Construction and use of two alpha-human atrial natriuretic peptide-fragment affinity chromatography columns in the isolation of C- and N-terminal epitope-specific antibodies for use in a prototype alpha-hANP biosensor.
Two alpha-human atrial natriuretic peptide (alpha-hANP) based affinity chromatography columns were produced by covalently immobilizing the C- and N-terminal epitopes of alpha-hANP. The stationary phase was made from a controlled-pore-glass bead solid support, which was silanized and treated with sulphosuccinimidyl 4-(maleimidomethyl)cyclohexyl carboxylate before the individual fragments were immobilized by substitution at their thiol groups. These columns were used to isolate alpha-hANP-specific antibodies from a goat anti-alpha-hANP serum, which were then further sorted according to their epitope specifity. These C- and N-terminal epitope-specific antibodies were in turn used as components in the construction of an alpha-hANP biosensor based on an enzyme-linked immunosorbent assay (ELISA) sandwich principle. Initial in vitro testing of the sensor using a physiological alpha-hANP solution showed a reproducible response to the peptide. There is to date no other equally fast, sensitive and precise method available to detect this peptide. This alpha-hANP sensor may prove to be an invaluable aid in human medicine as a monitor of patient status during transplant surgery, for example, an area inaccessible to radioimmunoassay and normal ELISA techniques.
Ferris
R
R
Institut für Allgemeine Biochemie, Universität Wien, Vienna, Austria.
Missbichler
A
A
Hartter
E
E
Mann-Buxbaum
E
E
Schalkhammer
T
T
Urban
G
G
Pittner
F
F
D000595:Amino Acid Sequence; D000906:Antibodies / Q000302:isolation & purification*; D009320:Atrial Natriuretic Factor; D015374:Biosensing Techniques; D002846:Chromatography, Affinity / Q000295:instrumentation*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments
D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D009320:Atrial Natriuretic Factor