RESUMO
Catecholamine derivatives were synthesized with potential applications as coating antigens in biosensors or in the raising of specific antibodies. Thioether-bridged derivatives of the catecholamines dopamine, norepinephrine, and epinephrine that attach carboxylic acid functionalities directly to the aromatic ring via an easily incremented linker chain were synthesized by an electrochemical method. These derivatives were purified by convenient ion-exchange chromatography, exact positions of conjugation determined by NMR, and a dopamine derivative immobilized in situ in a BIAcore surface plasmon resonance (SPR) biosensor and its antibody binding studied in comparison with immobilization via the catecholamine primary amine. Binding of an antibody raised to an amine-conjugated protein conjugate showed clear distinction between conjugations at different positions on the catecholamine, illustrating the importance of rational conjugate design in immunosensing of the catecholamines.
Assuntos
Catecolaminas/química , Catecolaminas/imunologia , Aminas/química , Anticorpos/imunologia , Catecolaminas/síntese química , Cromatografia por Troca Iônica , Ésteres/química , Estrutura Molecular , Compostos de Sulfidrila/química , Ressonância de Plasmônio de SuperfícieRESUMO
Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.
Assuntos
Anticorpos/imunologia , Estradiol/química , Estradiol/imunologia , Estrona/química , Estrona/imunologia , Ressonância de Plasmônio de Superfície/métodos , Anticorpos/química , Estradiol/análise , Estrona/metabolismo , Etilenoglicol/química , ImunoensaioRESUMO
Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.
Assuntos
Progesterona/análise , Ressonância de Plasmônio de Superfície , Anticorpos Monoclonais/química , Coloide de Ouro/química , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície/métodosRESUMO
A series of progesterone-4-ovalbumin (OVA) conjugates with different length linkers (4-, 11-, and 18-atoms long) were synthesized by successive aminocaproic acid homologation of 3-(pregn-4-ene-3,20-dione-4-yl)thiopropanoic acid (1) before conjugation to ovalbumin. The performance studies of these progesterone-4-ovalbumin conjugates showed that the effects of the length of linker on the antibody binding are dependent upon different immunoassay formats. In a rapid flow biosensor surface, on a BIAcore Surface Plasmon Resonance (SPR) instrument, antibody-binding capacities and response rate were dramatically increased for progesterone-4-ovalbumin conjugates when the length of the linker was incremented from 4 atoms to 11 or 18 atoms. Thus, highly sensitive SPR-based immunoassays for progesterone over a range of 0.1-50 ng ml(-1) were developed using biosensor surfaces immobilized with progesterone-ovalbumin conjugates having extended linkers. The SPR-based assays were fully competitive with conventional enzyme-linked immunosorbant assay (ELISA) but much more rapid and simple. However, there were little changes in antibody-binding performance using a conventional ELISA for the same conjugates. The progesterone-4-ovalbumin conjugate (1-OVA) had better antibody binding than its progesterone-7alpha-ovalbumin analog (2-OVA) in the SPR-based assay, but with a conventional ELISA there was no significant difference between these two isomeric conjugates.