Development Of Immunsorbent Assay For The Determination Of The Herbicide Dicamba And Glyphosate

With the development of modern agriculture technology, herbicides are more and more widely used, and result in serious residues in agricultural products and threats to food safety. The residues of herbicides not only have enormous influence on human heath, but also on environment. For this reason, it is a task hanging over our head to set up a simple, rapid, sensitive method to detect herbicide residues, which will have significant social benefits and good prospects of application. Dicamba (DIC) and glyphosate (GLY) are two extensively applied herbicides in agricultural production, and there is an upward trend in application amounts in recent years. A great number of researches have proved both of them have teratogenicity and mutagenesis effects. In addition, dicamba and glyphosate both are agricultural and urban pollutant, which are focused monitored by many countries including China. Therefore, in order to offer technical supports for testing two herbicide residues in agricultural product and environment, we prepared the monoclonal antibodies of dicamba and polyclonal antibodies of glyphosate, and established quick and sensitive immunoassay means for dicamba and glyphosate detection..The diimine carbonization methods were used to synthesize immunogen and coating antigen by dicamba and glyphosate being conjugated to ovalbumin (OVA) and bovine serum albumin (BSA), respectively. The UV spectrophotometer detection showed the coupling ratios of DIC to BSA and OVA were34:6and20:1. The results indicated the coupling ratios of GLY to BSA and OVA were11:1and9:1by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).One hybridoma cell line capable of secreting monoclonal antibodies (MAbs) against dicamba was obtained by fusing mouse myeloma cells (SP2/0) with spleen cells taken from the BALB/c mice having been immunized by the synthetised DIC-OVA. The MAbs could specifically react with DIC. The ascites were purified by caprylic acid-ammonium sulfate, of which concentration was4.53mg/mL detected by a UV spectrophotometer. The enzyme-linked immunosorbent assay (ELISA) titre of ascitic fluids of theMAbs against dicamba was1:1.024x105. The percentages of the antibody cross reactivity with other analogues of dicamba (2,3,5-trichlorobenzoic acid,2,3,6-trichlorobenzoic acid and2-amino-3,5-dichlorobenzoic acid) were all less than0.01%. An indirect competitive ELISA (ciELISA) was developed for dicamba detection, with coating antigen and monoclonal antibody diluted at1:10000and1:16000. The lineal range of the assay for detecting dicamba was0.05to5000ng/mL, with the detection limit (IC20)0.026ng/mL. The regression equation was y=4.0236lnx+34.753, R2=0.9968, and IC50value was44.230ng/mL。When dicamba add value was10and20μg/kg in the recovery tests, the recoveries of dicamba in corn meal blank samples were102.06%å'Œ115.32%, while108.41%and100.31%in wheat flour blank samples, respectively. Compared with the reported researches about ciELISA detections of dicamba residues by polyclonal antibodies, the ciELISA detection with our prepared monoclonal antibodies against dicamba was more sensitive, and had a wider test range.The polyclonal antibodies against glyphosate were produced through immunizing New Zealand white rabbits with the GLY-OVA. The titer of antibody against glyphosate was1:1x107by the test of the ELISA. The glyphosate polyclonal antibodies did not cross-react with glyphosine and N-(Phosphonomethyl) iminodiacetic An ciELISA was developed for glyphosate detection, which has a detection limit(IC10) of1.15μg/mL and a linear working range of0.78-100μg/mL with an IC50value of14.35μg/mL. The regression equation was y=15.851lnx+7.7805, R2=0.9938. When5and10μg/g glyphosate was added in the recovery tests, the recovery ratios of glyphosate were104.12%and81.37%in corn meal samples, while118.94%and109.39%in wheat flour blank samples, respectively. The experiment results revealed that the ciELISA on the basis of the prepared antibodies against glyphosate was able to effectively detect the glyphosate residues in corn meal and wheat flour samples.