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Fluorimetric immunoassay for multianalysis of aflatoxins.

Kanungo L, Bhand S - J Anal Methods Chem (2013)

Bottom Line: The linear range of the immunoassay was found to be 6.25-50 pg/mL.Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1.The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling.

View Article: PubMed Central - PubMed

Affiliation: Biosensor Lab., Department of Chemistry, BITS, Pilani-K. K. Birla Goa Campus, Goa 403726, India.

ABSTRACT
A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25-50 pg/mL. AFM1 as low as 1 pg/mL was detected by this method with assay volume 40  μ L. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling.

No MeSH data available.


Optimization of incubation time for FITC conjugated 2°Ab. 1°Ab dilution, 1 : 16000; FITC conjugated 2°Ab dilution, 1 : 64000; excitation time, 0.1 s.
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fig4: Optimization of incubation time for FITC conjugated 2°Ab. 1°Ab dilution, 1 : 16000; FITC conjugated 2°Ab dilution, 1 : 64000; excitation time, 0.1 s.

Mentions: In order to achieve the optimal binding of analyte, the incubation time varied from 10 min to 180 min. Figure 4 shows the optimization result of incubation time of AFM1 and FITC labeled 2°Ab complex with capture 1°mAb. It is apparent from the graph that around 100–120 min, the stable signal was obtained. So, the incubation time was chosen at 120 min in further experimental analysis.


Fluorimetric immunoassay for multianalysis of aflatoxins.

Kanungo L, Bhand S - J Anal Methods Chem (2013)

Optimization of incubation time for FITC conjugated 2°Ab. 1°Ab dilution, 1 : 16000; FITC conjugated 2°Ab dilution, 1 : 64000; excitation time, 0.1 s.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3755385&req=5

fig4: Optimization of incubation time for FITC conjugated 2°Ab. 1°Ab dilution, 1 : 16000; FITC conjugated 2°Ab dilution, 1 : 64000; excitation time, 0.1 s.
Mentions: In order to achieve the optimal binding of analyte, the incubation time varied from 10 min to 180 min. Figure 4 shows the optimization result of incubation time of AFM1 and FITC labeled 2°Ab complex with capture 1°mAb. It is apparent from the graph that around 100–120 min, the stable signal was obtained. So, the incubation time was chosen at 120 min in further experimental analysis.

Bottom Line: The linear range of the immunoassay was found to be 6.25-50 pg/mL.Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1.The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling.

View Article: PubMed Central - PubMed

Affiliation: Biosensor Lab., Department of Chemistry, BITS, Pilani-K. K. Birla Goa Campus, Goa 403726, India.

ABSTRACT
A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25-50 pg/mL. AFM1 as low as 1 pg/mL was detected by this method with assay volume 40  μ L. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling.

No MeSH data available.