Human ACH(Acetylcholine) ELISA Kit

Cat: ELK8634
For research use only. Not intended for diagnostic use.

Sensitivity: 5.31 pg/mL

Detection Range: 15.63-1000 pg/mL
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.

Please refer to the outer packaging label of the kit for the specific shelf life.

KIT Components
Quantity
Reagents Storage Condition
48T 96T
Pre-Coated Microplate 6 strips x 8 wells 12 strips x 8 wells 4°C/-20°C
Standard (Lyophilized) 1 vial 2 vials 4°C/-20°C
Biotinylated-Conjugate (100×) 30 μL 60 μL 4°C/-20°C

Streptavidin-HRP (100×) 60 μL 120 μL 4°C/-20°C

Standard/Sample Diluent Buffer 10 mL 20 mL 4°C/-20°C
Biotinylated-Conjugate Diluent 5 mL 10 mL 4°C/-20°C
HRP Diluent 6 mL 12 mL 4°C/-20°C
Wash Buffer (25×) 10 mL 20 mL 4°C/-20°C
TMB Substrate Solution 6 mL 10 mL 4°C/-20°C (store in dark)
Stop Reagent 3 mL 6 mL 4°C/-20°C
Plate Covers 1 piece 2 pieces RT

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Special Explanation
1. *If the kit is opened, Store the whole kit at 4°C. If the kit is not used up in 1 week. Store the
Pre-Coated Microplate, Standard ,Biotinylated-Conjugate and Streptavidin-HRP at -20°C, the rest
reagents at 4°C, please used up within 6 months.
*If the kit is not opened, store the whole kit: 4°C(short time storage, valid for 6 months); -20°C
(long-term storage, valid for 1 year). Avoid repeated freeze-thaw cycles
2. Do not use the kit beyond the expiration date.
3. If the whole kit is stored at -20°C, place the kit at 4°C the day before the experiment.
4. After opening the package, please check that all components are complete.
5. The cap must be tightened to prevent evaporation and microbial contamination. The reagents
volume is slightly more than the volume marked on labels, please use accurate measuring
equipment and do not pour directly into the vial.
All kit components have been formulated and quality control tested to function successfully. Do not mix
or substitute reagents or materials from other kits, detection effect of the kit will not be guaranteed if
utilized separately or substituted.

Materials Required, Not Supplied

1. Microplate reader capable of measuring absorbance at 450 ± 10 nm.
2. High-speed centrifuge.
3. Electro-heating standing-temperature cultivator.
4. Absorbent paper.
5. Double distilled water or deionized water.
6. Single or multi-channel pipettes with high precision and disposable tips.
7. Precision pipettes to deliver 2 μL to 1 mL volumes.

Safety Notes
1. This kit is only used for lab research and development and should not be used for human or animals.
2. Reagents should be regarded as hazardous substances and should be handled carefully and
correctly.
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3. Gloves, lab coats, and goggles should always be worn to avoid skin and eyes coming into contact
with Stop Reagent and TMB. In case of contact, wash thoroughly with water.

Test Principle
This assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate
provided in this kit has been pre-coated with Human ACH protein. Standards or samples are added to
the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Human ACH.
Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated.
After TMB substrate solution is added. The enzyme-substrate reaction is terminated by the addition of
sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of
450nm ± 10nm. The concentration of Human ACH in the samples is then determined by comparing the
OD of the samples to the standard curve.

