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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.


Ochratoxin A estimation from natural cereal samples.∗ represents W4 (wheat), M44 and M63 (Maize) samples with OTA contamination within range of 3.3–4.6 μg Kg-1 and were found positive for HPLC but showed negative by s-dot ELISA.
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Figure 7: Ochratoxin A estimation from natural cereal samples.∗ represents W4 (wheat), M44 and M63 (Maize) samples with OTA contamination within range of 3.3–4.6 μg Kg-1 and were found positive for HPLC but showed negative by s-dot ELISA.

Mentions: A total of 195 cereal [wheat (55), maize (80), and rice (60)], samples were analyzed for occurrence of OTA by the s-dot ELISA method developed in this study as well as HPLC method. Twenty out of 55 wheat samples contained OTA in a range of 4.604 – 12.1 μg Kg-1, with a mean level of 8.61 μg Kg-1. Forty out of 80 maize samples were found to be OTA positive, ranging from 3.3 to 27.021 μg Kg-1 with a mean level of 13.67 μg Kg-1. Fifteen out of 60 rice samples were positive for OTA, ranging from 4.9 to 9.67 μg Kg-1, with a mean level of 6.65 μg Kg-1 (Figure 7). W4 (wheat), M44 and M63 (Maize) samples (represented as ∗ in Figure 7) were contaminated with OTA within range of 3.3–4.6 μg Kg-1 and were found positive for HPLC. But these samples were found negative by s-dot ELISA probably due to the limited yield of the OTA extraction protocol.


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)

Ochratoxin A estimation from natural cereal samples.∗ represents W4 (wheat), M44 and M63 (Maize) samples with OTA contamination within range of 3.3–4.6 μg Kg-1 and were found positive for HPLC but showed negative by s-dot ELISA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Ochratoxin A estimation from natural cereal samples.∗ represents W4 (wheat), M44 and M63 (Maize) samples with OTA contamination within range of 3.3–4.6 μg Kg-1 and were found positive for HPLC but showed negative by s-dot ELISA.
Mentions: A total of 195 cereal [wheat (55), maize (80), and rice (60)], samples were analyzed for occurrence of OTA by the s-dot ELISA method developed in this study as well as HPLC method. Twenty out of 55 wheat samples contained OTA in a range of 4.604 – 12.1 μg Kg-1, with a mean level of 8.61 μg Kg-1. Forty out of 80 maize samples were found to be OTA positive, ranging from 3.3 to 27.021 μg Kg-1 with a mean level of 13.67 μg Kg-1. Fifteen out of 60 rice samples were positive for OTA, ranging from 4.9 to 9.67 μg Kg-1, with a mean level of 6.65 μg Kg-1 (Figure 7). W4 (wheat), M44 and M63 (Maize) samples (represented as ∗ in Figure 7) were contaminated with OTA within range of 3.3–4.6 μg Kg-1 and were found positive for HPLC. But these samples were found negative by s-dot ELISA probably due to the limited yield of the OTA extraction protocol.

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.