| Rice false smut is a disease caused by the pathogenic fungus Villosiclava virens Tanaka and Tanaka by infecting rice spikes,which not only results in severe losses of yield and quality of rice,but also generates mycotoxins that are poisonous to humans and animals.Three kinds of mycotoxins(i.e.ustiloxins,ustilaginoidins and sorbicillinoids)have been isolated and identified from false smut pathogen.Immunoassays have become increasingly popular as fast,sensitive,economical and efficient screening methods for mycotoxin analysis,which are wildly used to guarantee food and feed safety.Studies on the preparation of monoclonal antibodies and immunoassays have not been reported yet.In this study,ustiloxins and modified precursor compounds of ustilaginoidins are coupled with carrier proteins to synthesize immunogens,respectively.Specific monoclonal antibodies(mAbs)separately against ustiloxins and ustilaginoidins were screened and prepared from the hybridoma cell lines.The variable region genes of three mAbs against ustiloxins were cloned.The emzyme-linked immunosorbent assay(ELISA)and colloidal-gold lateral flow immunoassay strips were established based on the mAbs.Immunoaffinity chromatography was developed and was use to purify ustiloxin.The distribution of ustiloxins was visualized by immunohistochemical localization.The main results are as follows.(1)Ustiloxin A(UA)and ustiloxin B(UB)were separately coupled with carrier proteins to synthesize complete antigens via glutaraldehyde method.After immunizing mice and screening from positive hybridoma cells,a mAb 2D3G5 which was specific to UA,a mAb 1B5A10 against UB,and a broad specific mAb 4C4F11 with equal reactivity against UA and UB were obtained,respectively.(2)From the hybridoma cell lines(2D3G5,1B5A10,and 4C4F11)which secreted different specific mAbs against ustiloxins,the variable region genes were cloned and sequenced,which provided the basis to identify the molecular recognization mechanism of three different specific mAbs.In addition,it also provided the genetic materials for the construction of genetic engineering antibody to ustiloxins.(3)An indirect competitive emzyme-linked immunosorbent assay(icELISA)method for the specific detection of UA was established based on the mAb 2D3G5 with IC50 of 13.8 ng/mL and working range of 2.8 to 72 ng/mL.The recoveries of rice false smut balls and rice were at 92-117%and 92-107%,respectively.The content of UA in rice samples tested by established icELISA agreed well with HPLC analysis,indicating that the established method could be used for rapid and reliable screening UA in rice samples.Based on mAb 1B5A10,an icELISA method for the specific detection of UB was established with IC50 value of 18.0 ng/mL and working range of 2.5-107.4 ng/mL.The UB content in rice samples detected by icELISA were generally higher than that by HPLC analysis,which might be related to the low cross-reactivity against UA.A direct competitive emzyme-linked immunosorbent assay(dcELISA)which could test the total content of UA and UB was developed using mAb 4C4F11.The recoveries were between 80%and 120%in rice samples spiked with UA or UB.The relative standard deviation(RSD)values of inter-well and inter-plate difference were all less than 15%.The test results by dcELISA agreed well with HPLC results.It can be used to detect the total amount of main ustiloxins in rice samples.(4)Based on the mAbs 2D3G5,1B5A10,and 4C4F11,colloidal-gold lateral flow immunoassay strips for specific detection of UA,specific detection of UB,and simultaneous detection of UA and UB were separately established.The indicator ranges were all at 50-100 ng/mL.The semi-quantitative results of the established strips were basically consist with the results by instrumental analysis and ELISA methods,which indicated that the established strips can be used for on-site and high-throughput screening of ustiloxins contamination in rice samples.(5)Two monomeric naphtha-y-pyrones namely hemiustilaginoidins F and D.which were considered as the precursors of ustilaginoidins(bis-naphtha-?-pyrones),were selected as the starting compounds.The hptens were successfully synthesized by structural modification of hemiustilaginoidins F and D.Two mAbs 4A12C6 and 5F4F6,which recognized the main ustilaginoidins,were prepared.The mAb 4A12C6 mainly recognized ustilaginoidin A and other ustilaginoidins with monomethyl substitution at 2-C and 3-C(or 2'-C and 3'-C)position.Differently,mAb 5F4F6 mainly recognized ustilaginoidin D and other ustilaginoidins with bis-methyl substitution at 2-C and 3-C(or 2'-C and 3'-C)position.Two icELISA methods were established based on the mAbs 4A12C6 and 5F4F6,respectively,for the detection of the major ustilaginoidins.The IC50 value and working range of the icELISA based on mAb 4A12C6 was 0.76 ng/mL and 0.2-2.8 ng/mL,respectively.Based on mAb 5F4F6,the IC50 value and working range of the icELISA was 25 ng/mL and 4.2-166 ng/mL,respectively.In the preparation of antibody to ustilaginoidins,this study innovatively used the monomers of the dimeric compounds for hapten derivatization and synthesis,which made it possible to prepare antibodies against mycotoxins with the complex structures.(6)The mAb 2D3G5 was coupled with CNBr-activated sepharose 4B to prepare an immunoaffinity chromatography column for UA purification.Different eluted solutions were performed on the immunoaffinity chromatography column.The results showed that the eluted solution with increasing ionic strength had the highest efficiency for UA purification with 70%adsorption and 45%elution efficiency.(7)Rice false smut balls and different strains of V.virens were used for fluorescence immunohistochemical localization based on the mAb 4C4F11.A brighter fluorescent band was observed in the mycelial layer of the rice false smut ball,indicating that the content of ustiloxins was higher.Especially in the part of the mycelium near the endosperm,an obvious fluorescent line was observed,predicting where ustiloxins were accumulated.Two different strains of V.virens were fermented in potato sucrose agar(PSA)and potato sucrose broth(PSB),respectively,and observed through fluorescence immunohistochemical localization.It was found that the fluorescence intensity of ustiloxins in strain LN-2010-1-1 was significantly brighter than that in strain P1.The fluorescence intensity of ustiloxins in LN-2010-1-1 hyphae fermented in PSA was much brighter than that fermented in PSB,which might be related to fermentation time and ustiloxins secretion.The research prepared the mAbs against ustiloxins and ustilaginoidins,cloned the variable region genes of three mAbs against ustiloxins,which provided the basis for the study of the molecular recognition mechanism of different specificity,developed a sensitive,rapid,and convenient method for the detection of ustiloxins and ustilaginoidins,provided a rapid and highly efficient immunoaffinity chromatography for the isolation and purification of UA,preliminary explored the possible biosynthesis and distribution of ustiloxins.The study results provide the basis for food and feed safety guaranteeing and the research on the production,secretion and distribution of ustiloxins and ustilaginoidins. |
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