problem
Serial number
possible reason
Solution
Light color development, low sensitivity
1
The kit is not fully balanced
After removing the kit from the 2 ～ 8 ℃ refrigerator, open the lid and equilibrate at room temperature for at least 20 minutes to ensure that all reagents have equilibrated to room temperature (about 25 ℃)
2
The sample is not suitable for ELISA test
Samples are generally selected from culture supernatant, serum and plasma. Other sample formats may not be suitable for ELISA testing or need to optimize the testing conditions (such as the composition of the diluent)
3
The microplate is too dry when it is dried after storage or washing
Prevent the board from being too dry
4
The coated antibody is destroyed
Gentle operation, the pipette can not touch the bottom of the well
5
Unsuitable sample and antibody incubation conditions
According to the conditions of the instructions, it is recommended to incubate during the entire shaking process.
6
Washing and soaking time is too long
Follow the instructions. Do not artificially increase the soaking time;
7
Contaminated with HRP reagent
Discard the reagent and reconfigure
8
Wrong dilution coupling HRP reagent
Discard the reagent and reconfigure
9
TMB substrate incubation time is too short, due to non-human factors, it is necessary to optimize the incubation time
Determine the appropriate incubation time: artificial judgment-the highest concentration of the standard appears dark blue; S4-5 has a light blue; the machine determines that the OD value at 620 nm wavelength reaches 0.6-0.65
10
High sample concentration or matrix effect
It is recommended to make different dilution ratios to confirm the best dilution ratio of the sample.
11
There is a problem with the filter setting of the microplate reader or the wavelength selection is wrong
Confirm the instrument settings and select the correct wavelength detection.
12
Microplate is not suitable for ELISA
Cell culture plates cannot be used, it is recommended to use ELISA special high-adsorption plates.
The background is deep and all are colored (usually Elisa background value OD <0.2)
1
Insufficient washing, not dry after washing, other components in the sample or enzyme markers remain
The lotion is accurately prepared; wash it thoroughly, let it stand for 15 seconds, and pat dry thoroughly. The filter paper for adding samples or adding enzyme to the plate should be discarded and not used repeatedly, otherwise it will cause pollution.
2
Substrate contamination, not protected from light, color appears
Discard the substrate and reconfigure
3
Sample dilution contamination
Discard the diluent and reconfigure
4
Reusable tips, not cleaned or disinfected thoroughly
Tips for single use as much as possible
5
Incorrect incubation time, temperature (especially substrate incubation time)
The most commonly used incubation temperature is 37 ℃ and room temperature, followed by 43 ℃ and 2 ~ 8 ℃. Completely follow the instructions.
6
Conjugated antibody (streptavidin-HRP) concentration is too high
Adjust to the best concentration according to the instructions
7
Incorrect storage of ELISA plates
Store microplate at 2-8 ℃; use Elisa plate with low binding force
8
Not properly closed
Add animal protein to the standard dilution or extend the blocking time
9
Severe hemolysis
It is recommended to use non-hemolyzed or slightly hemolyzed samples. Severe hemolyzed samples will cause a high background.
Poor repeatability, high CV value
1
Uneven samples
Thoroughly mix the sample before sample addition to ensure that the pipetting position is consistent;
2
The surface of the coated board is damaged
Try to be careful when washing and adding samples to avoid damaging the coated surface of the board
3
Cross contamination between holes
After incubation, remove the cover and operate carefully to avoid contamination of the hole and the hole; the sealing film cannot be reused
4
Sample volume is inconsistent
The sample should be mixed well before dilution. Use the same pipette as far as possible and install the nozzle tightly
5
Edge effect (the color of the peripheral hole is deeper than the central hole)
Rotate and mix as much as possible at 100 rpm during incubation
6
Inaccurate micro-sampling
Ensure the accuracy and uniformity of micro-sampling
7
Incorrect washing
The lotion is accurately prepared; wash it thoroughly, let it stand for 15 seconds, and confirm every step of washing
A white board appears, the positive control does not develop color
1
Deterioration of color developing solution
Replace with new developer
2
Wrong preparation of washing solution
Please prepare according to the dilution factor shown in the manual
3
No enzyme conjugate added but considered added
Be careful not to miss
4
The stop solution is mistakenly diluted as a washing solution or used as a substrate solution
Always read the label before adding liquid
5
The standard dilution method is wrong / or the standard is defective
Please configure the instructions as shown in the instructions, pay attention to the unit and dilution factor
6
Mix reagents in different kits or batches
It is recommended to use the same batch of kits. Do not mix reagents in different kits or batches.
Abnormal standard curve
1
The wrong way to resuspend the standard
Add the correct amount of solution to resuspend the standard, resuspend fully
2
Standard dilution error
Dilute the standard according to the instructions
3
Standard pollution
Use disposable sterilized tips, store the standard at 2-8 ° C
4
Wrong dilution steps
Dilute the standard according to the instructions
5
Wrong wavelength selection
Both TMB as a substrate and OPD as a substrate are used. The former has a colorimetric wavelength of 450 nm and the latter has a 492 nm. Dual wavelength colorimetric analysis is superior to single wavelength.
6
Standard mixed
Do not mix reagents of different batches
7
The kit was in transit for too long, the temperature was too high, and the standard was inactivated
Try to shorten the transportation time, ice cubes should be used to cool down in summer
8
Direct detection of 450nm and 620nm without termination of ELISA
TMB color development needs to be detected after termination, otherwise there will be a phenomenon that the reading of 620nm is higher than 450nm. (The value still has the gradient law)
Sample or standard OD is out of normal range
1
Wrong dilution of sample or standard
Fully diluted according to the instructions
2
Conjugated antibody concentration is too high
Adjust to the best concentration according to the instructions
3
TMB substrate incubation time is too long
Adjust to the appropriate incubation time
4
Sample or standard contamination
Avoid pollution
5
Wrong incubation time or temperature
Follow the instructions exactly
6
Uncovered during incubation leads to solution evaporation and contamination
Sealing or stamping
The tune is good, but the sample cannot calculate the value
1
Unsuitable selection
Choose the most suitable curve fitting;
2
The sample content is very low and cannot be detected below the sensitivity
Try to concentrate the sample or use high sensitivity ELISA kit
3
The sample content is very high, exceeding the standard score
It is recommended to conduct a pre-experiment to explore the dilution factor to ensure that the OD value of the sample falls within the calibration range
No obvious trend between high concentrations
1
If the absolute value of OD is reliable, but there is only no gradient, the color development is excessive
In the TMB color development system, it is recommended to judge based on the colors of S1 and S2. When S1 and S2 are dark blue, there is a clear gradient, and S5 is light blue, it can be terminated.
2
Only for single wavelength detection.
Because the reference wavelength reads non-specific tests, such as fingerprints, scratches, water stains, etc., it also affects the results. It is recommended that the final result be the most accurate with dual wavelengths.
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