Why Does Size Matter? Add a 3^rd Dimension to Your Immunoassay

ELISA is considered the gold standard immunoassay for quantitative protein analysis despite limitations stemming from the lack of size-based protein separation. As a CE-immunoassay platform, Simple Western™ provides sensitive, accurate, and reproducible quantitative immunoassay read-outs with the addition of several critical advantages provided by adding the dimension of molecular weight enabled by electrophoretic protein separation. While ELISA can only measure antibody binding as you vary experimental conditions (i.e., 2-dimensional), Simple Western measures antibody binding activity and provides molecular weight size information as you vary experimental conditions, adding a 3^rd dimension to your immunoassay.

Simple Western’s size separating abilities overcome problems with cross-reactivity (specificity), obstruction from endogenous proteins, and lack of accurate intact antigen quantitation, which are common problems with sandwich ELISA assay. With Simple Western, isoforms can be resolved by high-resolution size separation, and then pan-target antibodies can be used which bind to all isoforms in the sample. The molecular weight information paired with immunodetection provides specificity to distinguish isoforms, fragments, aggregates, degradation products, oligomers, and more.

In this article, we review how size-based separation on Simple Western provides greater analytical capacity (a large amount of information in a relatively short time) and size-based specificity for a broad spectrum of applications, including analytical development, clinical biomarkers, and pharmacodynamic/pharmacokinetic studies.