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

Pengyan Liu,Yunyun Chang,Yirong Guo,Shasha Jiao,Ying Liu,Yihua Liu,Mengli Chen,Guonian Zhu,Yuanhao Guo,Rubing Zou

doi:10.3390/ijms21186857

Abstract

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.

Highlights

As alternatives to the classical instrumental methods, immunoassays are recognized as rapid, simple, and cost-effective analytical techniques for on-site screening of small molecular contaminants in environmental and agricultural samples
In order to identify the recognition spectrum of the monoclonal antibodies (mAbs)-C4C4, low molecular weight (LMW) selectivity screening was performed by the direct Surface Plasmon Resonance (SPR) immunoassay
The results showed that the association–dissociation kinetic modes of the anti-thiacloprid mAb toward various compounds could be divided into three categories (Figure 1 and Figure S3): (1) Thiacloprid and acetamiprid belong to the “fast on and rare off” group (Figure 1A and Figures S3B), meaning that the formed immunocomplexes were hard to dissociate (Kd of 5–6 × 10−4 1/s) after the quick binding reaction

Summary

Introduction

As alternatives to the classical instrumental methods, immunoassays are recognized as rapid, simple, and cost-effective analytical techniques for on-site screening of small molecular contaminants in environmental and agricultural samples. It is well known that an accurate and reliable immunoassay depends on the core reagent, namely an antibody with high sensitivity, specificity, and stable performance. Neonicotinoid insecticides are among the most extensively used agrichemicals in the world. Since thiacloprid is one of the representative varieties of these insecticides, which has been banned for outdoor use in the EU since January 2020 [5], it is urgent to establish sensitive and quick analytical methods for reliable monitoring of thiacloprid residues in environmental and agricultural samples. The investigation of antibodies against thiacloprid is the priority for the immunoassay development

Methods

Results

Conclusion