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

Area of Science:

• Bioconjugation Chemistry
• Polymer Science
• Biomaterials

Background:

• Antibody-polymer conjugates are crucial for sensitive immunoassays.
• Cellulose acetate (CA) membranes offer a platform for immobilizing biomolecules.
• Controlling polymer properties like LCST is key to optimizing assay performance.

Purpose of the Study:

• To develop and evaluate a novel antibody-terpolymer conjugate for enhanced protein capture in CA membrane-based immunoassays.
• To investigate the impact of terpolymer composition and assay temperature on conjugate performance.
• To establish the relationship between polymer LCST and its binding efficiency on CA membranes.

Main Methods:

• Synthesis of an N-isopropylacrylamide (NIPAAm)/N-n-butylacrylamide (nBAAm)/N-acryloxysuccinimide (NASI) terpolymer with a low LCST (7-13°C).
• Conjugation of the terpolymer to an antibody.
• Evaluation of the conjugate in a CA membrane immunoassay, comparing it to an antibody-copolymer conjugate (NIPAAm/NASI, LCST = 32°C).
• Assessing performance at room temperature and elevated temperature (45°C).

Main Results:

• The NIPAAm/nBAAm/NASI terpolymer conjugate demonstrated superior performance at room temperature compared to the NIPAAm/NASI copolymer conjugate.
• At 45°C, both terpolymer and copolymer conjugates exhibited equivalent performance.
• The study highlights the significant role of the polymer's LCST in its adsorption and binding characteristics on the CA membrane.

Conclusions:

• The developed terpolymer conjugate offers improved protein binding in CA membrane immunoassays, particularly at room temperature.
• The lower critical solution temperature (LCST) of the polymer is a critical factor influencing conjugate performance.
• Tailoring polymer properties, such as LCST, is essential for optimizing antibody-polymer conjugates in immunoassay applications.