Four Hapten Spacer Sites Modulating Class Specificity: Nondirectional Multianalyte Immunoassay for 31 ß-Agonists and Analogues.

Immunoassay methods are important for monitoring ß-agonists illegally used for reducing animal fat deposition in livestock. However, there is no simultaneous screening surveillance immunoassay for detecting various ß-agonist chemicals that are possibly present in food. In this study, through the use of an R-(-)-salbutamol derivative as the immunizing hapten, an antibody recognizing 31 ß-agonists and analogues was generated for the first time. Three-dimensional quantitative structure-activity relationship (3D QSAR) revealed that strong steric and hydrophobic fields around the hapten spacer near C-2, as well as a chirality at C-1', dominantly modulated the class specificity of the raised antibody. However, a hapten spacer linked at C-2' or C-1 would lead to a narrow specificity, and the spacer charge at C-6 could affect the raised antibody specificity spectrum. A class specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) was established with an ideal recovery ranging from 81.8 to 118.3% based on the obtained antibody. With a good agreement to the HPLC/MS method, the proposed ciELISA was confirmed to be reliable for the rapid surveillance screening assay of ß-agonists in urine. This investigation will contribute to the rational design and control of the immunoassay specificity.

Four Specific Hapten Conformations Dominating Antibody Specificity: Quantitative Structure-Activity Relationship Analysis for Quinolone Immunoassay.

Antibody-based immunoassay methods have been important tools for monitoring drug residues in animal foods. However, because of limited knowledge about the quantitative structure-activity relationships between a hapten and its resultant antibody specificity, antibody production with the desired specificity is still a huge challenge. In this study, the three-dimensional quantitative structure-activity relationship (3D QSAR) was analyzed in accordance with the cross-reactivity of quinolone drugs reacting with the antibody raised by pipemidic acid as the immunizing hapten and compared with the reported cross-reactivity data and their hapten structures. It was found that the specificity of a quinolone antibody was strongly related to the conformation of the hapten used and that hapten conformations shaped like the letters "I", "P", and "Φ" were essential for the desired high specificity with low cross-reactivity, but that the hapten conformation shaped like the letter "Y" led to an antibody with broad specificity and high cross-reactivity. Almost all of the antibodies against quinolones could result from these four hapten conformations. It was first found that the concrete conformations dominated the specificity of the antibody to quinolone, which will be of significance for the accurate hapten design, predictable antibody specificity, and better understanding the recognition mechanism between haptens and the antibodies for immunoassays.