How to Resolve Common ELISA Issues

Researchers rely on ELISA to detect and measure antigens in samples due to its robust nature, high degree of sensitivity, and ease of performance. However, it is possible to encounter certain problems that cause ELISA-based experiments to produce inaccurate or non-reproducible results. Accuracy is the degree of proximity between the experimental value obtained and the true value accepted under the experimental conditions. Reproducibility is the amount of variation between different samples within the same run of a test or the variation between different tests performed over several days or by different investigators.

The two main factors that affect precision and reproducibility are nonspecific binding and cross-reactivity. Nonspecific binding (NSB) occurs when substances other than the target reagents are absorbed into the plate, causing high background noise or false signals. Adding blocking buffer is a critical step to saturate unoccupied wells of the plate and prevent NSB. Cross-reactivity refers to any unexpected interaction between an antibody and non-specific analytes and is common among indirect ELISAs when the enzyme-labeled secondary antibody or enzyme-labeled avidin against the detection antibody reduces the selectivity of the test.

Here are some of the most common ELISA problems encountered by researchers, along with their potential causes and corresponding solutions:

Weak or Weak Signal Strength

Incorrect storage of components. Check the storage conditions on the label. Most ELISA test kits should be stored between 2 and 80 degrees Celsius.

Improper preparation or addition of reagents. Confirm the protocol to verify that the reagents were prepared at the correct dilution and added in the correct order.

Expired, degraded or contaminated reagents. Check the expiration dates on the reagents and do not use them after the expiration date. Replace all expired, degraded, or contaminated reagents with new reagents.

Reactive to the wrong temperature. All reagents should be kept at room temperature for at least 15-20 minutes before starting an assay.

Overexposure of fluorescent reagents. Intense light can cause photobleaching by breaking down the fluorophore (fluorescent dye or protein). Protect reagents from light exposure to generate effective signals.

Assay components require further optimization. An assay component may be at a limiting concentration which reduces the overall signal.

The substrate requires further optimization. The substrate may not be sensitive enough to detect the generated signal or the standard curve may not be appropriate for the given sample. Use a more sensitive substrate or concentrate the sample.

Scratched or damaged wells. Touching the wells with the pipette while dispensing or aspirating in and out of the wells or washing may scratch them. Follow correct pipetting technique.

Plate read at incorrect wavelength. Use the recommended wavelengths and filters and confirm that the plate reader is set for the substrate you are using in the assay.

Excessive signal strength

Poor preparation of dilutions. Confirm that you used the correct pipetting technique and double-check your calculations.

Excessive incubation time. Follow the recommended incubation time included in your ELISA test kit.

Well-to-well contamination. Reused plate sealers or failure to use plate sealers increases the risk of contamination in wells. Cover plates with plate sealers during incubation and replace with fresh sealers each time you open them.

High background noise

Exposure of the substrate to light. Store the substrate in a dark place and limit light exposure while running the test.

Inadequate concentration of blocking buffer. Blocking buffer should bind to all non-specific interaction sites while impacting the target epitope for antibody binding and detection. Optimize your blocking buffer solution by increasing the concentration.

Excessive amount of enzyme conjugate. Optimize the enzyme conjugate. You can achieve this by preparing different concentrations in the standard diluent according to the appropriate range for the substrate, applying an equal volume of each to the plate, performing the ELISA and checking the signal to noise ratio.

Bad linearity or dynamic range of the standard curve Low variation between replicates. This may result from a concentra