Fluorometric titration approach for calibration of quantity of binding site of purified monoclonal antibody recognizing epitope/hapten nonfluorescent at 340 nm.
A fluorometric titration approach was proposed for the calibration of the quantity of monoclonal antibody (mcAb) via the quench of fluorescence of tryptophan residues. It applied to purified mcAbs recognizing tryptophan-deficient epitopes, haptens nonfluorescent at 340 nm under the excitation at 280 nm, or fluorescent haptens bearing excitation valleys nearby 280 nm and excitation peaks nearby 340 nm to serve as Förster-resonance-energy-transfer (FRET) acceptors of tryptophan. Titration probes were epitopes/haptens themselves or conjugates of nonfluorescent haptens or tryptophan-deficient epitopes with FRET acceptors of tryptophan. Under the excitation at 280 nm, titration curves were recorded as fluorescence specific for the FRET acceptors or for mcAbs at 340 nm. To quantify the binding site of a mcAb, a universal model considering both static and dynamic quench by either type of probes was proposed for fitting to the titration curve. This was easy for fitting to fluorescence specific for the FRET acceptors but encountered nonconvergence for fitting to fluorescence of mcAbs at 340 nm. As a solution, (a) the maximum of the absolute values of first-order derivatives of a titration curve as fluorescence at 340 nm was estimated from the best-fit model for a probe level of zero, and (b) molar quantity of the binding site of the mcAb was estimated via consecutive fitting to the same titration curve by utilizing such a maximum as an approximate of the slope for linear response of fluorescence at 340 nm to quantities of the mcAb. This fluorometric titration approach was proved effective with one mcAb for six-histidine and another for penicillin G.
Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University , Chongqing 400016, China.
Xiaolan
X
Yang
Xiaolei
X
Hu
Bangtian
B
Xu
Xin
X
Wang
Jialin
J
Qin
Chenxiong
C
He
Yanling
Y
Xie
Yuanli
Y
Li
Lin
L
Liu
Fei
F
Liao
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002138:Calibration; D000939:Epitopes / Q000276:immunology*; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry / Q000379:methods*; D006241:Haptens / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006241:Haptens