The Research On Competitive And Noncompetitive Phage Enzyme-linked Immunosorbent Assay For Benzothiostrobin

Immunoassay,with the advantages of simple,rapid,sensitive,economic and high-throughput,has been widely studied in detection of pesticide residue.Heterologous and noncompetitive immunoassays are the two research highlights of immunoassay for pesticide residue.Phage display technology is a fast way to develop heterologous and noncompetitive immunoassays.In this article,phage-displayed peptidomimetic and anti-immunocomplex of benzothiostrobin have been prepared,and the immunoassays with higher sensitivity and stronger specificity have been developed.Using C18 reversed-phage chromatographic column and UV 230 nm detection wavelength,content of benzothiostrobin was measured with HPLC.In this method,the mobile phage was acetonitrile:water（65:35,v/v）.Within the concentration of 0.05 to 10.0 mg/L,the peak area and concentration of benzothiostrobin standard solution had good linear relationship.The average recoveries of benzothiostrobin were 80.83%-100.46%,the relative standard deviations（RSDs）were 0.25%-5.54%in tomato and rice samples.The results met the requirement of residual detection for pestiside.Twenty-three phage-displayed peptides,including twenty kinds of peptidomimetics and three kinds of phage anti-immunocomplex,that specifically bind to an anti-benzothiostrobin monoclonal antibody（mAb）in the absence or presence of benzothiostrobin were isolated from a cyclic 8-residue peptide phage library.The peptidomimetics had three consensus amino acid sequences,they were TPXGSL、GLAXFM and PXGAXH,respectively;while anti-immunocomplex had one consensus amino acid sequences,which was PXIWPXXW,where X represents different amino acids.The competitive phage enzyme linked immunosorbent assays（ELISAs）for benzothiostrobin were developed by using the prepared peptidomimetics.The optimized clone named C3-3 was selected,and experimental parameters of concentrations of coating antibodies and phage and PBS buffer were studied to improve the sensitivity.Under the optimal conditions,the half maximal inhibition concentration（IC₅0）and the limit of detection（LOD,IC₁0）of the competitive phage ELISA were 0.94 and 0.22 μg/L,respectively.The linear range was 0.22-3.94 μg/L.Compared to the conventional ELISA,the sensitivity was improved 8-fold.The cross-reactions with analog compounds were negligible except pyraclostrobin,which had 0.34%cross-reactivity.The average recoveries of benzothiostrobin were 67.6%-119.6%,the RSDs were 2.2%-12.8%in tomato,pear,cucumber and rice samples,which met the requirement of residual detection for pesticide.The detection results of competitive phage ELISA and HPLC for authentic samples showed high degree of correlations（R²=0.981,y=0.948x+0.437 in rice samples;R²= 0.981,y= 1.109x+11.88 in tomato samples）.These results indicated that a heterologous competitive immunoassay with high sensitivity can be developed for detection of benzothiostrobin in agricultural products based on the phage-displayed peptide,the detection results of which are accurate and reliable.The noncompetitive phage enzyme linked immunosorbent assays（ELISAs）for benzothiostrobin were developed based on the prepared anti-immunocomplex.The optimized clone named N6-18 was selected,and experimental parameters of concentrations of coating antibodies and phage and PBS buffer were studied to improve the sensitivity.Under the optimal conditions,the concentration of analyte producing 50%saturation of the signal（SC₅0）and the limit of detection（LOD,SC₁0）of the noncompetitive phage ELISA were 2.27 and 1.11 μg/L,respectively.The linear range was 1.11-4.62 μg/L.Compared to the conventional ELISAs,the sensitivity was improved 3-fold.The specificity was evaluated by measuring the cross reactivities（CRs）with some analogues of benzothiostrobin,and all the CRs were lower than 0.03%.The average recoveries of benzothiostrobin were 70.43%-128.57%,the RSDs were 1.79%-11.53%in tomato,pear,cucumber and rice samples,which met the requirement of residual detection for pesticide.The detection results of noncompetitive phage ELISA and HPLC analysis for authentic samples were similar.The correlation equations were y=1.073x-9.558 with R²=0.996 and y= 0.962x-7.429 with R = 0.997 in rice and tomato samples,respectively.These results indicated that a noncompetitive immunoassay with better sensitivity and specificity can be developed for detection of benzothiostrobin by using anti-immunocomplex,which is an alternative tool for detecting benzothiostrobin in envitonmental and agricultural samples.