Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk

Anfossi, Laura; Baggiani, Claudio; Giovannoli, Cristina; Biagioli, Francesca; D’Arco, Gilda; Giraudi, Gianfranco

doi:10.1016/j.aca.2013.02.020

Cited by 83 publications

(41 citation statements)

References 36 publications

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“…: the molar conjugation rate, cr, between OTA and BSA of the OTA-BSA conjugate). All those factors were demonstrated to play some role on determining the sensitivity of the assay in our previous observations [30] and according to several other authors [31][32][33][34].…”

Section: Optimization Of the Silver Enhanced Lateral Flow Immunoassaysupporting

confidence: 63%

“…Unlike expected, the hapten density that was accounted for the strongest influence on assay sensitivity for a gold-based LFIA system previously optimized by our group [30], played a minor role compared to the effects due to lowering the anti-OTA antibody or the sprayed conjugate (Fig 3 and Table 1). However, the lower the OTA quantity in the Test line (because of a lower OTA-BSA amount was sprayed or because less OTA was present in the OTA-BSA conjugate) the highest the sensitivity.…”

Section: Optimization Of the Silver Enhanced Lateral Flow Immunoassaymentioning

confidence: 66%

“…The saturating condition was judged as the lower antibody amount that prevented aggregation [35]. The amount of Ab rab able to completely cover GNP surface was also theoretically calculated as the ratio between the total surface area of GNPs and the size of an antibody molecule [30]. The experimental and the calculated values were in agreement (exp = 4.5 μg antibody per ml of GNP preparation, calc = 6.0 μg antibody per ml of GNP preparation).…”

Section: Optimization Of the Silver Enhanced Lateral Flow Immunoassaymentioning

confidence: 69%

“…High sensitivity in indirect lateral flow immunoassays for haptens is promoted from having the opportunity of tuning some key-factors, such as the amount of specific antibodies and of immobilized antigens in the Test line [30][31][32][33][34]. Specifically, the lower the amount of one or both of those components, the higher the sensitivity reached.…”

Section: Optimization Of the Silver Enhanced Lateral Flow Immunoassaymentioning

confidence: 99%

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Increased sensitivity of lateral flow immunoassay for ochratoxin A through silver enhancement

et al. 2013

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Silver nucleation on gold has been exploited for signal amplification and has found application in several qualitative and quantitative bio-sensing techniques, thanks to the simplicity of the method and the high sensitivity achieved. Very recently, this technique has been tentatively applied to improve performance of gold-based immunoassays. In this work, the exploitation of the signal amplification due to silver deposition on gold nanoparticles has been first applied to a competitive lateral flow immunoassay (LFIA). The signal enhancement due to silver allowed us to strongly reduce the amount of the competitor and of specific antibodies employed to build a LF device for measuring ochratoxin A (OTA), thus determining the attainment of the high sensitive assessment of OTA contamination, with a sensitive gain of more than 10-folds compared to the gold-based LFIA that used the same immunoreagents and to all previously reported LFIA for measuring OTA. In addition, a less sensitive "quantitative"-LFIA could be established, by suitably tuning competitor and antibody amounts, which was characterized by reproducible and accurate OTA determinations (RSD% 6-12%, recovery% 82-117%). The quantitative system allowed a reliable OTA quantification in wines and grape musts at the μg/l level requested by the European legislation, as demonstrated by agreeing results obtained through the "quantitative" silver enhanced-LFIA and a reference HPLC-FLD on 30 samples.

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Section: Optimization Of the Silver Enhanced Lateral Flow Immunoassaysupporting

confidence: 63%

Section: Optimization Of the Silver Enhanced Lateral Flow Immunoassaymentioning

confidence: 66%

Section: Optimization Of the Silver Enhanced Lateral Flow Immunoassaymentioning

confidence: 69%

Section: Optimization Of the Silver Enhanced Lateral Flow Immunoassaymentioning

confidence: 99%

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Increased sensitivity of lateral flow immunoassay for ochratoxin A through silver enhancement

et al. 2013

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“…[3][4][5] Once present in dairy products, AFM 1 poses a hazard to humans (especially infants) who consume them. …”

Section: Introductionmentioning

confidence: 99%

A qPCR aptasensor for sensitive detection of aflatoxin M1

et al. 2016

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. In addition, the aptasensor developed here exhibits high selectivity for AFM 1 over other mycotoxins and small effects from cross-reaction with structural analogs. The method proposed here has been successfully applied to quantitative determination of AFM 1 in infant rice cereal and infant milk powder samples. Results demonstrated that the current approach is potentially useful for food safety analysis, and it could be extended to a large number of targets. IntroductionAflatoxin M 1 (AFM 1 ), one of the most toxic contaminants in dairy products, is a metabolite produced by dairy cows as a result of being fed with feeds contaminated with Aflatoxin B 1 (AFB 1 ). [3][4][5] Once present in dairy products, AFM 1 poses a hazard to humans (especially infants) who consume them. Results and Discussion Optimization of the amplification of complementary ssDNAThe complementary ssDNA is applied as the subsequent PCR template. The Optimization of streptavidin and the biotinylated aptamerOther factors would affect the performance of this method including the concentrations of streptavidin, the biotinylated aptamer and the complementary ssDNA and these all should be optimized. Under a fixed concentration of complementary ssDNA at 10 nM, the concentrations of streptavidin and the 6 biotinylated aptamer were analyzed for the PCR amplification signal change (Fig. S3).The adsorptive power of streptavidin to PCR tubes and the binding ability of streptavidin-coated tubes to biotinylated aptamer was primarily detected using different concentrations of streptavidin (Fig. S3). This also shows that there was a clear difference of Ct values between the control group and streptavidin-coated tubes, which showed the intense adsorptive ability between biotinylated aptamer and streptavidin-coated tubes. In analysis of the Ct values at different concentrations of streptavidin, the Ct value reached the lowest level when the concentration of streptavidin was 2.5 ng mL -1 . This graph also shows that the Ct values decreased with the increase of the amount of aptamer when aptamer levels were below 10.0 nM andalso that the Ct values increased with the amount of aptamer above 10.0 nM mainly because of steric hindrance. Thus, 2.5 ng mL -1 of streptavidin and 10 nM of aptamer were the optimal conditions for RT-qPCR amplification. AFM 1 determinationUnder optimal conditions, the amplification curves and calibration curve of this aptasensor for different levels of AFM 1 with AFM 1 DNA1 were determined by RT-qPCR (Fig. 1). As is shown in Fig. 1(A), the cycle number increased with an increase in AFM 1 . More complementary ssDNA would be released when more AFM 1 is present in the reaction system, which leads to a decrease in the amount of the PCR template and an increase in cycle number. Meanwhile, a good linear relationshipbetween Ct values and AFM 1 levels in the range of 1×10 -4 to 1 µg L -1 was obtained as (Table 1). Specificity analysisThe specificity of the aptasensor plays an important role in the development and practicality of this method. In ...

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State of the art immunoassay developments and application to food chemical hazards

Wang

2014

Food Chemical Hazard Detection

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Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk

Cited by 83 publications

References 36 publications

Increased sensitivity of lateral flow immunoassay for ochratoxin A through silver enhancement

Increased sensitivity of lateral flow immunoassay for ochratoxin A through silver enhancement

A qPCR aptasensor for sensitive detection of aflatoxin M1

State of the art immunoassay developments and application to food chemical hazards

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