Development of antibodies for non-competitive detection of saxitoxin

Abstract

Saxitoxin (STX) is a highly potent natural alkaloid toxin produced in aquatic environments by certain dinoflagellates and freshwater cyanobacteria. It accumulates in the ecosystem e.g. in shellfish and crustaceans and prevails in the water for a long time after formation, therefore posing a risk for humans and livestock by causing variable levels of neural effects ranging from convulsions to even death. STX levels are expected to increase in the future due to climate change and eutrophication causing a shift upwards in the frequency of harmful algae and cyanobacterial blooms. Current competitive, reagent limited immunoassays do not fully answer to the need for an easy-to-use and fully capable assay, as they suffer from limited detection and quantification of STX with low concentrations, which can be solved with the use of a non-competitive assay format.

Primary anti-STX antibodies modified from a previously published paper were expressed in E. coli and mammalian cells, biotinylated and tested in order to construct an immunocomplex with STX. Anti-immunocomplex antibodies designed to act as a secondary antibody were isolated from a synthetic antibody library with the use of phage display antibody technology, and subsequently, expressed in E. coli. Most promising individual binders were determined with immunoassays.

Primary antibody 1E8 was verified specific and immunoreactive enough for the construction of the immunocomplex. Four anti-immunocomplex antibody clones isolated from phage display demonstrate potential for further development. Their specificity towards the immunocomplex was proven suitable, yet they need more testing regarding affinities. Moreover, further development with e.g. site-directed mutagenesis would be beneficial.