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Sample Collection and Storage
Serum - Samples should be collected into a serum separator tube. After clotting for 2 hours at room
temperature or overnight at 4°C, and then centrifuging at 1000 × g for 20 minutes. Assay freshly
prepared serum immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated
freeze-thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 1000 × g and
2-8°C for 15 minutes within 30 minutes of collection. Remove plasma and assay immediately or store
samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type.
1. Rinse the tissues in pre-cooled PBS to completely remove excess blood, and weigh them before
homogenization.
2. Mince the tissues to small pieces and homogenize them in fresh lysis buffer (different lysis buffer
needs to be chosen based on subcellular location of the target protein) (PBS can be used as the lysis
buffer of most tissues) (w:v = 1:9, e.g. 900 µL lysis buffer is added in 100 mg tissue sample) with a
glass homogenizer on ice (micro tissue grinders, too).
3. Ultrasound the obtained suspension with an ultrasonic cell disrupter until the solution is clear.
4. Then, centrifuge the homogenates for 5 minutes at 10000 × g and collect the supernatant and assay
immediately or store in aliquots at ≤ -20°C.
*Note: Tissue homogenates are recommended to be tested for protein concentration at the same time
to obtain a more accurate concentration of the test substance per mg of protein. For protein detection,
you can purchase our product: BC016, BCA Protein concentration determination kit.
Cell lysates - Cells need to be lysed before assaying according to the following directions.
1. Adherent cells should be washed by pre-cooled PBS gently, and then be detached with trypsin, and
collect them by centrifugation at 1000 × g for 5 minutes (suspension cells can be collected by
centrifugation directly).
2. Wash cells 3 times in pre-cooled PBS.
3. Then, resuspend the cells in fresh lysis buffer with concentration of 107 cells/mL. If it is necessary,
the cells could be subjected to ultrasonication until the solution is clear.
4. Centrifuge at 1500 × g for 10 minutes at 2-8°C to remove cellular debris. Assay immediately or store
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 in aliquots at ≤ -20°C.
Urine - Collect the first urine of the day (mid-stream) and discharge it directly into a sterile container.
Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤ -20°C. Avoid
repeated freeze-thaw cycles.
Saliva - Collect saliva using a collection device or equivalent. Centrifuge samples at 1000 × g at 2-8°C for
15 minutes. Remove particulates and assay immediately or store samples in aliquot at ≤ -20°C. Avoid
repeated freeze-thaw cycles.
Cell culture supernatants and other biological fluids - Centrifuge samples at 1000 × g for 20 minutes.
Collect the supernatant and assay immediately or store samples in aliquot at -20°C or -80°C for later use.
Avoid repeated freeze-thaw cycles.

Notes
1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C
(≤ 1 month) or -80°C (≤ 2 months) to avoid loss of bio-activity and contamination. Avoid repeated
freeze-thaw cycles.
2. Sample hemolysis will influence the result, so it should not be used.
3. When performing the assay, bring samples to room temperature.
4. If the concentration of the test material in your sample is higher than that of the Standard product,
please make the appropriate multiple dilutions according to the actual situation (it is recommended
to do preliminary experiment to determine the dilution ratio.

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Summary
1. After the kit is equilibrated at room temperature, add 50 μL of Standard Working
Solution (gradually dilution refers to Reagent Preparation) or 50 μL of sample to each well,
immediately add 50 μL of 1× Biotinylated-Conjugate Working Solution to each well, mix well,
incubate at 37°C for 60 minutes.

2. Discard the liquid in the plate, add 200 μL 1× Wash Buffer to each well, and wash the
plate 3 times. After pat it dry against clean absorbent paper, add 100 μL 1× Streptavidin-HRP
Working Solution to each well, incubate at 37°C for 60 minutes.

3. Discard the liquid in the plate, add 200 μL 1× Wash Buffer to each well, and wash the
plate 5 times. After pat it dry against clean absorbent paper, add 90 μL TMB Substrate
Solution to each well, incubate at 37°C for 20 minutes in the dark .

4. Add 50 μL Stop Reagent to each well, shake plate on a plate shaker for 1 minute to mix.
Record the OD at 450 nm immediately, calculation of the results.

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Reagent Preparation
1. Bring all kit components and samples to room temperature (18-25°C) before use.Make sure all
components are dissolved and mixed well before using the kit.
2. If the kit will not be used up in 1 time, please only take out strips and reagents for present
experiment, and save the remaining strips and reagents as specified.
3. Dilute the 25× Wash Buffer into 1× Wash Buffer with double distilled water.
4. Standard Working Solution - Centrifuge the Standard at 1000 × g for 1 minute. Reconstitute the
Standard with 1.0 mL of Standard Diluent Buffer, kept for about 10 minutes at room temperature,
shake gently (not to foam). The concentration of the standard in the stock solution is 1000 pg/mL.
Please prepare 7 tubes containing 0.5 mL Standard Diluent Buffer and use the Diluted Standard to
produce a double dilution series according to the picture shown below. To mix each tube thoroughly
before the next transfer, pipette the solution up and down several times. Set up 7 points of Diluted
Standard such as 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.63
pg/mL, and the last EP tubes with Standard Diluent is the Blank as 0 pg/mL. In order to guarantee
the experimental results validity, please use the new Standard Solution for each experiment. When
diluting the Standard from high concentration to low concentration, replace the pipette tip for each
dilution. Note: the last tube is regarded as the Blank and do not pipette solution into it from the
former tube.

