De novo assisted AFB1-Specific monoclonal antibody sequence assembly and comprehensive molecular characterization.

Despite their widely used and access as biological reagents in analytical methods, the detailed structural features for most of the antibodies were rarely known. Here, a new antibody for AFB1 with high specificity in constructing ELISA was studied in detail. The molecular structure and modification were elucidated mainly by nano-electrospray ionization mass spectrometry. The mass experiments, including MALDI-TOF MS, revealed complete and specific fragments, including antibody molecular weight, peptides, glycopeptide, and N-glycoform. By proteolytic treatment of pepsin and trypsin and high-resolution tandem-MS, the primary structure of the newly developed anti-AFB1 antibody was assembled by several rounds of Database search process assisted with the de novo results. The antibody CDR annotation and constraint-based multiple alignment tool were used to differentiate and align the sequences. The method uses only two proteases to generate numerous peptides for de novo sequencing. This artificial assembled AFB1-specific monoclonal antibody sequence was validated by comparison with the sequencing results of the immunoglobulin gene. The results showed that this method achieves full sequence coverage of anti-AFB1 monoclonal antibody, with an accuracy of 100% in the CDR regions of light chain and four amino acid mismatch in heavy chain. This simple and low-cost method was confirmed by treating a public dataset. The secondary structure information of intact antibody was also elucidated from the results of circular dichroism spectrum.

Development and application of lateral flow strip with three test lines for detection of deoxynivalenol in wheat.

Lateral flow strip was widely used and their qualitative and quantitative performance was in continuous improvement. However, the traditional strip was in a single-test-line format, which restricted operators to making a semi-quantitative judgment around a desired threshold concentration. Herein, a single strip with three test lines (TTLS) was developed for the semi-quantitative and quantitative determination of deoxynivalenol (DON). Four visual detection thresholds were obtained under optimized conditions and 35 wheat samples with DON content from 45 µg/kg to 2841 µg/kg were used to verify the method. The detection results were compared with that of the traditional strip and UPLC-MS/MS. In a three-test-line format, TTLS could reveal at least 200, 500, 1000, and 2000 µg/kg DON existed in different samples by the naked eye. The agreement analysis and statistical results indicated the new TTLS can be used as a useful tool for quantitative detection of DON with wide dynamic range.