Highly Efficient Screening And Immunoassay Technology For Monoclonal Antibodies Against Mycotoxins In Agricultural Products

Aflatoxin B₁（AFB₁）,the most harmful of the aflatoxins（AFs）,and T-2,the most toxic of the trichothecene toxins,both damage the human immune system.Low doses of the toxin can cause an inflammatory response,and long-term exposure can have a suppressive effect on the human immune system.Timely and accurate detection of fungal toxin contamination in agricultural products is an effective way to ensure the safety of agricultural products.Immunoassay techniques are widely used for rapid mycotoxin detection because of their high sensitivity and simple operation.Immunochromatographic assay（ICA）and Enzyme-linked immunosorbent assay（ELISA）kits are commonly used for rapid on-site testing of agricultural products.Food inspection methods issued by the General Administration of Market Regulation are used in the colorimetric and test strip detection methods.The core reagents of immunoassay technology are antibodies,and obtaining positive hybridoma cell lines that secrete high-quality monoclonal antibodies is a prerequisite for monoclonal antibody preparation.At present,the sensitivity of rapid detection products on the market is not high,prone to false positives,cumbersome operation steps,time-consuming and costly,and the quality of the key core reagents-monoclonal antibodies need to be improved.In response to the serious contamination of agricultural products with AFB₁ and T-2 toxins and the shortcomings of the current commonly used rapid immunoassay detection techniques,the main research results of this topic are as follows:（1）The AFB₁ monoclonal antibody was obtained by a distribution function-like vertex subcloning method combined with a three-step amplification sample assay and a multi-concentration standard assay.During the study,it was found that the distribution of the cell supernatant assay data with subcloning resembled a distribution function.Cell wells with high sensitivity and specificity near the top of the distribution function were selected for subcloning.As the subcloning proceeded,the concentration of the standard was gradually reduced to domesticate the cells to improve the sensitivity of the monoclonal antibody.However,the single standard concentration has some limitations for the detection of cell supernatants,and some good cells are overlooked when there are more antibodies in the supernatant.The cells screened under a single standard in a 96-well cell plate were amplified in a 24-well,6-well cell plate and culture flask,and the supernatant was detected by using multiple concentrations of standards,and the cells with consistent comparison data were the stable cell lines obtained by monoclonal culture best hybridoma cell line AFB₁-B56-0591A2-2,which stably secreted anti-AFB₁ monoclonal antibody,was eventually obtained.The semi-inhibitory concentration of the secreted monoclonal antibody MAb A2-2 was 0.13522 ng/m L,and the detection range was0.08518-0.21464 ng/m L.（2）Establishment of a dynamic,pseudo-homogeneous immunomagnetic bead-based indirect competition ELISA（MBs-ic ELISA）.The MBs-ic ELISA was constructed using the monoclonal antibody MAb A2-2 as the recognition element and MBs as the antigen carrier,which has a larger surface area than the plate to fix more antigen and transfer the reaction system from solid phase to liquid phase to make the reaction system more homogeneous.The addition of external conditions allows the MBs-ic ELISA to be dynamic,not only increasing the binding rate but also greatly reducing the detection time.Compared with existing ELISA,MBs-ic ELISA reduces the detection time by2/3 and increases the sensitivity.Under the optimal reaction conditions,the linear fitting equation in methanol solution（10%,z/o）was Y=-0.57lg X+2.64（R²=0.99396）,the linear detection range was 0.004-10 ng/m L,and the limit of detection（LOD）was 0.0013 ng/m L.（3）Construction of a fluorescence immunochromatographic assay（FIA）based on bioactivity protection and signal amplification.Most current immunochromatographic detection techniques are based on direct labeling with target antibodies,and the labeling process is prone to damage antibody activity and low signal acquisition efficiency,so in this study,an FIA was established for the detection of T-2toxin.The fluorescent probe（Ig G@Eu）was prepared by combining polystyrene fluorescent microspheres with sheep anti-mouse Ig G,which not only protected the monoclonal antibody activity,but also improved the binding rate of the fluorescent microspheres to the monoclonal antibody and thus amplified the fluorescent signal.the standard working equation for T-2 toxin in methanol solution（70%,z/o）is Y=-0.43022X+0.7308（R²=0.98593）.the LOD of T-2 toxin in methanol solution（70%,z/o）was 0.01 ng/m L with a linear range of 0.0625-50 ng/m L.the LOD in corn substrate and feed substrate was 0.052 ng/m L,0.071 ng/m L,and the recoveries of T-2 toxin ranged from 95.31%-119.03%,95.7%-110.33%with the relative standard deviations less than 11.38%and 16.02%.The method was validated by LC-MS/MS,and the results showed that the method has good accuracy.This study provides a theoretical basis for the innovation of hybridoma cell screening technology,and the established MBs-ic ELISA and ICA are expected to be transformed into products to be sold in the market,realizing on-site high-throughput rapid detection of agricultural products,and providing a theoretical basis and practical technical support for the construction of immunoassay methods.