Fig. 2. Hapten–protein conjugation chemistry (a) haptens (H) with a free carboxyl group form an active intermediate upon reaction with ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Introduction of the carrier protein (P) to this active complex allows for the primary amine on the protein to form an amide bond with the hapten. (b) Carrier proteins (P) have many surface amines, which can react with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) to functionalize maleimide groups onto the surface of the protein. Maleimide-activated proteins can react with haptens (H) containing a terminal sulfhydryl group in order to form a stable thioether bond.