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Acetylcholine (ACH) ELISA Kit

Citations(0)Uniprot : N/A
  • Cat.No.: AE64892GE

  • Reactivity: General

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48T 96T
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Product Details

Species Reactivity General
UniProt N/A
Abbreviation ACH
Alternative Names N/A
Range 31.2-2000 pg/mL
Sensitivity 15.6 pg/mL
Sample Type Serum, Plasma, Other biological fluids
Detection Method Sandwich
Analysis Method Quantitive
Assay Duration 1-4.5h
Sample Volume 1-200 μL
Detection Wavelengt 450 nm

Test principle

This assay employs a two-site sandwich ELISA to quantitate ACH in samples. An antibody specific for ACH has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any ACH present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for ACH is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of ACH bound in the initial step. The color development is stopped and the intensity of the color is measured.
 

Product Overview

Acetylcholine (Ach) is produced by the synthetic enzyme choline acetyltransferase which uses acetyl coenzyme A and choline as substrates for the formation of acetylcholine.Dietary choline and phosphatidylcholine serve as the sources of free choline for acetylcholine synthesis. Upon release, acetylcholine is metabolized into choline and acetate by acetylcholinesterase, and other nonspecific esterases. Acetylcholine release can be excitatory or inhibitory depending on the type of tissue and the nature of the receptor with which it interacts. Cholinergic receptors can be divided into two types, muscarinic and nicotinic, based on the pharmacological action of various agonists and antagonists. Muscarinic receptors originally were distinguished from nicotinic receptors by the selectivity of the agonists muscarine and nicotine respectively.
 

Components


Reagents

Quantity

Reagents

Quantity

Assay plate (96 Wells)

1

Instruction manual

1

Standard (lyophilized)

2

Sample Diluent

1 x 20 mL

Biotin-Conjugate (concentrate 100 x)

1 x 120 μL

Biotin-Conjugate Diluent

1 x 12 mL

Streptavidin-HRP  (concentrate 100 x)

1 x 120 μL

Streptavidin-HRP Diluent   

1 x 12 mL

Wash Buffer (concentrate 25 x)

1 x 20 mL

Substrate Solution

1 x 10 mL

Stop Solution

1 x 6 mL

Adhesive Films

4

 

Specificity

This assay has high sensitivity and excellent specificity for detection of Human ACH. No significant cross-reactivity or interference between Human ACH and analogues was observed.
 

Recovery

Matrices listed below were spiked with certain level of recombinant Human ACH and the recovery rates were calculated by comparing the measured value to the expected amount of Human ACH in samples.

Sample Type

Number

Recovery range (%)

Average(%)

Serum

10

90-101

96

EDTA plasma

10

89-97

93

Heparin plasma

10

91-99

95

 

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 forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%
 

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ACH and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample Type

1:2

1:4

1:8

1:16

Serum

78-89%

81-99%

92-103%

95-105%

EDTA plasma

91-101%

90-98%

93-101%

91-98%

Heparin plasma

92-103%

93-102%

92-99%

91-101%

 

Stability

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
 

Sample collection and storage

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
 

Kits storage instructions

Store at 2-8°C. Please refer to Instruction Manual.