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Development of sandwich dot-ELISA for specific detection of Ochratoxin A and its application on to contaminated cereal grains originating from India.

Venkataramana M, Rashmi R, Uppalapati SR, Chandranayaka S, Balakrishna K, Radhika M, Gupta VK, Batra HV - Front Microbiol (2015)

Bottom Line: In the present study, generation and characterization of a highly specific monoclonal antibody (mAb) against Ochratoxin A (OTA) was undertaken.Developed method was more specific toward OTA and no cross reactivity was observed with the other tested mycotoxins such as deoxynivalenol, fumonisin B1, or aflatoxin B1.Therefore, in conclusion, the developed s-dot ELISA is a better alternative for routine cereal based food and feed analysis in diagnostic labs to check the presence of OTA over existing conventional culture based, tedious analytical methods.

View Article: PubMed Central - PubMed

Affiliation: Division of Toxicology and Immunology, DRDO-BU Center for Life Sciences, Bharathiar University, Coimbatore India.

ABSTRACT
In the present study, generation and characterization of a highly specific monoclonal antibody (mAb) against Ochratoxin A (OTA) was undertaken. The generated mAb was further used to develop a simple, fast, and sensitive sandwich dot-ELISA (s-dot ELISA) method for detection of OTA from contaminated food grain samples. The limit of detection (LOD) of the developed enzyme-linked immunosorbent assay (ELISA) method was determined as 5.0 ng/mL of OTA. Developed method was more specific toward OTA and no cross reactivity was observed with the other tested mycotoxins such as deoxynivalenol, fumonisin B1, or aflatoxin B1. To assess the utility and reliability of the developed method, several field samples of maize, wheat and rice (n = 195) collected from different geographical regions of southern Karnataka region of India were evaluated for the OTA occurrence. Seventy two out of 195 samples (19 maize, 38 wheat, and 15 rice) were found to be contaminated by OTA by s-dot ELISA. The assay results were further co-evaluated with conventional analytical high-performance liquid chromatography (HPLC) method. Results of the s-dot ELISA are in concordance with HPLC except for three samples that were negative for OTA presence by s-dot ELISA but found positive by HPLC. Although positive by HPLC, the amount of OTA in the three samples was found to be lesser than the accepted levels (>5 μg/kg) of OTA presence in cereals. Therefore, in conclusion, the developed s-dot ELISA is a better alternative for routine cereal based food and feed analysis in diagnostic labs to check the presence of OTA over existing conventional culture based, tedious analytical methods.

No MeSH data available.


Specificity of s-dot ELISA. 1-OTA; 2-OTB; 3-AFB1; 4-FB1; 5-DON; 6-PBS (negative control).
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Figure 4: Specificity of s-dot ELISA. 1-OTA; 2-OTB; 3-AFB1; 4-FB1; 5-DON; 6-PBS (negative control).

Mentions: When assessed for cross-reactivity against other major mycotoxins, the developed s-dot ELISA method was specific to OTA, although, it exhibited weak cross reaction with OTB (Figure 4). OTA and OTB are structurally related mycotoxins, where OTA has just an extra chlorine atom. Although it is not uncommon that anti-OTA antibodies react with OTB, the weak reaction indicates that either the capture antibody or revealing antibody might bind to OTB. When assessed using OTB-BSA conjugate in dot-ELISA format, anti-OTA rabbit polysera reacted moderately with OTB whereas mAb reacted weakly (data not shown). On the other hand, no cross-reactivity was observed with other major mycotoxins such as AFB1, FB1, and DON. Sensitivity of the assay showed as a minimum limit of 5 ng/ml of standard OTA in dot ELISA (Figure 5).


Development of sandwich dot-ELISA for specific detection of Ochratoxin A and its application on to contaminated cereal grains originating from India.

Venkataramana M, Rashmi R, Uppalapati SR, Chandranayaka S, Balakrishna K, Radhika M, Gupta VK, Batra HV - Front Microbiol (2015)

Specificity of s-dot ELISA. 1-OTA; 2-OTB; 3-AFB1; 4-FB1; 5-DON; 6-PBS (negative control).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4443250&req=5

Figure 4: Specificity of s-dot ELISA. 1-OTA; 2-OTB; 3-AFB1; 4-FB1; 5-DON; 6-PBS (negative control).
Mentions: When assessed for cross-reactivity against other major mycotoxins, the developed s-dot ELISA method was specific to OTA, although, it exhibited weak cross reaction with OTB (Figure 4). OTA and OTB are structurally related mycotoxins, where OTA has just an extra chlorine atom. Although it is not uncommon that anti-OTA antibodies react with OTB, the weak reaction indicates that either the capture antibody or revealing antibody might bind to OTB. When assessed using OTB-BSA conjugate in dot-ELISA format, anti-OTA rabbit polysera reacted moderately with OTB whereas mAb reacted weakly (data not shown). On the other hand, no cross-reactivity was observed with other major mycotoxins such as AFB1, FB1, and DON. Sensitivity of the assay showed as a minimum limit of 5 ng/ml of standard OTA in dot ELISA (Figure 5).

Bottom Line: In the present study, generation and characterization of a highly specific monoclonal antibody (mAb) against Ochratoxin A (OTA) was undertaken.Developed method was more specific toward OTA and no cross reactivity was observed with the other tested mycotoxins such as deoxynivalenol, fumonisin B1, or aflatoxin B1.Therefore, in conclusion, the developed s-dot ELISA is a better alternative for routine cereal based food and feed analysis in diagnostic labs to check the presence of OTA over existing conventional culture based, tedious analytical methods.

View Article: PubMed Central - PubMed

Affiliation: Division of Toxicology and Immunology, DRDO-BU Center for Life Sciences, Bharathiar University, Coimbatore India.

ABSTRACT
In the present study, generation and characterization of a highly specific monoclonal antibody (mAb) against Ochratoxin A (OTA) was undertaken. The generated mAb was further used to develop a simple, fast, and sensitive sandwich dot-ELISA (s-dot ELISA) method for detection of OTA from contaminated food grain samples. The limit of detection (LOD) of the developed enzyme-linked immunosorbent assay (ELISA) method was determined as 5.0 ng/mL of OTA. Developed method was more specific toward OTA and no cross reactivity was observed with the other tested mycotoxins such as deoxynivalenol, fumonisin B1, or aflatoxin B1. To assess the utility and reliability of the developed method, several field samples of maize, wheat and rice (n = 195) collected from different geographical regions of southern Karnataka region of India were evaluated for the OTA occurrence. Seventy two out of 195 samples (19 maize, 38 wheat, and 15 rice) were found to be contaminated by OTA by s-dot ELISA. The assay results were further co-evaluated with conventional analytical high-performance liquid chromatography (HPLC) method. Results of the s-dot ELISA are in concordance with HPLC except for three samples that were negative for OTA presence by s-dot ELISA but found positive by HPLC. Although positive by HPLC, the amount of OTA in the three samples was found to be lesser than the accepted levels (>5 μg/kg) of OTA presence in cereals. Therefore, in conclusion, the developed s-dot ELISA is a better alternative for routine cereal based food and feed analysis in diagnostic labs to check the presence of OTA over existing conventional culture based, tedious analytical methods.

No MeSH data available.