ELISA Troubleshooting Tips, Common Issues & Solutions

Problem 1: High Background or Non-specific Binding

Problem Description	Possible cause	Solution
After the reaction is terminated, the whole plate appears uniform yellow or light yellow; or the standard curve is linear but the background is too high.	Wrong reagents were added.	Before the assay, check the reagents and the lot numbers to confirm that all reagents come from the same kit. Do not mix use the reagents from different kits or different lots of reagents.
	Insufficient plate washing.	Ensure that the amount of wash buffer added to each well during the washing process is consistent. After washing, invert the plate onto absorbent paper and gently tap it a few times to remove any remaining buffer.
	Longer incubation time than recommended.	Strictly follow the instructions.
	HRP-avidin or HRP-conjugate contaminates the pipette tip and the container for TMB substrate, or positive control contaminates the wells.	When aspirating different reagents, the pipette tip should be replaced. When configuring different reagents, different containers should be used. Please use pipette during operation.
	High concentration of detection antibody or HRP-avidin.	Check if the concentration calculation is correct or perform further dilution before use.
	Exposure or contamination of TMB substrate before use.	TMB substrate should be stored in the dark before adding to wells.
	Longer color reaction time than recommended.	Strictly follow the instructions to control the color reaction time.
	Incorrect filtering was used when reading the absorbance value.	When TMB is used as the substrate, the absorbance value should be read at 450nm wavelength, and 540nm or 570nm should be used as the correction wavelength.

Problem 2: No Color

Problem Description	Possible cause	Solution
After the color reaction, all the wells of the plate have no color. Positive control does not develop color.	Reagents were mix used.	When preparing or using reagents, check the label of reagents carefully.
	During the process of plate washing and sample addition, HRP-avidin or HRP-conjugate lost activity.	Ensure that the container for wash buffer is clean and free of contamination.
	A reagent or a step was missed.	Carefully review the instructions and strictly follow the operation steps.

Problem 3: Weak Signal

Problem Description	Possible cause	Solution
The color of all plate wells is light, including the standard and the sample.	The kit has expired or was not stored properly.	Confirm that the kit is within the validity period and store it in the storage conditions recommended in the protocol to avoid contamination.
	Reagents and samples were not balanced before use.	Bring all reagents to room temperature (18-25°C) for 30min before use.
	The aspirating capacity of the pipette is not high, the aspiration and discharge of the pipette are too fast, and there is too much liquid hanging on the inner wall of the pipette tip or the inner wall is not clean.	Calibrate the pipette, the pipette tip should be matched and fit tightly. The liquid transfer should not be too fast, and the discharge should be complete. The inner wall of the pipette should be clean, and it is best to use it once.
	Incubation time too short.	Accurate timer timing.
	The color reaction time was insufficient.	The color reaction time is generally 15-30min, 20min is better.
	The order of adding the substrate(A and B) was reversed.	Strictly follow the instructions.
	Too many washing times, and the dilution factor of concentrated wash buffer was wrong.	Strictly follow the instructions to dilute the concentrated wash buffer, and accurately record the washing times and dosage.
	Unqualified distilled water.	Ensure that the distilled water required for reagent preparation is free of contamination.
	During the process of plate washing and sample addition, HRP conjugate and HRP-avidin lost activity.	Ensure that the container for wash buffer is clean and free of contamination.
The standard curve is normal, but the sample develops light color.	The sample was stored with NaN₃, which inhibits the enzyme reaction.	Samples cannot be stored with NaN₃.
	The sample may not contain strong positive analyte, so the test result may be normal.	Repeat the assay if any doubts.
The results seem to be normal, but the reading value of the plate reader is low.	Incorrect filtering was used when reading the absorbance value.	When TMB is used as the substrate, the absorbance value should be read at 450nm wavelength, and 540nm or 570nm should be used as the correction wavelength.

