A Robust and Scalable Method to Crosslink Antibodies and Proteins

Abstract

Abstract The conjugations of biopolymers are critical tasks in life science field. A traditional method is to use SMCC, but SMCC-modified protein is unstable and easy to cause self-crosslink if the protein contains thiol group. Another known method is based on S-HyNic and Sulfo-S-4FB chemistry, but catalyst is required and the procedure is tedious and long. We developed a Buccutite method to crosslink antibodies to biopolymers (e.g. enzymes and ligonucleotides) or nano particles (such as Qdots and latex beads). This new technique is based on a pair of crosslinkers: Buccutite FOL and MTA. One protein is labeled with MTA, and the other is pre-activated with FOL; when two parts are mixed, the conjugation occurs promptly without any catalyst required. The Buccutite method was validated with the conjugation of antibody with HRP or phycobiliproteins (PE, APC or their tandems). ELISA assays showed the Buccutite antibody-HRP has the same sensitivity as traditional antibody-HRP conjugate. Flow cytometry result also showed the Buccutite conjugates performed as well as the ones prepared with traditional SMCC method when evaluated with human lymphocytes CD antibodies conjugates. Pre-activated proteins (e.g., PE-FOL) were stable for 12 months without any decreasing in performance. Buccutite conjugation was very efficient: there was less than ~10% of the free antibody left, while there was still more than 40% free antibody remaining for the traditional SMCC method. In conclusion, Buccutite method has demonstrated a way to prepare conjugate at high efficiency in short reaction time and mild reaction conditions with the same performance. It is a superior and effective alternative to the existing methods for crosslinking biopolymers to their targets.