A new approach for quantitative analysis of l-phenylalanine using a novel semi-sandwich immunometric assay

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.