I. Principle of Acetylcholine ELISA
This blog synthesizes information from several sources regarding acetylcholine (ACh), acetylcholinesterase (AChE), and Enzyme-Linked Immunosorbent Assay (ELISA) technology.
ACh is a critical neurotransmitter involved in a wide range of physiological processes,
including memory, attention, and autonomic functions. Its breakdown is primarily
regulated by AChE. Dysregulation of the cholinergic system, particularly AChE activity,
is implicated in the pathogenesis and progression of numerous neurodegenerative
diseases such as Alzheimer's Disease (AD), Parkinson's Disease (PD),
Huntington's Disease (HD), Multiple Sclerosis (MS),
Amyotrophic Lateral Sclerosis (ALS), Olivopontocerebellar Atrophy (OPCA),
Spinocerebellar Ataxia (SCA), Progressive Supranuclear Palsy (PSP), and
Depressive Disorders.
ELISA kits are widely used diagnostic and research tools for detecting and quantifying
ACh, AChE, and related components like ACh receptor antibodies in biological samples.
While traditional AChE inhibitors (e.g., rivastigmine, donepezil, galantamine,
huperzine A) primarily offer symptomatic relief in neurodegenerative diseases,
emerging research suggests their potential in disease modification by influencing
inflammation, oxidative stress, and pathological protein aggregation. Furthermore,
multi-target directed ligands (MTDLs) and natural plant extracts are being investigated
for their multi-faceted therapeutic benefits, including AChE inhibition.
The detection of acetylcholine typically employs a quantitative sandwich ELISA method.
1. A micro-ELISA strip plate is pre-coated with an antibody specific to acetylcholine.
2. Standards or samples containing ACh are added to the wells, allowing any ACh present to bind to the coated antibody.
3. A Horseradish Peroxidase (HRP)-conjugated antibody, also specific for acetylcholine, is then added and incubated, forming a "sandwich" complex.
4. Unbound components are washed away.
5. A TMB (3,3',5,5'-tetramethylbenzidine) substrate solution is added. In wells containing the HRP-conjugated antibody bound to ACh, the substrate reaction produces a blue color.
6. A stop solution is added, which changes the color from blue to yellow.
7. The optical density (OD) is measured spectrophotometrically at 450 nm using an ELISA plate reader.
8. The OD value is directly proportional to the concentration of acetylcholine in the sample. The concentration is calculated by comparing the sample's OD to a standard curve generated from known concentrations of ACh standards.
Alternatively, for smaller molecules, a competitive ELISA format can be used, where a higher concentration of ACh in the sample leads to less binding of enzyme-linked antibody to antigen coated in the well, resulting in a weaker signal.
II. Sample Collection and Preparation
Acetylcholine ELISA kits can detect ACh in various biological samples including serum, plasma, urine, tissue homogenates, cell culture supernates, saliva, and feces. Proper sample handling is crucial for accurate results.
• General Recommendations:
◦ Assay freshly prepared samples immediately or store in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
◦ Bring samples to room temperature (20-25°C) before assaying.
◦ Centrifuge samples (e.g., 5 min at 10,000-15,000 rpm in a microfuge for serum) to remove particulate matter, especially if cloudy.
◦ Hemolytic specimens should not be used as hemolysis can influence results.
• Specific Sample Types:
◦ Serum: Use a serum separator tube. Allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1,000×g (11). Serum is highly recommended over plasma.
◦ Plasma (EDTA or Heparin): Centrifuge samples for 15 minutes at 1,000×g at 2-8°C within 30 minutes of collection.
◦ Tissue Homogenates: Rinse in ice-cold PBS, weigh, mince, and homogenize in lysis buffer (e.g., 1:20-1:50 w:v) on ice. Sonicate until clarified, then centrifuge for 5 minutes at 10,000×g. Collect supernates.
◦ Cell Lysates: Wash adherent cells with cold PBS, detach with trypsin, and centrifuge at 1,000×g for 5 minutes. Wash cells three times in cold PBS. Resuspend cells in fresh lysis buffer at 10^7 cells/mL. Sonicate if necessary, then centrifuge at 1,500×g for 10 minutes at 2-8°C to remove cellular debris.
