title: Development of enantioselective chemiluminescence flow- and sequential-injection immunoassays for alpha-amino acids.
abstract: The development of an enantioselective flow-through chemiluminescence immunosensor for amino acids is described. The approach is based on a competitive assay using enantioselective antibodies. Two different instrumental approaches, a flow-injection (FIA) and a sequential-injection system (SIA), are used. Compared to the flow-injection technique, the sequential injection-mode showed better repeatability. Both systems use an immunoreactor consisting of a flow cell packed with immobilized haptens. The haptens (4-amino-L- or D-phenylalanine) are immobilized onto a hydroxysuccinimide-activated polymer (Affi-prep 10) via a tyramine spacer. Stereoselective antibodies, raised against 4-amino-L- or D-phenylalanine, are labeled with an acridinium ester. Stereoselective inhibition of binding of the acridinum-labeled antibodies to the immobilized hapten by amino acids takes place. Chiral recognition was observed not only for the hapten molecule but also for a series of different amino acids. One assay cycle including regeneration takes 6:30 min in the FIA mode and 4:40 min in the SIA mode. Using D-phenylalanine as a sample, the detection limit was found to be 6.13 pmol/ml (1.01 ng/ml) for the flow-injection immunoassay (FIIA) and 1.76 pmol/ml (0.29 ng/ml ) for the sequential-injection immunoassay (SIIA) which can be lowered to 0.22 pmol/ml (0.036 ng/ml) or 0.064 pmol/ml (0.01 ng/ml) by using a stopped flow system. The intra-assay repeatability was found to be about 5% RSD and the inter-assay repeatability below 6% (within 3 days).
authors.affiliation: Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, Karl-Franzens University Graz, Universitätsplatz 1, A-8010 Graz, Austria.
authors.forename: Hossein
authors.initials: H
authors.lastname: Silvaieh
authors.forename: Martin G
authors.initials: MG
authors.lastname: Schmid
authors.forename: Oliver
authors.initials: O
authors.lastname: Hofstetter
authors.forename: Volker
authors.initials: V
authors.lastname: Schurig
authors.forename: Gerald
authors.initials: G
authors.lastname: Gübitz
mesh_terms: D000166:Acridines; D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry; D004867:Equipment Design; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D008163:Luminescent Measurements; D010649:Phenylalanine / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D013388:Succinimides
chemical_list: D000166:Acridines; D000596:Amino Acids; D013388:Succinimides; D010649:Phenylalanine; C053333:4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium-9-carboxylate