Analytical development groups need to consider FDA guidelines when developing quantitative, validated assays.
Researchers in these groups need quantitative assays using inherently complex sample types, often with limited material available for analysis. While ELISAs have the advantages of sensitivity and specificity, matrix effects can occur when analyzing complex sample types like tissue homogenates. In addition, custom development of sandwich ELISAs can also be challenging owing to the need to identify and validate a pair of two antibodies rather than just a single antibody reactive against the target of interest.
Overestimation of analyte molecules due to crossreactivity is still a problem with ELISA. ELISA lacks the separation profiles needed to characterize off-target binding and protein isoforms, like full-length and cleaved protein targets, or non-functional and enzymatically activated prodrugs. ELISAs also use capture antibodies to bind sample analytes directly in the presence of the sample matrix which can increase the matrix effect.
This application note from Bio-Techne shows that Simple Western analysis of human endothelial nitric oxide synthase (eNOS) in brain whole tissue lysate is less susceptible to matrix effect than a leading commercial eNOS ELISA kit, allowing for more accurate quantification in brain tissue homogenates.
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