Stock
Standard

pg/mL 1000 500 250 125 62.5 31.25 15.63

5. 1× Biotinylated-Conjugate and 1× Streptavidin-HRP Working Solution - Briefly spin or centrifuge

the stock Biotinylated-Conjugate and Streptavidin-HRP before use. Dilute them to the working
concentration 100-fold with Biotinylated-Conjugate Diluent and HRP Diluent, respectively. For

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 example, 10 μL of Streptavidin-HRP with 990 μL of HRP Diluent.
6. TMB Substrate Solution - Aspirate the needed dosage of the solution with sterilized tips and do not
dump the residual solution into the vial again.

Notes
1. After receive the kit, please store the reagents according to the instructions. The plates can be
disassembled to single strips. Please use it in batches on demands.
2. The test tubes, pipette tips and reagents used in the experiment are all disposable and are strictly
prohibited from being reused; otherwise the experiment results will be affected. Kit reagents of
different batches cannot be mixed (except TMB, Washing Buffer and Stop Reagent).
3. Lyophilized Standards, Biotinylated-Conjugate, and Streptavidin-HRP are small in volume and may be
scattered in various parts of the tube during transportation. Please centrifuge at 1000 × g for 1
minute before use. Then, carefully pipette 4-5 times to mix the Solution. Please configure the
Standard, 1× Biotinylated-Conjugate and 1× Streptavidin-HRP Working Solution according to the
required amount, and use the corresponding Dilution Solution, cannot be mixed used.
4. Bring all reagents to room temperature (18-25°C) before use. If crystals form in the concentrate
(25×), it is a normal phenomenon. Heat it to room temperature (the heating temperature should not
exceed 40°C), gently Mix until crystals are completely dissolved.
5. Prepare to dissolve Standard within 15 minutes before the test. This Standard Working Solution can
only be used once. If the dissolved Standard is not used up, please discard it. The sample addition
needs to be rapid. Each sample addition should preferably be controlled within 10 minutes. To
ensure experimental accuracy, it is recommended to test duplicate wells, and when pipetting
reagents, keep a consistent order of additions from 1 well to another, this will ensure the same
incubation time for all wells.
6. During the washing process, the residual washing liquid in the reaction well should be patted dry on
absorbent paper. Do not put the paper directly into the reaction well to absorb water. Before
reading, pay attention to remove the residual liquid and fingerprints at the bottom, so as not to
affect the microplate reader reading.
7. TMB Substrate Solution is light-sensitive, avoid prolonged exposure to light. Dispense the TMB

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 Substrate Solution within 15 minutes following the washing of the microtiter plate. In addition,
avoid contact between TMB Substrate Solution and metal to prevent color development. TMB is
contaminated if it turns blue color before use and should be discarded. TMB is toxic, avoid direct
contact with hands.
8. Bacterial or fungal contamination of either samples or reagents or cross-contamination, between
reagents may cause erroneous results.

Samples Preparation
1. Equilibrate all materials and prepared reagents to room temperature prior to use. Prior to use, mix
all reagents thoroughly taking care not to create any foam within the vials.
2. The user should calculate the possible amount of the samples used in the whole test. Please reserve
sufficient samples in advance.
3. Please predict the concentration before assaying. If values for these are not within the range of the
standard curve, users must determine the optimal sample dilutions for their particular experiments.

Assay Procedure
1. Determine wells for Diluted Standard, Blank and Sample. Prepare 7 wells for Standard, 1 well for
Blank. Add 50 µL of Standard Working Solution (please refer to Reagent Preparation) or Sample into
each well (Blank is Standard Diluent). Then, add 50 µL of Biotinylated-Conjugate (1×) to each well
immediately. Mix well, cover with the Plate Cover. Incubate for 1 hour at 37°C. Note: solutions
should be added to the bottom of the micro ELISA plate well, avoid touching the inside wall and
causing foaming as much as possible.
2. Pour out the liquid of each well. Aspirate the solution and wash with 200 μL of 1× Wash Solution to
each well and let it sit for 1-2 minutes. After the liquid has been decanted, completely remove the
remaining liquid from all wells by snapping the plate onto absorbent paper. Totally wash 3 times.
Complete removal of liquid at each step is essential for good performance. After the last wash invert
the plate and blot it against clean paper towels to remove excess liquid.
Notes: (a) When adding Washing Solution, the pipette tip should not touch the wall of the wells to a
void contamination.

