Precise and sensitive detection of non-hemolytic enterotoxin based on recombinant antibody: Lateral flow immunochromatography assays and exploring recognition mechanisms.

Bacillus cereus (B. cereus), an important foodborne pathogen, can produce multiple enterotoxins. It is essential to detect food with enteropathogenic potential as rapidly and accurately as possible to ensure food safety. In this study, the recombinant antigen non-hemolytic enterotoxin A (Nhe A) was expressed, purified, and then used to immunize mice to obtain monoclonal antibodies (mAbs). Furthermore, double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) based on paired mAbs 5C1-8G7 was established to detect Nhe A with limits of detection (LOD) of 0.35 ng/mL in milk samples. Then, the variable region of the heavy-chain (VH) and light-chain (VL) genes of mAbs 5C1, 8G7, and 13E12 were amplified, sequenced, and inserted into expression vectors. The highest yield of recombinant antibodies (rAbs) was achieved when the ratio of heavy- to light-chain plasmids was 1:1.5, and the highest antibody production reached 100 mg/L. Next, lateral flow immunochromatography assays (LFIA) were constructed to detect Nhe A in milk and vegetables with the cLOD of 2.6, and 3.28 ng/mL. Additionally, molecular docking and molecular dynamics (MD) simulation revealed that hydrogen bonds played important roles in the antigen-antibody interaction. Overall, the platform demonstrated its suitability for detecting B. cereus in food samples.