Analysis of structural specificity in antibody—antigen reactions by capillary electrophoresis with laser-induced fluorescence detection

A rapid and simple procedure for screening antibodies for binding to an antigen is proposed. A fluorescent hapten —dye conjugate was prepared by labeling the amino moiety of the hapten with a commercially available reactive cyanine dye, Cy5 (excitation maximum: 650 nm, emission maximum: 670 nm). A fixed amount of the Cy5—hapten was titrated with serial dilution of the antibody. Each of the titration mixture was analyzed by capillary electrophoresis (25 cm × 20 μm column) monitored by laser-induced fluorescence (laser: 10 mW helium-neon, 632.8 nm). Free and antibody-bound Cy5-hapten were analyzed simultaneously on the electropherogram. Competitive immunoassay of hapten was demonstrated with low-end sensitivity of 5 · 10⁻⁸ M, about 10× more sensitive than the present drug screening methods. Using morphine as an example, the screening of various antibodies (from different vendors) and cross-reactivity of morphine analogues using the present procedure will be discussed.