Problem 4: Poor Standard Curve and Repeatability

Problem Description	Possible cause	Solution
Poor Repeatability	Standard solution was not prepared correctly.	Strictly follow the instructions, Only use the recommended diluent for standard dilution.
	Inappropriate storage.	Ensure the kit is stored according to recommendations. Do not leave the reconstituted components at room temperature for too long.
	Dilute each working component too early.	Please prepare each working component 10 minutes before use and add it to the micro-well immediately.
	The sample was not mixed well after addition.	When adding multiple reagent components at the same time, mix thoroughly on the mixer after adding the sample. Pay attention to stable handling and placement to prevent external splashing.
	The plate reader has poor repeatability.	Calibrate the plate reader.
	Inconsistency in incubation time, washing and color development conditions.	Repeat the assay, and keep the reaction conditions as consistent as possible with the last time.
	Incorrect washing.	Aspirates the required volume of wash buffer accurately and injects it into the well without overflowing. Do not block the wells when washing by plate washing machine. Ensure sufficient washing.
	Incubation temperature not constant.	Ensure that the temperature is constant and avoid local temperatures being too high or too low.
	Too much residual solution on the wall of the plate well.	When adding solution, the pipette tip should be suspended as much as possible without touching the bottom or wall of the well.
	Reuse of consumables.	When aspirating different reagents, the pipette tip should be replaced. When configuring different reagents, different containers should be used.
	Scratches or dirt on the bottom of the wells.	Be careful and avoid touching the bottom during operation. Wipe the bottom of the plate to remove dirt or fingerprints.
	When determining the qualitative result, it is either negative or positive near the threshold.	Run triplicated wells for the same sample, and take two (including two or more) same results as the standard.
	Cross-well contamination.	Replace the pipette tip when adding samples to avoid cross contamination.
Random drifted wells appear and test values are abnormal.	Cross contamination when patting the plate.	When patting the plate, use a suitable suction paper, do not put any substances into the wells. Pat at the same location to avoid cross contamination.
	The liquid filling head of the washing machine is blocked.	Dredge the liquid filling head of the plate washer.
	Insufficient centrifugation of the sample caused coagulation in the well or interference with sediment or residual cellular components.	The stored samples should be thoroughly centrifuged.
	The sample was stored for a long time and was contaminated.	The samples should be kept fresh or kept at low temperature to prevent contamination.
	Wash buffer prepared incorrectly or concentrated wash buffer misused directly.	Follow the instructions.

What quality controls and performance evaluation do you do for every ELISA kit?

Linearity range, linearity, lower limit of detection, precision, recovery, stability, specificity and natural samples detection are carefully evaluated under strict QC standards to ensure good performance. Click here to know more about quality control.

Do you offer standards or controls in every ELISA kit?

We offer positive and negative controls in qualitative ELISA kits, while we offer standards which can work as quality control in quantitative ELISA kits. Kit standards and the entire reagent system are matched. The customer can draw a good standard curve through the experiment, which proves that the whole system including the standards works well, so the standards can play a role of quality control. We suggest you run duplicates for the standards.

What kind of species is your ELISA kit specific to? Can I use your ELISA kit for the species which is not mentioned in the manual?

Different ELISA kit is designed for different species varying from human, rat, horse, rabbit, mouse, sheep, pig, bovine, dog, chicken, fish, monkey, plant, etc. Please refer to each protocol for species instruction. For different species, the antibody's specificity is different, and there is matrix interference. We can recommend the right kit if you have difficulty to find the kit of species you need.

How many times do I have to run the ELISA standards and samples in each test?

It is recommended to run duplicated wells both for the standards and samples.

Is it possible to mixedly use the reagents of two different ELISA kits? Can I only purchase the standard/antibody in the kit?

No, the reagents in the ELISA kits with different lot numbers cannot be mixed and we do not sell the components in the kit separately. Hope you could understand.

Do you add BSA and preservative in your kit? What is your stop solution?

Some components in the ELISA kits contain BSA. We use commercial Proclin 300 as our preservative and 2N sulfuric acid as stop solution in most of ELISA kits.

Can I incubate at room temperature?

Incubation is performed at 37℃ for all ELISA kits except for food safety & drug residues ELISA kits as it is the best temperature for combination of antigen and antibody. We do not recommend the room temperature since it is not stable.

What is TMB? Why should I use dual-spectrum to test my sample using TMB system?

TMB(3,3',5,5'-Tetramethylbenzidine) is chromogenic substrate used in ELISA. TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase. The reaction will be terminated and the color developed in the wells will turn from blue to yellow upon addition of the stop solution. The absorbance value should be read at 450 nm. We recommend you use dual-spectrum to test your sample to avoid the error caused by interference and scratch in the bottom.

What is ELISA? What is the common procedure of ELISA?

ELISA is the abbreviation of the enzyme-linked immunosorbent assay used to identify the presence of specific proteins and to determine their concentrations. There are three kinds of ELISA including ”sandwich”, competitive and indirect ELISA. The common procedures are Storage, Reagents preparation, ELISA plate, Samples or reagents adding, Incubation, Washing and Reading. Please refer to our manual for details.

Why is yellow or light yellow color developed in the wells of the whole plate when stop solution is added?

a. Incorrect reagents added. You should check the components and Lot No. of reagents to ensure correct reagents of the kit.

b. Insufficient plate washing. You should be sure all wells are filled with buffer with the same volume during every wash step. After final wash, blot plate forcefully on paper towel to remove residual buffer.

c. Contamination of the pipette tips and chromogenic agent container with the enzyme conjugate or contamination of blank wells with positive control. You should change pipette tips between reagents and use separate reservoirs for each reagent. Use pipette during operation.

d. Substrate exposed to light or contaminated prior to use. You should keep substrate in the dark until ready to dispense into wells.

e. Concentration of detection antibody or avidin-HRP is too high. You should check calculations or try again after further dilution.

f. Incubation time or color developing time is too long. You should follow the kit protocol strictly.

g. Wrong wavelength used when taking readings. Wavelength should be 450nm with a 650nm wavelength correction for TMB.

