title: A fluorescent-photochrome method for the quantitative characterization of solid phase antibody orientation.
abstract: A fluorescent-photochrome method of quantifying the orientation and surface density of solid phase antibodies is described. The method is based on measurements of quenching and rates of cis-trans photoisomerization and photodestruction of a stilbene-labeled hapten by a quencher in solution. These experimental parameters enable a quantitative description of the order of binding sites of antibodies immobilized on a surface and can be used to characterize the microviscosity and steric hindrance in the vicinity of the binding site. Furthermore, a theoretical method for the determination of the depth of immersion of the fluorescent label in a two-phase system was developed. The model exploits the concept of dynamic interactions and is based on the empirical dependence of parameters of static exchange interactions on distances between exchangeable centers. In the present work, anti-dinitrophenyl (DNP) antibodies and stilbene-labeled DNP were used to investigate three different protein immobilization methods: physical adsorption, covalent binding, and the Langmuir-Blodgett technique.
authors.affiliation: Interdepartmental Research Center "E. Piaggio", via Diotisalvi 2, Pisa, 56126, Italy.
authors.forename: Arti
authors.initials: A
authors.lastname: Ahluwalia
authors.forename: Danilo
authors.initials: D
authors.lastname: De Rossi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Giusto
authors.forename: Oren
authors.initials: O
authors.lastname: Chen
authors.forename: Vladislav
authors.initials: V
authors.lastname: Papper
authors.forename: Gertz I
authors.initials: GI
authors.lastname: Likhtenshtein
mesh_terms: D000327:Adsorption; D000906:Antibodies / Q000032:analysis* / Q000737:chemistry; D001666:Binding Sites, Antibody; D019295:Computational Biology; D004136:Dinitrobenzenes / Q000276:immunology; D005456:Fluorescent Dyes / Q000378:metabolism*; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010834:Phytochrome / Q000378:metabolism*; D012821:Silanes / Q000378:metabolism; D013267:Stilbenes / Q000276:immunology
chemical_list: D000906:Antibodies; D004136:Dinitrobenzenes; D005456:Fluorescent Dyes; D007454:Iodides; D012821:Silanes; D013267:Stilbenes; D010834:Phytochrome