabetes, and AD (22). The Human Acetylcholinesterase/ACHE Immunoassay is a 4.5 hour solid phase ELISA designed to measure ACHE levels in cell culture supernates, tissue lysates, serum, and plasma. It contains CHO cell-expressed recombinant human ACHE and antibodies raised against the recombinant protein. Results obtained for naturally occurring human ACHE showed linear curves that were parallel to the standard curves obtained using the Human ACHE Immunoassay standards. These results indicate that this kit can be used to determine relative mass values for natural human ACHE

Standard Curve