Reverse design of haptens based on antigen spatial conformation to prepare anti-capsaicinoids&gingerols antibodies for monitoring of gutter cooking oil.

Rapid simultaneous detection of multi-component adulteration markers can improve the accuracy of identification of gutter cooking oil in edible oil, which is made possible by broad-spectrum antibody (bs-mAb). This study used capsaicinoids (CPCs) and gingerol derivatives (GDs) as adulteration markers, and two broad-spectrum haptens (bs-haptens) were designed and synthesized based on a reverse design strategy of molecular docking. Electrostatic potential (ESP) and monoclonal antibodies (mAbs) preparation verified the strategy's feasibility. To further investigate the recognition mechanism, five other reported antigens and mAbs were also used. Finally, the optimal combination (Hapten 5-OVA/1-F12) and key functional groups (f-groups) were determined. The half maximal inhibitory concentration (IC50) for CPCs-GDs was between 88.13 and 499.16 ng/mL. Meanwhile, a preliminary lateral flow immunoassay (LFIA) study made practical monitoring possible. The study provided a theoretical basis for the virtual screening of bs-haptens and simultaneous immunoassay of multiple exogenous markers to monitor gutter oil rapidly and accurately.

Activates B lymphocytes and enhanced immune response: A promising adjuvant based on PLGA nanoparticle to improve the sensitivity of ZEN monoclonal antibody.

In preparing monoclonal antibodies by hybridoma cell technology, the quality of B lymphocytes used for cell fusion directly affects the sensitivity of monoclonal antibodies. To obtain B-lymphocytes producing high-quality specific antibodies for cell fusion during the immunization phase of the antigen, we prepared a TH2-Cell stimulatory delivery system as a novel adjuvant. Astragalus polysaccharide has a good ability to enhance antigenic immune response, and it was encapsulated in biocompatible materials PLGA as an immunostimulatory factor to form the delivery system (APS-PLGA). The preparation conditions of APSP were optimized using RSM to attain the highest utilization of APS. Immunization against ZEN-BSA antigen using APSP as an adjuvant to obtain B lymphocytes producing ZEN-specific antibodies for cell fusion. As results present, APSP could induce a stronger TH2 immune response through differentiating CD4 T cells and promoting IL-4 and IL-6 cytokines. Moreover, it could slow down the release efficiency of ZEN-BSA and enhance the targeting of ZEN-BSA to lymph nodes in vivo experiments. Ultimately, the sensitivity of mouse serum ZEN-specific antibodies was enhanced upon completion of immunization, indicating a significant upregulation of high-quality B lymphocyte expression. In the preparation of monoclonal antibodies, the proportion of positive wells for the first screening was 60%, and the inhibition rates of the antibodies were all similar (>50%). Then we obtained the ZEN monoclonal antibody with IC50 of 0.049 ng/mL, which was more sensitive than most antibodies prepared under conventional adjuvants. Finally, a TRFIAS strip assay was preliminarily established with a LOD value of 0.246 ng/mL.

Production of AFB1 High-Specificity Monoclonal Antibody by Three-Stage Screening Combined with the De-Homologation of Antibodies and the Development of High-Throughput icELISA.

To achieve accurate detection of AFB1 toxin content in agricultural products and avoid false-positive rates in the assays, the specificity of mAbs is critical. We improved the specificity of the prepared monoclonal antibodies by modifying the traditional limiting dilution subcloning method. The traditional finite dilution method was modified with three-stage screening (the trending concentration of standards used in the screening is low-high-low) to achieve high specificity in pre-cell screening and increased the number of subclones to 10 to achieve the de-homologation of antibodies. A modified limiting dilution obtained a highly specific AFB1 monoclonal cell line, ZFG8, with a 50% inhibition concentration (IC50) of 0.3162 ng/mL. Notably, it exhibited the highest specificity compared to anti-AFB1 monoclonal antibodies prepared by other investigators. The maximum cross-reactivity of the mAb with structural analogues for AFB2, AFG1, AFG2, and AFM1 was 0.34%. The results showed that this type of screening improves the monoclonal antibodies' specificity. Based on this ZFG8 monoclonal antibody, an icELISA assay was established with an IC50 of 0.2135 ng/mL for AFB1. The limit of the linear detection range of icELISA is 0.0422-1.29267 ng/mL with reasonable specificity and precision. The recoveries of AFB1 in samples of corn flour and wheat meal ranged from 84 to 107%, with CVs below 9.3%. The recoveries of structural analogues (AFB2, AFM1, AFG1, and AFG2) were less than 10% in both corn flour and wheat meal. The results showed that the prepared AFB1 monoclonal antibody could accurately and specifically recognize AFB1 residues in agricultural products while ignoring the effects of other structural analogues.