Dot-ELISA Affinity Test: An Easy, Low-Cost Method to Estimate Binding Activity of Monoclonal Antibodies

Abstract

Selecting the “right” monoclonal antibody (mAb) for an immunoaffinity-based application can be tricky, as many mAb producers offer a wide range of mAb clones against molecular structures of interest. Since there are significant differences in the quality of mAb clones, and particularly in their binding activity, an easy method for quick and low-cost comparison of various mAb clones was developed. The dot-ELISA affinity test is a simple, versatile and instrumentally no demanding technique, since it requires no expensive equipment (such as an ELISA reader or chemiluminescence/fluorescence imaging system) and can be performed in any biochemical laboratory. This method is based on a previously described dot-ELISA technique that is improved with a chaotropic step using different concentrations of ammonium thiocyanate in the range 0-2 M. In this work, the dot-ELISA affinity test was optimized on Aβ peptide as antigen and anti-Aβ mAb. Such protocol was then applied to a panel of eight anti-EpCAM (epithelial cell adhesion molecule) mAbs which should be subsequently used for preparation of magnetic immunosorbent to capture circulating tumor cells (CTCs).