Target-triggered catalytic hairpin assembly activation of CRISPR/Cas12a for amplified detection of therapeutic monoclonal antibody

Abstract

It is of great significance to monitor therapeutic antibodies in human serum to obtain the pharmacokinetic data for assessing their therapeutic efficacy at the dosage of administration. Here, based on the target-triggered proximity binding approach, as well as the catalytic hairpin assembly (CHA) and CRISPR/Cas12a cleavage dual signal amplification strategy, we describe the establishment of a highly sensitive method for detecting panitumumab, a cancer therapeutic monoclonal antibody, in diluted human serums. The two proximity probes bind the target panitumumab simultaneously to form initiation sequences for subsequent CHA for the cyclic yield of many dsDNAs, which further activate the specific trans-cleavage activity of Cas12a to digest lots of fluorescently quenched ssDNAs to restore drastically enhanced fluorescence for detecting panitumumab with a 0.62 pM detection limit. Besides, the sensing approach can selectively discriminate panitumumab against other monoclonal antibodies and realize low levels of panitumumab detection in diluted serums. Moreover, by simple replacement of the proximity recognition probes, the established method can be easily expanded for the development of versatile sensing platforms for different biomolecules in addition to antibodies.