Preparation Of Monoclonal Antibodies And Immunochromatographic Test Strip For The Staphylococcal Enterotoxin B Determination

Staphylococcal enterotoxin B(SEB)is a serious threat to human health because it is superantigenic,the most virulent and heat-resistant of the SEs,the most resistant to gastrointestinal proteases,and extremely difficult to remove during food processing.In this study,SEB was used as the immunogen,and Balb/C mice were immunized with a series of operations,including serum titer determination,cell fusion,screening of positive hybridoma cells,subcloning and antibody pairing,and finally two cell lines were obtained(named 15H11 and 5D6,respectively)that could stably secrete anti-SEB monoclonal antibodies,and the two antibodies could form a stable"sandwich"structure with SEB."Both anti-SEB monoclonal antibodies were Ig G1 subtypes with affinity constants of 2.08×10~9 M and 1.81×10~9 M,respectively,and did not cross-react with the other four common enterotoxins SEA,SEC,SED and SEE.Next,colorimetric-fluorescent bifunctional nanospheres(J-AIE@Au NBs)with Janus structure were synthesized by microemulsion method using oleyl ammonia-modified gold nanoparticles(Au NPs@OA),red aggregation-induced emission fluorescent dyes(AIEgens)as colorimetric and fluorescent functional materials and 1-octadecene maleic anhydride polymer(PMAO)as filler.Due to the great difference in solubility of Au NPs@OA and AIEgens in the polymer PMAO,an obvious phase separation of Au NPs@OA and AIEgens occurred during the chloroform evaporation process,resulting in the automatic nucleation of AIEgens and the formation of a shell layer by PMAO,while Au NPs@OA was distributed on one side of the PMAO layer,thus forming the Janus-structured.Since Au NPs were physically spaced from AIEgens and were on the AIEgens side,the energy transfer between them was effectively eliminated and the fluorescence internal filtering effect was reduced,thus greatly preserving the fluorescence signal of AIEgens.The immunoprobe was subsequently obtained by labeling J-AIE@Au NBs with monoclonal antibody 15H11,and the immunochromatographic test strip(J-AIE@Au-ICA)was prepared by spraying antibody 5D6 on the NC membrane for colorimetric qualitative analysis and fluorescence quantitative analysis of SEB.Under the optimal conditions,the linear range of SEB in whole milk was 0.39 ng/m L～400 ng/m L,the Limit of detection(LOD)was 0.09 ng/m L,and the sensitivity was 70 times higher than that of traditional ELISA method.At the same time,the minimum detection limit of SEB by colorimetric method with naked eyes was 1.56 ng/m L.The intra-and inter-spiked recoveries of the test strips for the determination of SEB in whole milk ranged from 86.02%to 115%,with coefficients of variation ranging from 4.97%to 12.86%,and has no cross-reactivity with other four common enterotoxins.In addition,the LOD of SEB in raw milk and skim milk was 0.19 ng/m L and 0.19 ng/m L,respectively.Subsequently,the test strips were used to detect whole milk,raw milk and skim milk samples contaminated with SEB,and the results showed that the recoveries were between 91.04%and 118.82%,and the coefficient of variation was between 3.91%and 12.86%.These results indicate that the colorimetric and fluorescent dual-function test strips developed in this study can achieve rapid and accurate quantification of SEB in a wide range of milk.