Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods.

Nanobodies (Nbs) have great potential in immunoassays due to their exceptional physicochemical properties. With the immortal nature of Nbs and the ability to manipulate their structures using protein engineering, it will become increasingly valuable to understand what structural features of Nbs drive high stability, affinity, and selectivity. Here, we employed an anti-quinalphos Nb as a model to illustrate the structural basis of Nbs' distinctive physicochemical properties and the recognition mechanism. The results indicated that the Nb-11A-ligand complexes exhibit a "tunnel" binding mode formed by CDR1, CDR2, and FR3. The orientation and hydrophobicity of small ligands are the primary determinants of their diverse affinities to Nb-11A. In addition, the primary factors contributing to Nb-11A's limited stability at high temperatures and in organic solvents are the rearrangement of the hydrogen bonding network and the enlargement of the binding cavity. Importantly, Ala 97 and Ala 34 at the active cavity's bottom and Arg 29 and Leu 73 at its entrance play vital roles in hapten recognition, which were further confirmed by mutant Nb-F3. Thus, our findings contribute to a deeper understanding of the recognition and stability mechanisms of anti-hapten Nbs and shed new light on the rational design of novel haptens and directed evolution to produce high-performance antibodies.

Two binding epitopes modulating specificity of immunoassay for ß-agonist detection: Quantitative structure-activity relationship.

A growing number of ß-agonists are illegally using for reducing animal fat deposition in animals, but the development of analytical methods always lags behind the emergence of new illegal compounds. Therefore, class specificity antibody-based immunoassays that can detect a great many ß-agonists are important for timely supervision. In this study, a competitive inhibition enzyme-linked immunosorbent assay (ciELISA) based on a clenbuterol monoclonal antibody was developed to recognize 23 ß-agonists and analogues. Holographic and three-dimensional quantitative structure-activity relationship (HQSAR and 3D QSAR) revealed that there are two critical binding epitopes on ß-agonist hapten affecting antibody specificity, and these epitopes have been further validated using a ractopamine antibody with narrow specificity. Tert-butyl at C-2' epitope is needed to generate class specific antibodies, and different characteristics of substituents at benzene ring epitope would adjust antibody specificity. This investigation could provide reference for future design of ß-agonist haptens.

Conformational adaptability determining antibody recognition to distomer: structure analysis of enantioselective antibody against chiral drug gatifloxacin.

Enantioselective antibodies have great potential to separate and detect chiral compounds. However, cross-reactivity of enantioselective antibodies to the distomer may limit the application. An in-depth understanding of interactions between antibodies and chiral drugs could be helpful to investigate antibody recognition to the distomer. In this study, a monoclonal antibody against chiral quinolone S-(-)-gatifloxacin (S-GAT) was produced and its Fab fragment was prepared by proteolysis. The S-GAT Fab exhibited 10% cross-reactivity against the R-enantiomer compared to that of the S-enantiomer in an indirect competitive enzyme-linked immunosorbent assay (icELISA). The crystal structures of the S-GAT Fab apo form and complex with S-GAT were analyzed, and molecular docking of the R-enantiomer was carried out. The ligand conformation was further studied using molecular dynamics simulations. The results showed that the distomer R-enantiomer could enter the chiral center recognition region of the antibody by adjusting the piperazine ring conformation. Meanwhile, the antibody binding cavity had obvious conformational adaptability during ligand binding. It demonstrated that conformational change of both ligand and antibody was the key reason why antibodies recognize the distomer. Restricting conformational adaptability could improve the enantioselective recognition ability of antibodies. This study provided a new explanation for the cross-reactivity of enantioselective antibodies to the distomer, and could help to modulate antibody enantioselectivity for immunoassay of chiral drugs.

Broad-specificity ELISA with a heterogeneous strategy for sensitive detection of microcystins and nodularin.

A highly sensitive and broadly specific competitive indirect enzyme-linked immunosorbent assay (ciELISA) method was developed for the simultaneous detection of nine microcystins (MCs) and nodularin (NOD) using MC-LR-keyhole limpet hemocyanin (KLH) for New Zealand white rabbit immunization to produce antibodies. The MC-LR-bovine serum albumin (BSA) and NOD-BSA coating antigens were compared and heterogeneous coating strategy was found to significantly improve the sensitivity of detection, as evident from the appropriate structure. Comparison of the half-maximum inhibitory concentration (IC50) with MC-LR and MC-LR-BSA coating techniques (0.29 ng/mL) revealed the superior performance of 0.054 ng/mL for NOD-BSA coating. NOD-BSA was selected as the coating antigen, because it showed ultrahigh sensitivity for the detection of MC-LR with a limit of detection (LOD) of 0.0016 ng/mL, which was below the maximum residue level (MRL) of 1 ng/mL. In addition, high reproducibility, good stability, and acceptable spiked sample detection, as validated by liquid chromatography tandem mass spectrometry (LC-MS/MS), indicated the possible application of this method for the analysis of MCs and NOD in water sample.

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.

Broad-Specificity Immunoassay for Simultaneous Detection of Ochratoxins A, B, and C in Millet and Maize.

Ochratoxins A, B, and C (OTA, OTB, and OTC) can be found in cereals and feeds; the simultaneous detection of these ochratoxins holds a great need in food safety. In this study, four antibodies raised from two ochrotoxin haptens and two coating antigens were compared, and then a sensitive and broad-specificity enzyme-linked immunosorbent assay (ELISA) was established for the simultaneous determination of three ochratoxins, where the detection limits were 0.005, 0.001, and 0.001 ng/mL for OTA, OTB, and OTC, respectively, and recoveries of three ochratoxins were between 84.3% and 111.7%. This developed method had been successfully applied to detect ochratoxins in both millet and maize. Molecular modeling revealed that the broad-specificity was related with the chlorine electronegativity on OTA and OTC and the potential of the acetyl ester group on OTC. The proposed ELISA can be used for simultaneous detection of three ochratoxins.

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.