title: A new approach for quantitative analysis of L-phenylalanine using a novel semi-sandwich immunometric assay.
abstract: Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, "biotin linker conjugate of FC-Phe N-succinimidyl ester" (FC(Biotin)-NHS), was synthesized to convert L-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized L-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new "semi-sandwich" immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1-20 μM were attained using a standard L-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6% of the coefficient of variation; inter-day variation was 0.1%. The recovery rates were from 92.4 to 123.7%. This is the first report of the quantitative determination of L-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.
authors.affiliation: Quality Management Department, Tokai Plant Ajinomoto Co., Inc., 1730, Hinaga-cho, Yokkaichi, Mie, 510-0885, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kubota
authors.forename: Toshimi
authors.initials: T
authors.lastname: Mizukoshi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Miyano
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D005260:Female; D006241:Haptens / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D051379:Mice; D008807:Mice, Inbred BALB C; D010649:Phenylalanine / Q000097:blood* / Q000276:immunology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000911:Antibodies, Monoclonal; D006241:Haptens; D010649:Phenylalanine