Carol-Ann Cole

APPLICATION OF A THERMALLY-REVERSIBLE POLYMER-ANTIBODY CONJUGATE IN A NOVEL MEMBRANE-BASED IMMUNOASSAY

We have developed a novel method to immobilize antibodies onto a cellulose acetate membrane using a conjugate of an N-isopropylacrylamide polymer covalently bound to the antibody. When compared with the unconjugated antibody, over 30-fold increase in retention of the antibody on the membrane was observed when it was conjugated to poly (N-isopropylacrylamide). Studies of the polymer-membrane interaction suggest a combination of hydrophobic and ionic forces, especially the former, is responsible for the high retention. We applied this novel immobilization technology in the development of a membrane-based immunoassay.

Activated, N-substituted acrylamide polymers for antibody coupling: application to a novel membrane-based immunoassay.

A room-temperature-precipitable, activated terpolymer consisting of N-isopropylacrylamide (NIPAAm)/N-n-butylacrylamide(nBAAm)/N-acryloxysuccinimide (NASI) (LCST = 7-13 degrees C) at a monomer feed ratio of 60:40:2.5, respectively, was prepared and conjugated to an antibody. The conjugate was evaluated in a novel cellulose acetate (CA) membrane-based immunoassay which utilizes the especially strong physical attachment of the polymer to CA to bind and concentrate the polymer attached protein onto the membrane. When compared in the CA membrane immunoassay to the antibody-poly(NIPAAm) conjugate prepared via anhydrous copolymerization of NIPAAm and NASI at the monomer feed ratio of 40:1, respectively, the performance of the NIPAAm/nBAAm/NASI terpolymer was superior to that of the NIPAAm/NASI copolymer (LCST = 32 degrees C) when the studies were carried out at room temperature. However, the terpolymer and copolymer gave equivalent performance when the assay mixture was heated to 45 degrees C. These results indicate the importance of the LCST of the polymer component of the Ab-polymer conjugate to its adsorption and binding on the CA membrane.