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Optimizing sample preparation and dilution is a key step in the validation of any ELISA assay. It is crucial that sample dilutions are utilized that guarantee a) that the analyte concentration within a given sample will fall within the range of the assay – ideally well above the lowest concentrated standard and well below the highest; and b) matrix interference can be excluded. Matrix interference describes when components of the sample that are not the analyte (i.e. the sample matrix) influence the optical densities, leading to false positive or false negative infuences on signal intensities. Certain samples require significant dilution to eliminate matrix interference. The absence of matrix interference is validated through confirmation of dilution linearity. A representative or pooled sample is serially diluted and the various dilutions are measured with the ELISA assay. Only dilutions that show a linear correlation between sample dilution and determined concentration should be used, with the lowest dilution factor in the linear range of dilutions being considered to be the minimal required sample dilution. Samples that don’t achieve dilution linearity within the assay range must be extracted, meaning the sample should be purified to enrich the analyte and eliminate sample matrix components. Depending on the sample type and analyte in question, various extraction methods may be utilized.