Elisa is conducted to identify infectious or antibody agents in the unknown sample.

Is ELISA giving you wrong results? Problems may include high background, unexpected ELISA data, Interference, etc. In ELISA troubleshooting, the first step to be followed is coating. It is the process in which a suitably diluted antibody or antigen is incubated until it is adsorbed to the well's surface. Hydrophobic interactions between the amino acids side chains on the antigen or antibody is used for coating and the plastic surface. It depends on the pH, temperature and time of the coating buffer and concentration of the coating agent.

In typical ELISA plate coating protocol condition, there is an addition of 50-100 ul coating buffer which contains antibody or antigen at a 1-10 ug/ml concentration. It is incubated overnight for 1-3 hours at 37 C or 4 C.

On the other side, times, coating agent concentrations, temperatures, and buffers can be used and tested by doing experiments. Maintenance of the plated in a moist environment is important in order to minimize evaporation.

There is a range of concentrations of coating agent and it is best to test them as higher concentrations of antigen or antibody have a negative effect on coating which leads over wells saturation that can inhibit antibody binding due to steric hindrance.

On the contrary, when crude antibody or antigen preparations are utilized for coating, it can make low effective antigen/antibody concentration and may contaminate proteins that make the distinct assay signal too low to be useful. A sandwich assay is more suitable in this case.

The stabilization of the antibody or antigen is done by coating buffers. This is used to coat the ELISA multiwell plate that boosts the adsorption to the plate and to optimize interactions with the detection antibody. No other proteins are added in the coating buffer as these will clash with the antigen for binding to the plate.

In ELISA troubleshooting, the most common buffers used are bicarbonate buffer at pH 9.6 or PBS. There is the availability of specialist coating buffers which are optimized for ELISA such as BUF030A. It has been developed to stabilize the adsorbed protein, to protect the antigenic regions and to allow greater binding reactivity to enhance the specific signal.

ELISA plate coating protocol for assays are sensitive to interfering substances. You have to use samples with interfering substances. If the concentration of your antigen of interest is high, use low detectable dilution of your sample.