◦ Urine: Collect first urine of the day (mid-stream) into a sterile container. Centrifuge to remove particulate matter.
◦ Saliva: Collect using a device. Centrifuge samples at 1,000×g at 2-8°C for 15 minutes. Remove particulates.
◦ Feces: Collect dry feces (>50 mg). Wash three times with PBS (1:9 w:v), sonicate or mash, then centrifuge at 5,000×g for 10 minutes. Collect the supernatant.
• For Cell Lines (Special Considerations):
◦ Culture in Serum-Free Medium: Fetal bovine serum (FBS) contains high amounts of acetylcholine, so cells must be cultured in serum-free medium to accurately measure ACh in the supernatant (19).
◦ Inhibit Acetylcholinesterase (AChE): ACh is quickly degraded by AChE, so it is essential to inhibit this enzyme to measure the ACh produced by cells.
▪ Add an AChE inhibitor such as neostigmine (e.g., 100 µM) to the cell culture medium for a specified time (e.g., 4 hours at 37°C) before collecting samples.
▪ Optimize the concentration of the inhibitor as high concentrations can be toxic and affect ACh levels. For example, 100-200 µM neostigmine was found optimal for A549 lung cancer cells, while 300 µM was toxic.
◦ Post-Collection Processing: Supernatants are typically collected, spun, then frozen at -80°C and lyophilized overnight to concentrate the sample. The lyophilizate can then be reconstituted with a smaller volume of autoclaved water (e.g., 1/5 original volume), snap-frozen in liquid nitrogen, and stored at -80°C for later analysis.
III. Reagent Preparation
Ensure all reagents are at room temperature (20-25°C) for at least 30 minutes before use.
• Wash Solution: Dilute concentrated wash buffer (e.g., 30-fold concentrate) with pure/DI water according to kit instructions.
• Standards: Prepare a series of dilutions from the provided high standard. For example, using a stock of 225 pg/ml, serially dilute to concentrations like 150, 100, 50, 25, 12.5 pg/ml, with sample diluent serving as the zero standard/blank.
IV. Assay Procedure
(Based on a typical quantitative sandwich ELISA, e.g., AFG Scientific kit:
Day 1 (for kits requiring overnight incubation):
1. Sample-AChR Mixture (if applicable to the specific kit): If the kit involves pre-incubation of samples with a receptor mixture, pipette 100 µL of samples (calibrators, controls, test sera) into individual microcentrifuge tubes. Add 25 µL of the foetal and adult type AChR mixture, seal, vortex gently, and incubate overnight (16-20 hours) at 2–8°C.
Day 2 (or immediately if no overnight incubation):
1. Plate Setup: Gently mix sample-AChR mixtures (if pre-incubated). Pipette duplicate 50 µL of each sample-AChR mixture (or just samples/standards if no pre-incubation) into the AChR MAb1 coated wells.
2. Sample Incubation: Cover the plate and incubate at 37°C for 40 minutes (or as per kit instructions).
3. Washing: Aspirate the wells and wash five times with the diluted wash solution (e.g., 300 µL per wash). Tap inverted wells gently on absorbent tissue to remove excess wash solution. Insufficient washing is a common cause of high background or poor replicate data.
4. HRP-Conjugate Addition: Pipette 50 µL of the HRP-conjugated antibody into each well (except blank wells). Cover the plate and incubate at 37°C for 40 minutes.
5. Second Washing: Repeat the washing step five times.
6. Substrate Addition: Add 50 µL of Chromogen Solution A, followed by 50 µL of Chromogen Solution B to each well (including blanks). Avoid light exposure during this step.
7. Color Development: Cover the plate and incubate in the dark at 37°C for 20 minutes (or as per kit instructions).
8. Stop Reaction: Add 50 µL of Stop solution to each well (including blanks) and gently shake for approximately 5 seconds.
9. Read Absorbance: Within 30 minutes of adding stop solution, read the absorbance of each well at 450 nm using an ELISA plate reader. Blank the reader against wells containing only TMB and stop solution.
