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 x 20 microns 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 10x 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.
Evangelista
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Advanced Technology Center, Beckman Instruments Inc., Fullerton, CA 92634.
Chen
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D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D001666:Binding Sites, Antibody; D002197:Capillary Action; D002232:Carbocyanines / Q000737:chemistry / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006241:Haptens / Q000737:chemistry / Q000378:metabolism; D007834:Lasers; D009020:Morphine / Q000032:analysis / Q000276:immunology*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002232:Carbocyanines; D005456:Fluorescent Dyes; D006241:Haptens; C085321:cyanine dye 5; D009020:Morphine