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Other 171 views Jun 04, 2018
ELISA Principle and Key Troubleshooting Areas

Enzyme-linked immunosorbent assays (ELISA) work on principles which are same as immunoassay technologies. In order to bind the target antigen and detect the presence and quantity of an antigen binding, the principle of ELISA test depends on specific antibodies. To increase the precision and sensitivity of the assay, the plate must be coated carefully with high-affinity antibodies.

Here Are Some Key ELISA Troubleshooting Areas:

Poor Standard Curve - In ELISA standard curve troubleshooting, a poor standard curve can show unpublished results if they are prepared correctly. A poor mixture of reagents leads to degraded standard or pipetting errors.

High Signal - Insufficient washing of plate, the addition of too much detection reagent or not stopping the reaction could lead to a high signal. This can result in incorrect data or false positives.

Out of Range - Out of range can occur due to the incorrect preparation of dilutions or insufficient washing which are based on samples. This also can result in loss of data due to no or negative results.

High Variation - High Variation can occur due to mistakes in sample preparation, inconsistencies, pipette errors, insufficient plate agitation among other issues. Variation in data can skew the real results and leads to inconsistencies in data.

Background is High - This may occur from cross-reactivity of contamination or samples or inadequate washing steps. Again this may result in negative data or false results and affect your results.

No Signal - In the principle of ELISA Test, no signal may occur from assay issues and a number of the sample including target detection of the assay, wash buffer contains azide, or avidin-HRP was not added. It may lead to no results from precious samples.

ELISA test can be difficult, it includes multiple intervening steps when it comes to measuring the concentration of protein in heterogeneous samples. In the overall process, the most difficult step is detection in which multiple layers of antibodies can be used to expand signal.