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 (b) Pay attention to pouring the washing liquid directly to ensure that the washing liquid does not
contaminate other wells.
3. Add 100 µL of Streptavidin-HRP Woking Solution (1×) to each well, cover the wells with the Plate
Cover and incubate at 37°C for 60 minutes.
4. Repeat the aspiration, wash process for total 5 times as conducted in step 2.
5. Add 90 µL of TMB Substrate Solution to each well. Cover with a new Plate Cover. Incubate for 20
minutes at 37°C (Don't exceed 30 minutes) in the dark. The liquid will turn blue by the addition of
TMB Substrate Solution. Preheat the Microplate Reader for about 15 minutes before OD
measurement. Avoid placing the plate in direct light.
6. Add 50 μL of Stop Reagent to each well. The liquid will turn yellow by the addition of Stop Reagent.
Mix the liquid by tapping the side of the plate. The insertion order of the Stop Reagent should be
the same as that of the TMB Substrate Solution.
7. Wipe off any drop of water and fingerprint on the bottom of the plate and confirm there is no
bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at
450 nm immediately.
Note: Samples may require dilution (please refer to Sample Preparation section).

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Calculation of Results
This assay employs the competitive inhibition enzyme immunoassay technique, so there is an
inverse correlation between Human ACH concentration in the sample and the assay signal intensity.
Average the duplicate readings for each standard, control, and samples. Create a standard curve with the
Human ACH concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line
through the standard points and it can be determined by regression analysis. If samples have been
diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Using
some plot software, for instance, curve expert.
Concentration (pg/mL) OD
1000 0.197
500 0.31
250 0.557
125 0.846
62.5 1.152
31.25 1.511
15.63 1.799
0 2.325

Note: this graph is for reference only

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Precision
Intra-assay Precision (Precision within an assay)：CV%<8%
Three samples of known concentration were tested twenty times on one plate to assess intra-assay
precision.
Inter-assay Precision (Precision between assays)：CV%<10%
Three samples of known concentration were tested in forty separate assays to assess inter-assay
precision.

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.

Matrix Recovery range Average

Serum (n = 5) 82-95% 88%
EDTA plasma (n = 5) 84-96% 90%
Heparin plasma (n = 5) 87-101% 90%

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 1:2 1:4 1:8 1:16

Serum (n = 5) 90-99% 96-105% 87-98% 93-105%
EDTA plasma (n = 5) 82-93% 96-105% 87-98% 85-97%
Heparin plasma (n = 5) 87-96% 85-97% 89-101% 85-97%

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Declaration
1. The kit may not be suitable for special experimental samples where the validity of the experiment
itself is uncertain, such as gene knockout experiments.
2. Certain natural or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins,
may not be detected because they do not match the detection antibody and capture antibody used
in this product.
3. This kit is not compared with similar kits from other manufacturers or products with different
methods to detect the same object, so inconsistent test results cannot be ruled out.

Analysis of Common Problems and Causes of ELISA Experiment

High background/Non-specific staining

Description of
Possible reason Recommendations and precautions
results
The yellowing of the whole plate Check the components and lot numbers of the
may be caused by wrong addition of reagents before the experiment, and confirm
other reagents that all components belong to the
corresponding kit. Reagents from different kits
or different lot numbers cannot be mixed.
ELISA plate was not washed Make sure that the same amount of Washing
sufficiently Solution is added to each microwell during the
After
washing process. After washing, press the
termination, the
ELISA plate firmly on the absorbent paper to
whole plate
remove the residual buffer.
results show a
Incubation time too long Please strictly follow the steps of the manual
uniform yellow
Streptavidin-HRP contaminates the When absorbing different reagents, the tips
or light color; or
tip and TMB container or positive should be replaced. When configuring different
the Standard
control contaminates the Pre-coated reagent components, different storage vessels
curve is linear
Microplate should be used. Please use a pipette during
but the
operation.
background is
Biotinylated-Conjugate or Check whether the concentration calculation is
too high
Streptavidin-HRP concentration correct or use after further dilution.
too high
Substrate exposure or Store in the dark at all times before adding
contamination prior to use substrate.
Color development time is too long Please strictly follow the steps of the manual.
The wrong filter was used when the When TMB is used as the substrate, the
absorbance value was read absorbance should be read at 450 nm.
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 NO color plate

Description of results Possible reason Recommendations and precautions

Mixed use of component Please read labels clearly when
reagents preparing or using

In the process of plate washing Confirm that the container holding the
and sample addition, the ELISA plate does not contain enzyme
After the color development
enzyme marker is inhibitors (such as NaN3, etc.), and
step, all wells of the ELISA plate
contaminated and inactivated, confirm that the container for
are colorless; the positive
and loses its ability to catalyze preparing the Wash Solution has been
control is not obvious
the color developing agent washed.

