Two Novel Enzyme-Linked Immunosorbent Assays For The Detection Of Ochratoxin A And Staphylococcal Enterotoxin A

Ochratoxin A（OTA）and Staphylococcal Enterotoxin A（SEA）are the most common biotoxins.Enzyme-Linked Immunosorbent Assays（ELISA）has been widely used for the screening detection of OTA and SEA in various food samples because of its unique advantages of simple operation,low cost,good specificity,and high throughput.However,the conventional ELISA based on horseradish peroxidase catalyzed the tetramethyl benzidine to generate the colorimetric substance commonly suffers from the relatively low sensitivity because of the low molar extinction coefficient of colorimetric substance,and the single-color colorimetric signal of conventional ELISA is also unsuitable for naked-eye interpretation.In addition,the amounts of OTA and organic reagents are required during the preparation of competing antigen,which poses a major threat to the environment and related practitioners.pELISA based on the AuNPs aggregation and growth as the signal output has a higher sensitivity and is also suitable for naked eye interpretation because the AuNPs can show different color based on their different morphology and size,whereas its stability of signal transducer is not good.It is demonstrated that the usage of ssDNA mediated AuNPs growth as the signal report can greatly improve the stability of signal transducer of pELISA.Up to date,the DNA configuration on the morphology of growth AuNPs and the sensitivity of pELISA has not yet reported.In this chapter,six of DNA sequences with different configuration have been designed,and assessed on the sensitivity of pELISA,and the results showed that the the ssDNA with hairpin structure mediated AuNPs growth can achieve the best sensitivity of pELISA.On this basis,a difunctional M13 phage(Biotin-M13_OTA)expressed with the mimotope of OTA at the p III protein,and chemically modified with biotin molecules at the p VIII protein was used as the competing antigen to establish a novel pELISA for the sensitive and visual detection of OTA.In this assay,the ssDNA can be cleaved into fragments by the·OH generated from oxidation of H₂O₂by Fenton reagents,and the biotin-M13_OTAis also used as the container of catalaze to amplify the H₂O₂consumption,and thereby adjusting the morphology of growth AuNPs.Under the optimal conditions,the visual detection of limit（v LOD）of proposed pELISA was achieved at 20 pg/mL,and the dynamic linearity for OTA quantitative determination was in a range of 1 pg/mL to 200 pg/mL,which could be expressed with the following equation of y=17.063 ln（x）+118.56（R²=0.9907）.The LOD was calculated at 1.58 pg/mL,which was 97-fold lower than that of conventional ELISA.The inter-or intra-assays showed that the average recoveries for OTA spiked corn samples ranged from 85%to 110%,with the coefficient of variation less than 15%,indicating a good accuracy and precision of the proposed pELISA for OTA quantitative detection in real corn samples.Secondly,a fluorescent ELISA（FELISA）was established for the ultrasensitive detection of SEA in milk by using the mercaptopropionic acid capped CdTe quantum dots（MPA-QDs）as signal transducers,where the fluorescent signal of MPA-QDs is extremely sensitive to H₂O₂ and p H.In this assay,the glucose oxidase was used to oxidize the glucose to generate the H₂O₂ and gluconic acid,and thereby quenching the fluorescent signal of MPA-QDs.Under the optimal conditions,the proposed FELISA showed a very high sensitivity for the SEA detection in skim（whole）liquid milks with the LOD values at 6.65 pg/mL and 3.4 pg/mL,respectively,and the LOD value for the SEA detection in milk powder is 0.292 ng/g,which is 678-fold lower than that of conventional ELISA.The inter-or intra-assays of this proposed FELISA showed the average recoveries for the SEA spiked milk were in a range of 92.65%～109.73%and 86.35%～101.75%,respectively,and the CV values for all assays were less than 15%,indicating that the proposed pELISA has a good accuracy and precision for SEA quantitative detection in real milk samples.In addition,the proposed method for blindly detecting milk samples showed a highly consistent with those of the traditional ELISA.However,the proposed method shows a higher sensitivity,and can be used for the trace SEA detection in the solution,and can be also used for the SEA detection in complex food samples.