Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing.
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.
Mitchell
John S
JS
Bioengineering Sector, HortResearch, East Street, Private Bag 3123, Hamilton, New Zealand.
Wu
Yinqiu
Y
Cook
Christian J
CJ
Main
Lyndsay
L
D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D004958:Estradiol / Q000032:analysis / Q000737:chemistry* / Q000276:immunology*; D004970:Estrone / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D019855:Ethylene Glycol / Q000737:chemistry; D007118:Immunoassay; D020349:Surface Plasmon Resonance / Q000379:methods*
D000906:Antibodies; D004970:Estrone; D004958:Estradiol; D019855:Ethylene Glycol