Capture antibodies reduction

Disulfide reduction and elution of captured antibody-protein complex... 

The capture antibody is optimized to bind Ag, which is detected by a constant amount of Abx (from animal species X). The competition involves the reaction of the Ag with antisera from species Y (which should not interact with the conjugate anti-Abx), in the liquid phase. The remaining Ag is then trapped and titrated with the Abx and the conjugate. A reduction in the expected OD for the system without any Aby represents competition. 

The first consideration when setting up an AlphaScreen assay is the choice of an assay format In most cases, the decision will depend on the interaction partners under investigation and on the biological tools available for their detection. The interaction partners can be coupled to the beads directly via reductive amination of reactive aldehyde groups, similar to the immobilization on a Biacore sensor chip (see above). The usefulness of this approach is limited by the reaction conditions, which may not be appropriate for maintaining the biologically active conformation of the biomolecule. Therefore the biomolecule of interest is usually not coupled to the beads directly, but instead captured via an antibody, also preventing steric hindrance. While not strictly necessary, it is often convenient to use a biotinylated molecule which can be captured by streptavidin-coated donor beads. 

---