A Synthetic Trivalent Hapten that Aggregates anti-2,4-DNP IgG into Bicyclic Trimers

This paper describes the synthesis of the trivalent hapten molecule 1, containing three 2,4-dinitrophenyl (2,4-DNP) groups, and the use of this molecule to aggregate three molecules of anti-2,4-DNP IgG into a complex with 3:2 stoichiometry (IgG312). The equilibrium product IgG312 was generated in ∼90% yield upon mixing IgG and 1; during incubation, thermodynamically unstable, high-molecular-weight aggregates (>104 nm in diameter) form first and convert subsequently to IgG312. The thermodynamics and the kinetics of the formation of aggregates were studied using size-exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC). An analytical model based on multiple species in equilibrium was developed and used to interpret the SE-HPLC data. The aggregate IgG312 was more stable thermodynamically and kinetically than monomeric aggregates of this IgG with monomeric derivatives of 2,4-DNP; this stability suggests potential applications of these aggregates in biotechnology.