Temperature-Responsive Bioconjugates. 3. Antibody-Poly(N-isopropylacrylamide) Conjugates for Temperature-Modulated Precipitations and Affinity Bioseparations

Immunoglobulin G (IgG) has been modified by poly(N-isopropylacrylamide) (PIPAAm) to create a novel bioconjugate which exhibits reversible phase transition behavior at 32 degrees C in aqueous media. A terminal carboxyl group introduced into PIPAAm molecule by polymerization of IPAAm with 3-mercaptopropionic acid was used for conjugation to IgG via coupling reaction of activated ester with protein amino group. These conjugates exhibited rapid response to changes in solution temperature and significant phase separation above a critical solution temperature corresponding to that for the original PIPAAm. These conjugates bound to antigen quantitatively in aqueous system, and antigen-bound complex also demonstrated phase separation and precipitation above a critical temperature. Precipitate was reversibly redissolved in cold buffer. Though particular conjugate which includes 12 molecules of PIPAAm with 6,100 molecular weight suppressed more than 95% of Fc-dependent binding with protein A, it retained approximately 60% of original specific antigen binding activity. It was manifested that polymer content of conjugate was 20-30 wt% for the case of 6,100 molecular weight of PIPAAm to demonstrate specific antigen binding activity most effectively and to reduce Fc-dependent binding with protein A. IgG-PIPAAm conjugates were soluble in water and formed antigen-bound complex in homogeneous solution system below a critical temperature. These conjugates were separated from solution and other solutes corresponding to PIPAAm nature and scarcely bound to antigen above a critical temperature. It is revealed that temperature-responsive PIPAAm conjugated to biomolecule operated as a switching molecule. These phenomena are attractive for not only reversible bioreactors and protein separations but also carrier substrate to localize biomolecules such as drugs, peptides and hormones in a living body.