Method of kinetic characterization of immunoreagents for development of express high-sensitive assays for detection of ochratoxin A and heart fatty acids binding protein

Development of rapid and sensitive immunoassays is a task of great importance in a variety of fields ranging from clinical practice and urgent diagnostics to food quality control and environmental monitoring. High attention of researches is paid to methods of screening, selection, and kinetic characterization of antibodies that enable fast, specific, and effective formation of immunocomplexes. Herein, we present a method for direct investigation of kinetics of immunoreagents during developments of express high sensitive lateral flow assays. As model biomolecules to be detected, the following substances were tested: ochratoxin A (OTA), which is one of the most dangerous mycotoxins naturally present in many vegetable raw materials; and heart fatty acids binding protein (hFABP), which is a cardiac marker used in differential diagnosis of acute myocardial infarction. The kinetic constants of association (k(on)) and dissociation (k(off)) with monoclonal antibodies are determined along with the corresponding equilibrium constants (K(A) and K(D)). The obtained values are as follows: for the anti-OTA antibodies – k(on) = 4.54*10(3) M(−1)s(−1); k(off) = 3.32*10(−4) s(−1); K(A) = 1.37*10(7) M(−1); K(D) = 7.31*10(−8) M; and for the anti-hFABP antibodies – k(on) = 7.28*10(3) M(−1)s(−1); k(off) = 1.97*10(−4) s(−1); K(A) = 3.70*10(7) M(−1); K(D) = 2.70*10(−8) • Investigation of immunoreagent kinetics for development of express high sensitive lateral flow assays; • Kinetic characterization of monoclonal antibodies against OTA and hFABP for their rapid and sensitive detection; • Both kinetic and equilibrium constants of association and dissociation are determined.