Antibodies directed against L and D isovaline using a chemical derivatizing reagent for the measurement of their enantiomeric ratio in extraterrestrial samples: First step-production and characterization of antibodies
Résumé
Determining the enantiomeric ratio of amino acids in meteorites requires very sensitive and precise measurements. In this study, an immunochemical approach, combined with new chemical derivatizing agents, was investigatedfor the measurement of the enantiomeric ratio of isovaline. In the initial step, L and D isovaline were derivatized with ε-benzyloxycarbonyl-L-lysine-(t-butyl ester)-chloroethylnitrosourea (Z-L-Lys-(OtBu)-CENU). The Z group was hydrolyzed and the resulting isovaline derivatives (i.-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) were conjugated with protein using glutaraldehyde and reduced with sodium borohydride. Rabbits were immunized with the immunogenic conjugates thus obtained. Antibodies were characterized using many compounds, both derivatized and underivatized, in competitive ELISA tests. These competition experiments performed enabled us to establish the following results: 1) unconjugated L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline were poorly recognized; 2) all related L-Lys(OtBu)-α-hydrogenated amino acids (L and n) were not recognized at all, which eliminates the possibility of the measurements being distorted by contamination; 3) only conjugated L-Lys(OtBu)-α-amino-isobutyric acid (AIB) was recognized by the antibody, 4) the enantiomeric discrimination of L and D isovaline through their derivatives (diastereoisomeric L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) was in accordance with the measurement of their enantiomeric ratio. Immunopurification was shown to enhance antibody specificity. The strategy employed shows potential for the quantification of meteoritic amino acids