When analyzing samples in the ELISA platform, there are a variety of components that are affecting the test results. Two of the most important are the intra (within) and inter (between) assay variation and the specificity of the assay. A highly specific assay with a low variation will give a test result close to the true value.
The intra assay variation is dependent on the properties of the assay. A poorly optimized assay with low sensitivity will give high intra assay variations. The inter assay variation is mainly dependent on the robustness of the assay. An ELISA sensitive to changes in the assay environment or production conditions will contribute to a high inter assay variation. Both the intra and inter assay variations are dependent on user handling and performance of the assay.
The variation in the ELISA format is often expressed in terms of Coefficient of Variation (%CV), which is a relative standard deviation.
The reproducibility between assays should be monitored by the use of controls. Analyzing controls along with unknowns gives reliable evaluations and conclusions, although the material has been analyzed at different times.
There are substances in serum and plasma samples that may affect the interaction between the antibodies and antigen in the ELISA. These substances can be complement, rheumatoid factors or heterofilic antibodies. It is important to block these proteins from interfering with the assay. Studies of recovery upon antigen addition or sample dilution show if there are any problems with interfering agents.
Other specificity issue could potentially occur if there are substances in the sample that are similar to the analyte of interest. Such homologous proteins must be considered during assay development in order to try to avoid their impact on the result. A specific assay will give results close to the true value.