Cell-Free Competition Binding Assays

Binding affinities were determined in a cell-free competitive binding assay (Table 1). Surprisingly, affinities of the esters (biphenyls and heteroaryl mannosides) were slightly improved compared to the corresponding acids, probably due to reduced desolvation costs. 

A number of different competitive cell-free and cell-based binding assays under static and hydrodynamic flow conditions have been used to obtain affinity data for selectin ligands. In addition to the fact that different positive controls have been used, this makes a direct comparison of reported binding affinities difficult. Therefore, in this chapter, we quote relative affinities wherever possible (Section 16.4.3). Another problem that has a negative effect on assay reliabihty has been encountered in cases where acidic ion exchange resins are used in the final step of the antagonist synthesis [170]. Small amounts of polyanions released from the resin were found to be potent selectin inhibitors, especially for P-selectin. These polyanions are difficult to remove and are not detectable by routine analysis. As a result, published assay data for P-selectin antagonists should be considered with caution. 

Garthwaite et al. [29] developed a competitive ELISA based on rabbit antibodies and a breve-toxin/peroxidase conjugate, hi this assay, antibodies were coated on the plate, and free toxin in the sample competed with peroxidase-labeled toxin for antibody binding. Upon addition of enzyme substrate, a concentration-dependent reduction in color development was observed. Initial assay performance was modest, but careful optimization of assay parameters later resulted in reduced assay time (1-2 h) and a working range of approximately 0.5-17 ng/mL. This assay has been used extensively in New Zealand to evaluate toxin production in G. breve cell cultures as well as regulatory screening of shellfish. 

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