Computer-aided profiling of a unique broad-specific antibody and its application to an ultrasensitive fluoroimmunoassay for five N-methyl carbamate pesticides.

Pollution of N-methyl carbamate (NMC) pesticides is threatening the non-target organisms' survival. Thus, broad-specific antibodies and class-selective immunoassays are demanding for multiple NMCs determination. In this study, we employed a molecular docking-based virtual screening strategy to fast profile antibody spectrum, based on a designed chemical pool containing 17 compounds. A monoclonal antibody (mAb)-6G against carbofuran was used as the objective. The recombinant full-length IgG was successfully expressed to validate the antibody sequences for homology modeling. After docking, we manually categorized the antibody-chemical binding strength into three groups. Non-competitive surface plasmon resonance (SPR) demonstrated the mAb-6G affinitive binding toward five NMCs (carbofuran, isoprocarb, propoxur, carbaryl and carbosulfan), which were classified into strong and moderate binding categories. Antibody binding properties were confirmed again by ic-ELISA and lateral flow immunochromatographic strip. Subsequently, an ultrasensitive indirect competitive fluoromicrosphere-based immunoassay (ic-FMIA) was established with the IC50 (half-maximal inhibitory concentration) values of 0.08-3.37 ng/mL. This portable assay presented a 30-230-fold improved sensitivity than traditional ic-ELISA and was applied in European surface water analysis. Overall, our work provides an efficient platform integrating in-silico and experimental methodologies to accelerate the characterization of hapten-specific antibody binding properties and the development of high-sensitive immunoassays for multi-pollutants monitoring.

Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody.

Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC50 values of 0.73 and 0.46 µg/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH-π interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties.

Up-Converting Nanoparticle-Based Immunochromatographic Strip for Multi-Residue Detection of Three Organophosphorus Pesticides in Food.

Organophosphorus (OP) pesticides are widely used to control pests because of their high activity. This study described a rapid and sensitive lateral flow immunochromatographic (LFIC) assay based on up-converting nanoparticles (UCNPs) for multi-residue detection of three OP pesticides. The developed assay integrated novel fluorescent material UCNPs labeled with a broad-specific monoclonal antibody. Based on the competitive platform by immobilized antigen in the test zone, the optimized UCNPs-LFIC assay enabled sensitive detection for parathion, parathion-methyl, and fenitrothion with IC50 of 3.44, 3.98, and 12.49 ng/mL (R 2 ≥ 0.9776) within 40 min. The detectable ability ranged from 0.98 to 250 ng/mL. There was no cross-reactivity with fenthion, phoxim, isocarbophos, chlorpyrifos, or triazophos, even at a high concentration of 500 ng/mL. Matrix interference from various agricultural products was also studied in food sample detection. In the spiked test, recoveries of the three OP pesticides ranged from 67 to 120% and relative standard deviations were below 19.54%. These results indicated that the proposed strip assay can be an alternative screening tool for rapid detection of the three OP pesticides in food samples.

Binding properties of broad-specific monoclonal antibodies against three organophosphorus pesticides by a direct surface plasmon resonance immunosensor.

In this study, heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) was introduced into the screening of hybridomas for the development of broad-specific monoclonal antibodies (mAbs) against organophosphorus (OP) pesticides. After immunization, two formats of icELISA based on the homologous hapten antigen and four heterologous hapten antigens were conducted for hybridoma screening. Two mAbs 2G6 and 7B2 with good recognition toward three OP pesticides (parathion, methyl-parathion, and fenitrothion) were produced. Results of the icELISA showed that the two mAbs exhibited high sensitivity against three OP pesticides, with IC50 ranging from 2.93 to 19.71 ng mL-1. Moreover, a non-competitive surface plasmon resonance (SPR) immunosensor was used for characterizing the binding properties of the mAbs to OP pesticides. After kinetic analysis, equilibrium dissociation constant (KD) values of mAbs 2G6 and 7B2 were calculated as 1.45 × 10-9 M and 4.26 × 10-9 M for parathion, 6.75 × 10-9 M and 4.17 × 10-9 M for methyl-parathion, and 2.44 × 10-8 M and 1.19 × 10-8 M for fenitrothion, respectively. Whereas, both icELISA and SPR-based immunoassay indicated that the two mAbs could not recognize other five OP analogs. Since SPR-based immunoassay provides comprehensive information of two molecules directly interacting with each other, it is a valuable tool during the development and characterization of broad-specific mAbs. Graphical abstract ᅟ.