How to overcome ELISA issues

ELISA (Enzyme-Linked Immunosorbent Assay) is a technique used to detect the presence of peptides, proteins, and other antibodies in the blood. ELISA assay is widely used to quantify biological molecules. It’s a common test that is used to diagnose diseases such as HIV, Lyme, pernicious anemia, rotavirus, syphilis, and so on. The experiment is simple and easy to perform but it’s exceptionally sensitive.
The test identifies the presence of specific molecules and it determines their concentration. The detection is accomplished by the interaction of antibody and antigen. It’s also detected by monitoring the enzyme activity through incubation with a substrate.

The common ELISA experiment issues

The results of the experiment are based on the color of the substrate. Reliability and accuracy of ELISA experiments depend on the usage of the proper technique and attention to detail. Like in any biological assay, there might be trouble in interpreting the results in ELISA as well. Highly concentrated samples or diluted samples will have a huge impact on the test results. Preparing different concentrations and blocking solutions, using standard diluent solutions, and choosing sample substrate based on the amount of antigen can optimise the experiment. However, at times the experiment fails to give optimal results and the ELISA controls will indicate a problem.
The problems with ELISA are categorised as a low signal, no signal, high background signal, inconsistent result, and samples out of range. There's a high probability of committing errors that will result in false negatives or false positives. Error in plate handing, plate-wash instrumentation, buffers, using reagents, and test procedures are likely to cause ELISA experiment issues.
Here are the possible reasons along with some solutions to perform ELISA experiment under uniform conditions.

1. Poor usage of reagent

If the result is blank or has minimal signal, it could be due to poor usage of reagent or a procedural error. A poorly mixed reagent or adding too much detection reagent results in a poor standard curve or high background signal. 
Avoid using reagents from different batches because each reagent kit is optimised for the specific ELISA kit. Reusing them can affect the detection ability, resulting in inconsistent values.
Mixing or substituting reagents from different kits can affect the quality of the assay.
Ensure reagents are fresh by avoiding the use of contaminated glassware during reagent preparation.
Reagents must be diluted properly to ensure accurate results.
To maintain consistency among all the test wells, the reagents and samples should be mixed well before pipetting onto the plates.
Reagents should be stored at room temperature to avoid edge effects.

2. Unequally balanced incubation

Stacking plates in the incubator can lead to edge effects that contribute to the variation in the signal. Edge effects happen when the outer wells in the plates are exposed to a different temperature than the inner wells. Stacking plates leave them unequally incubated resulting in inconsistent values. It does not allow the even distribution of temperature across the wells and there will be differences in the evaporation rate.
Incubating the substrate in the light can affect the assay. It is important to carry out the substrate incubation process in the dark.
Longer incubation times are a major problem. Adhere to the rules provided in the manual and ensure the incubation time do not exceed.
Not using plate sealers during incubation can cause a problem too. It is important to cover the assay plates with plate sealers. Ensure a new sealer is used every time to prevent wells from contamination.
Incorrect assay temperature is another problem. The temperature should not exceed 37 degree Celsius.

3. Too Much or Too Less of Residual Fluid

ELISA test instructs to invert the plate and remove the excess residual fluid on any absorbent material. Extra fluid must be carefully removed while making sure that the plate is not completely dry. If the well is completely dry, the components on the plate will be inactive and can result in inconsistent value.
Reusing sealers and reagents can increase the presence of residual fluids. This will result in a high range value. To avoid this issue, use fresh plate sealer and reagent for every procedure.
Insufficient washing can cause a problem. Follow the washing step given in the manual by allowing the wells to drain under normal condition and by removing the excess fluid.

4. Pipette technique and cross-contamination

Verifying the pipetting technique will help you avoid multichannel pipette errors and incorrect standard curve during the procedure. Pipette calibration is essential when there’s inconsistency in the reading. Don’t let cross-contamination ruin the test results.
Change the pipette tips when working on different samples. Take proper care when reusing the same pipette tips. It shouldn’t touch the reagents on the plate.
The pipette should pick up the right amount of liquid and release it. Ensure the tips are sealed properly to avoid cross-contamination.
To avoid contamination, prevent the pipette from reaching to the bottom of the well. The tip of the pipette should be on the sides without reaching the bottom.
Using multichannel pipettes can avoid cross-contamination of wells. You can avoid touching the reagents on the plate while using pipettes.

5. Low-standard ELISA kits

Elisa kits are made of different materials that include polyethylene, polyacrylamide, silicone, glass, and so on. The Elisa plate should be transparent, and have a low black value and good adsorption.
Choose a kit that does not affect the colour reaction. Check the quality of the kit by adding human IgG and anti-human IgG antibodies before and after washing the wells. Measure the dilution value of every well and calculate the average of all values. The mean difference between individual values should be less than 10%
ELISA plates come with detachable tube stripes in 8 or 12. Use only the required amount of tubes and store the rest for later. Do not allow the reagents and the kit to numerous freeze cycles. Use a detachable SBS footprint tube strip that works with all ELISA machines. Prepare single time reagent to ensure a consistent result.


Implementing the solutions discussed above will help you fix small variations in the pipette, manage reagents effectively, and fix incubation issues. However, it is difficult to deal with inconsistent results in all samples. Sometimes, the controls and kits will pass through the quality test but the samples will be problematic. Awareness of sample handling and test procedures, however, can ensure signal accuracy.

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