Why is weak color developed in all the wells of standards and samples?

a. Reagents expired or improperly stored. You should keep the kit well stored according to the kit protocol and use it before expiration date. Avoid contamination.

b. Reagents and samples were not brought to room temperature prior to use. You should put all the reagents and samples under room temperature for 30 minutes.

c. Pipette suction of fluids insufficient, pipette emissions too fast, fluids on the tip wall too much or tip wall unclean. You should use calibrated pipettes to ensure the pipette tips firmly matched and pipette slowly. Single use is recommended.

d. Insufficient incubation time. You should set the timer accurate.

e. Insufficient color development time. You should develop color in 15-30 minutes. 20 minutes is the best unless otherwise instructed.

f. Color development reagent added in incorrect order. You should follow the kit protocol strictly.

g. Too much washing or improper dilution of concentrated wash buffer. You should reduce washing impact force: dilute the concentrated wash buffer, control the washing time, and record the washing times and dosage according to the manual.

h. Unqualified distilled water. You should check and ensure the pH is neutral when preparation of wash buffer is finished.

i. Chromogenic agent inactive with the contamination of enzyme conjugate during wash or sample addition steps. You should ensure no enzyme inhibitor in the enzyme conjugate container. Check and ensure the washing buffer container clean. Ensure the purified water no contamination.

Why does the standard curve look fine but weak color is developed in the wells of samples?

a. NaN3 preservatives from the samples inhibit the reaction of enzyme. You should avoid use this perservative in samples.

b. No strong positive samples on the detected ones, the result is normal. You should repeat the assay if you have any doubt.

Why does it read low with normal results by eyes?

Wrong filter is used when taking readings. Wavelength should be 450nm with a 650nm wavelength correction for TMB.

Why is repeatability poor?

a. Improper storage of the kit or poor storage environment. You should store all components as recommended on data sheet rather than room temperature for excess time.

b. Incorrect preparation of standard. You should reconstitute standard strictly with the recommended diluent according to the kit protocol. You should prepare reagents in 10 minutes prior to use and add them to wells promptly.

c. Insufficient mixing after adding samples. You should fully mix reagents in the vortex mixer when adding several reagents at the same time. Be careful when holding reagents to avoid splashing.

d. Poor repeatability of the plate readings. You should calibrate the plate reader.

e. Inconsistent incubation time, washing condition, color development condition and operators. You should repeat the assay of standard. Ensure consistent reactivity condition and operators.

f. Improper washing. You should add 200μL of wash buffer or fully fill into every well with pipette but no overflows are allowed. Check that all ports of the plate washer are unobstructed to ensure sufficient washing.

g. Uneven temperature. You should keep constant temperature during incubation to avoid temperature fluctuations.

h. Too much residual on the wall of the wells when adding or the bottom of wells scratched with pipette tip. You should lower the pipette tips along the wall of wells when adding slowly and carefully. Do not touch the bottom of wells.

i. Reused materials. You should change pipette tips between samples and reservoirs between reagents.

j. Occasional positive and negative values close to the cut off value. You should set 3 duplicates for the same sample and the same result over 2 samples.

k. Cross contamination when adding samples. You should avoid cross contamination when adding samples.

Why is abnormal color developed?

a. Cross contamination during manual washing. You should reduce the cross contamination by promptly removing the liquid in wells after 3 times of filling washing buffer during manual washing and then setting the soak time the next times.

b. Cross contamination when patting the plate. You should use proper paper towels when patting the plate. Do not bring unrelated materials into the plate. Do not pat at the same place to avoid cross contamination.

c. Contamination due to long storage of samples. You should keep samples fresh or store them under low temperate to avoid contamination.

d. Abnormal developed color due to insufficient filling or too much residual when the plate washer is obstructed. You should fully fill into every well with pipette but no overflows are allowed. Check that all ports of the plate washer are unobstructed to ensure sufficient washing.

e. Coagulationor interference of precipitates or residual cell caused by incomplete centrifugation of samples. You should complete centrifugation of serum and plasma.

f. Wrong preparation of wash buffer or misuse of concentrated wash buffer. You should prepare wash buffer as manual required.

Are the plates pre-coated? How can I store the remaining wells or plates if I can not use in one time?

Yes. The strips are movable and it’s recommended to separate those wells needed for assay and store the rest at 2-8℃ in the dark without germ contamination.