-transparent.png){kind=logo}
We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
October 2003,
Organic letters,
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
September 1987,
Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme,
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
January 2016,
Journal of nanoscience and nanotechnology,
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
January 1971,
International archives of allergy and applied immunology,
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
January 2016,
Methods in enzymology,
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
January 2015,
Immunology,
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
January 2013,
Molecular imaging,
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
March 2010,
Bioconjugate chemistry,
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
December 2021,
Chemical communications (Cambridge, England),
R Bredehorst, and
G A Wemhoff, and
A W Kusterbeck, and
P T Charles, and
R B Thompson, and
F S Ligler, and
C W Vogel
April 2019,
Chimia,