V. Result Analysis
1. Standard Curve: Plot the calibrator concentrations on the x-axis (linear scale) against their corresponding absorbance values on the y-axis (linear scale). A 4-parameter curve fit is commonly used.
2. Sample Concentration: Determine the ACh concentration in test samples by interpolating their absorbance values on the standard curve.
3. Dilution Factor: If samples were diluted prior to the assay, multiply the calculated concentration by the dilution factor (e.g., multiply by 5 if a 5-fold dilution was performed). Note that some sera may not dilute linearly, and it's suggested to use the dilution giving a value closest to 50% inhibition for calculation.
4. Cut-off and Reference Ranges: Each laboratory should establish its own normal and pathological reference ranges for AChRAb levels (for autoantibodies).
VI. Troubleshooting and Accuracy Considerations
Several factors can affect the accuracy and reliability of ELISA results.
• Reagent Quality and Storage: Use high-quality, unexpired reagents stored according to manufacturer's instructions (most kits at 2-8°C).
• Pipetting and Dilutions: Ensure accurate pipetting and correct preparation of all dilutions to avoid weak/strong signals or poor standard curves/replicates.
• Washing Technique: Insufficient washing is a common issue leading to high background. Ensure thorough washing, completely draining wells after each step, and potentially increasing soak times.
• Incubation Conditions: Maintain consistent incubation temperatures and adhere to recommended incubation times. Use plate sealers to prevent evaporation and cross-contamination3233. Avoid stacking plates during incubation to ensure even temperature distribution.
• Interferences: Be aware of potential interferences from matrix components (e.g., from sample type), cross-reactivity with similar substances, or the presence of enzyme inhibitors (e.g., NaN3) in samples.
• Equipment: Ensure the ELISA plate reader is calibrated and set to the correct wavelength (e.g., 450 nm for TMB substrate) (26,34). Avoid scratching wells with pipette or washer tips.
References
AFG Scientific. (n.d.). Human acetylcholine (ACH) Elisa Kit. Retrieved [Current Date], from [Source content for "Human acetylcholine (ACH) Elisa Kit - AFG Scientific" provided in query].
Biocompare. (n.d.). Acetylcholine ELISA Kits. Retrieved [Current Date], from [Source content for "Acetylcholine ELISA Kits - Biocompare" provided in query].
Lau, J. K., Brown, K. C., & Dasgupta, P. (2013). Measurement of acetylcholine from cell lines. Bio-protocol, 3(24), e1007. doi:10.21769/bioprotoc.1007
MyBioSource. (n.d.). Factors affecting the accuracy and reliability of ELISA results. Retrieved [Current Date], from [Source content for "Factors affecting the accuracy and reliability of ELISA results - MyBioSource" provided in query].
RSR Limited. (n.d.). ELISA AChRAb Acetylcholine Receptor Autoantibody ELISA Kit - Instructions for use. Retrieved [Current Date], from [Source content for "ELISA AChRAb Acetylcholine Receptor Autoantibody Kit - Instructions for use - RSR Limited" provided in query].
Thermo Fisher Scientific. (n.d.). ELISA Troubleshooting Guide. Retrieved [Current Date], from [Source content for "ELISA Troubleshooting Guide | Thermo Fisher Scientific - ES" provided in query].
Walczak-Nowicka, Ł. J., & Herbet, M. (2021). Acetylcholinesterase inhibitors in the treatment of neurodegenerative diseases and the role of acetylcholinesterase in their pathogenesis. International Journal of Molecular Sciences, 22(17), 9290. doi:10.3390/ijms22179290
Wikipedia. (n.d.). ELISA. Retrieved [Current Date], from https://en.wikipedia.org/w/index.php?title=ELISA&oldid=1292958058
Yan, C., Li, W., Song, J., Feng, X., Xi, J., Lu, J., Zhou, S., & Zhao, C. (2019). Cell-based versus enzyme-linked immunosorbent assay for the detection of acetylcholine receptor antibodies in Chinese juvenile myasthenia gravis. Pediatric Neurology, 98, 74–79. doi:10.1016/j.pediatrneurol.2019.01.016
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