Revealing the Kinetic Advantage of a Competitive Small-Molecule Immunoassay by Direct Detection

Abstract

Small-molecule detection in an immunoassay format generally employs competition or labeling. A novel direct-detection label-free primary immunoassay utilizing second harmonic generation (SHG) has been developed and the utility of the method has been demonstrated for several small-molecule narcotics. Specifically, the binding of morphine, methadone, and cocaine to antimorphine, antimethadone, and anticocaine antibodies was measured by SHG, allowing binding affinities and rates of dissociation to be obtained. The SHG primary immunoassay has provided the first kinetic measurements of small-molecule hapten interactions with a receptor antibody. The kinetics reveal for the first time that competitive immunoassays achieve their selectivity by taking advantage of the kinetics of association and dissociation of the labeled and unlabeled target and nontarget small-molecule to the capture antibody. In particular, the induced fit of the target small-molecule to their antibody pairs prolongs their residence time, while the nontarget small-molecule dissociate rapidly in comparison.

Cocaine Detection by a Laser-Induced Immunofluorometric Biosensor

“…Several excellent reviews have been published to give a broad insight into conventional and emerging techniques for the detection of illegal drugs and their metabolites. Reviews or other papers covering the detection of drugs, e.g., via the testing of hair [ 6 , 7 , 8 , 9 ], the use of molecularly imprinted polymers [ 10 , 11 ], aptamers [ 12 ], ion mobility spectrometry [ 10 , 11 ], mass spectrometry [ 13 , 14 , 15 , 16 , 17 ], hybrid methods [ 18 ], electrochemical methods [ 19 , 20 ], biosensors [ 12 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ], and immunoassays [ 35 , 36 , 37 ] have been published. Quite popular in analytical chemistry was the detection of cocaine on banknotes [ 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ].…”

Cocaine Detection by a Laser-induced Immunofluorometric Biosensor

“…Several excellent reviews have been published to give a broad insight over conventional and emerging techniques for the detection of illegal drugs and their metabolites. Reviews or other papers covering the detection of drugs, e.g via the testing of hair [3][4][5][6], the use of molecularly imprinted polymers [7,8], aptamers [9], ion mobility spectrometry [10,11], mass spectrometry [12][13][14][15][16], hybrid methods [17], electrochemical methods [18,19], biosensors [9,[20][21][22][23][24][25][26][27][28][29][30][31][32][33] and immunoassays [34][35][36] have been published. Quite popular in analytical chemistry was the detection of cocaine on banknotes [37][38][39][40][41][42][43][44].…”

Ultrasensitive Label-Free Detection of Protein–Membrane Interaction Exemplified by Toxin-Liposome Insertion

“…In addition, SHS as presented here does not require any planar substrate and can utilize free-floating liposomes in an aqueous environment. Moreover, the obtained SHS data are quantifiable and could be extended in the following ways: Obtained values for the binding constant, the second-order surface susceptibility of water, and the electrostatic surface potential 23 could be connected with MD simulations as is currently done for X-ray crystallography and cryo-electron microscopy. In fact, if MD-predicted molecular conformations would have been used here, it would have been possible to explicitly compute the SHS response, rather than using a more simplified assumption as was done here.…”

“…Likewise, QCM and SPR are indirect methods that require a specific substrate, which restricts the application mostly to planar-supported model membranes, and calorimetry lacks specificity to a binding event. Resonant reflection second harmonic generation (SHG) has recently been successfully employed and suggested as an alternative to immunoassays , since the SH photons emerge directly from an interface and thus facilitate direct detection of interfacial events. In these experiments, the SH photons were generated via a total internal reflection scheme from a UV resonant interaction (266 nm).…”