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  • 04 Jun 2018
    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Troubleshooting Areas: Poor Standard Curve - In ELISA standard curve troubleshooting, a poor standard curve can show unpublished results if they are prepared correctly.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.
    478 Posted by Jacob brown
814 views Oct 10, 2018
Understanding the Protocol for ELISA Assay

Have you ever heard of Sandwich ELISA test? Enzyme-linked Immunosorbent assay includes the addition of a capture antibody to a microplate. Then there is an addition of the samples which contain an unknown amount of the analyte and target protein of interest and bind the capture antibody. Then to eliminate the unbound substance from the microplate, washing steps are followed and HRP conjugate is added for detection during ELISA sample preparation.

For the antigens quantification, it is important to follow the protocol for the ELISA assay. Different types of ELISA tests are flexible to high-throughput screening as its results are consistent rapid, and relatively easy to examine. Sandwich ELISA test provides the best results as it uses pure, pre-matched capture and detection antibodies. The data provided by resulting signal is highly specific and sensitive. However, you can find ready to use ELISA kits which are available for biological molecules and investigated proteins.

Protocol for ELISA assay starts with a capture antibody, a protein of interest which is coated on the microplates wells. Samples which include control specimens, a standard containing protein of interest and unknowns are added into these wells. In the first incubation, the protein antigen is bonded to the capture antibody. After washing steps, there is the addition of the detection antibody to the wells which further binds to the restrained protein in capture during the first incubation. After getting rid of the excess of detection antibody, a secondary antibody such as HRP conjugate is added which binds with the detection antibody.

After a third incubation and washing, an excess of the HRP conjugate is removed, there is the addition of a substrate solution which is converted to a detectable form by the enzyme. The colored product intensity is directly related to the concentration of an antigen which is present in the original specimen during Elisa sample preparation.