Missing a reagent or a step Review the manual in detail and

strictly follow the operating steps

Light color

Description of results Possible reason Recommendations and precautions

The sample uses NaN3 Samples cannot use NaN3
preservative, which inhibits the
reaction of the enzyme
The sample to be tested may In case of doubt, please test again.
The Standard is normal, the
The standard is normal, the
color of the sample is light
color of the sample is lightnot
contain strong positive
samples, so the result may be
normal
The visual result is normal, Wrong filter used for When TMB is used as the substrate, the
but the reading value of the absorbance reading absorbance should be read at 450 nm.
microplate reader is low

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 Description of
Possible reason Recommendations and precautions
results
Insufficient incubation time Timer accurate timing
Insufficient color reaction Usually 15 - 30 minutes
The number of washings Reduce the impact of washing, dilute the
increases, and the dilution ratio concentrated lotion and washing time according to
of the concentrated lotion does the manual, and accurately record the washing
not meet the requirements times and dosage.
Distilled water quality problem The prepared lotion must be tested to see if the pH
value is neutral.
In the process of plate washing Confirm that the container holding the ELISA plate
and sample addition, the does not contain enzyme inhibitors (such as NaN3,
enzyme marker is contaminated etc.), confirm that the container for preparing the
and inactivated, and loses its Washing Solution has been washed, and confirm
All wells, including
ability to catalyze the color that the purified water for preparing the Washing
Standard and
developing agent. Solution meets the requirements and is not
Samples, are
contaminated.
lighter in color
The kit has expired or been Please use it within the expiration and store it in
improperly stored accordance with the storage conditions
recommended in the manual to avoid
contamination.
Reagents and samples are not All reagents and samples should be equilibrated at
equilibrated before use room temperature for about 30 minutes.

Insufficient suction volume of To calibrate the pipette, the tips should be

the pipette, too fast discharge matched, each time the tips should fit tightly, the
of pipetting suction, too much pipetting should not be too fast, and the discharge
liquid hanging on the inner wall should be complete. The inner wall of the tips
of the tip or the inner wall is should be clean, and it is best to use it once.
not clean.

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 Description of results Possible reason Recommendations and precautions

Incubation temperature Keep the temperature constant to avoid the local

constant temperature temperature being too high or too low
effect is not good
When adding liquid, too When adding liquid, the tip should try to add liquid
much remains on the along the bottom of the medial wall of wells without
medial wall of wells touching the bottom of the hole.
Reuse of consumables The tips should be replaced when different reagents
are drawn, and different storage vessels should be
Poor repeatability used when configuring different reagent components.
The bottom of the Be careful when operating, be careful not to touch the
microwell is scratched or bottom and wipe the bottom of the microplate to
there is dirt remove dirt or fingerprints.
Technical repetition of the same sample for 3 times,
including more than 2 approximate values.
Cross-contamination Try to avoid cross-contamination when adding samples
during sample addition
Cross-contamination When washing the plates by hand, the first 3 injections
from manual plate of the lotion should be discarded immediately, and the
washing soaking time should be set for the next few times to
The color of plate is reduce cross-contamination.
chaotic and irregular Cross-contamination Use a suitable absorbent paper towel when clapping
when clapping the plate, do not pat irrelevant substances into the well
of the plate, and try not to pat in the same position to
avoid cross-contamination.

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 Description of
Possible reason Recommendations and precautions
results
The liquid filling head of the Unblock the liquid addition head, so that each well
plate washer is blocked, resulting is filled with washing liquid when washing the plate
in unsatisfactory liquid addition and the residual amount should be small when
or large residual amount of liquid aspirating liquid.
suction, resulting in the color of
plate is chaotic and irregular
Incomplete centrifugation of the Serum plasma should be fully centrifuged at 3000
The color of plate
sample, resulting in coagulation rpm for more than 6 minutes
is chaotic and
in the reaction well or
irregular
interference of sediment or
residual cellular components
The sample is stored for too long Samples should be kept fresh or stored at low
time, resulting in contamination. temperature to prevent contamination
Incorrect preparation of Washing Please configure according to the manual
Solution or direct misuse of
concentrated Washing Solution

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