General Troubleshooting Guide for ELISA

Q: 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.

Q: 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.

Q: 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.

Q: 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.

Q: 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.

Q: 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.

Q: 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.

Q: 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.

Q: 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.

Q: 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.

Q: Why is there no color developed in the wells of the whole plate including the positive control when color development is finished?

a. Reagents were wrongly used. You should check the label carefully when preparing or using.

b. 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.

c. A reagent or a step of the procedure taken by mistake. You should read the kit protocol carefully and follow each step strictly.

Q: 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.

Q: 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.

Q: 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.

Q: 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.

Q: 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.

Q: 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.

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