ABSTRACT
Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 microg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 microg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.
Subject(s)
Animals , Female , Mice , Animal Feed/analysis , Antibodies, Fungal/analysis , Antibodies, Monoclonal/analysis , Chemistry Techniques, Analytical/methods , Enzyme-Linked Immunosorbent Assay/methods , Food Contamination/analysis , Fusarium/immunology , Imidazoles/chemistry , Magnetics/methods , Mice, Inbred BALB C , Mycotoxins/analysis , Nanoparticles/chemistry , Ovalbumin/chemistry , Trichothecenes/analysisABSTRACT
The principal agents of Fusarium head blight in the main cropping area of Argentina were investigated in heavily infected samples. The ability of the isolates to produce trichothecenes was determined by GC and HPLC. Fusarium graminearum was the predominant species and of 33 isolates, 10 produced deoxinivalenol (DON) (0.1- 29 mg kg-1), 13 produced both deoxinivalenol (1.0- 708 mg kg-1) and nivalenol (0.1- 6.2mg kg-1), 12 produced 3-acetyldeoxinivalenol (0.1- 14 mg kg-1), 13 produced 15-acetyldeoxinivalenol (0.1- 1.9 mg kg-1), 10 produced Fusarenone X (0.1- 2.4 mg kg-1) and 7 produced zearalenone (0.1- 0.6 mg kg-1). These results suggest that F. graminearum strains isolated from the wheat growing regions in Argentina belong to DON chemotype. Although some strains produced both deoxinivalenol and nivalenol, nivalenol was produced in lower levels. The natural occurrence of nivalenol in wheat affected by head-blight collected in the main production area during two years (2001-2002) was also determined. From 19 samples 13 were contaminated with deoxinivalenol in a range of 0.3 to 70 mg kg-1and 2 samples with both deoxinivalenol (7.5 and 6.7 mg kg-1) and nivalenol (0.05 and 0.1 mg kg-1), respectively. This is the first report of natural occurrence of nivalenol in wheat cultivate in Argentina.
O principal causador de giberela no trigo na Argentina e sua capacidade de produzir tricotecenos foram estudados por GC e HPLC em amostras altamente infectadas. A espécie predominante foi Fusarium graminearum, sendo que de um total de 33 isolados, 10 produziram deoxinivalenol (0,1-29 mg kg -1), 13 produziram deoxinivalenol (1,0-708 mg kg-1) e nivalenol (0,1-6,2 mg kg-1), 12 produziram 3-acetildeoxinivalenol (0,1-14 mg kg-1), 13 produziram 15-acetildeoxinivalenol (0,1-1,9 mg kg-1), 10 produziram fusarenona X (0,1- 2,4 mg kg-1) e 7 produziram zearalenona (0,1- 0,6 mg kg-1). Esses resultados sugerem que as cepas de F. graminearum isoladas de trigo cultivado na Argentina pertencem ao quimiotipo DON. Embora algumas cepas tenham produzido tanto DON quanto NIV, NIV foi produzido em quantidade inferior ao DON. A ocorrência natural de nivalenol em trigo afetado pela giberela coletado na principal área de produção durante dois anos (2001-2002) foi também determinada. De 19 amostras, 13 estavam contaminadas com deoxinivalenol na faixa de 0,3 a 70 mg kg-1 e 2amostras continham tanto deoxinivalenol (7,5 e 6,7 mg kg-1) quanto nivalenol (0,05 e 0,1 mg kg-1), respectivamente. Esse é o primeiro relato da ocorrência de nivalenol em trigo cultivado na Argentina.
Subject(s)
Fusarium/isolation & purification , Gibberella/isolation & purification , In Vitro Techniques , Mycotoxins/analysis , Toxicogenetics , Triticum , Trichothecenes/analysis , Chromatography, High Pressure Liquid , Food Samples , MethodsABSTRACT
Testes para confirmaçäo da idade de alguns tricotecenos (deoxinivalenol, diacetoxiscirpenol, toxina T-2, roridina A, verrucarina A) em cromatografia de camada delgada säo descritos, envolvendo(a) uma reaçäo de acetilaçäo antes do desenvolvimento cromatográfico e (b) o uso de 5 reveladores (ácido sulfúrico, sulfato de cério, 2,4 dinitrofenil hidrazina, p-anisaldeído, ácido cromotrópico) após o desenvolvimento cromotográfico. Nivalenol näo respondeu aos reveladores e näo pode ser incluido na presente proposta. O sistema foi testado em oito amostras de milho inoculadas e incubadas com cepas toxigênicas de Fusarium e que produziram diacetoxiscirpenol e toxina T-2, e os resultados, comparados com os obtidos com espectrometria de massa, demonstraram a utilidade e facilidade de aplicaçäo do sistema para confirmaçäo destes tricotecenos em camada delgada.