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

M Venkataramana,K Balakrishna,M Radhika,H V Batra,R Rashmi,Siva R Uppalapati,S Chandranayaka,Vijai K Gupta

doi:10.3389/fmicb.2015.00511

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.

Highlights

The incidence of microfungi and their secondary metabolites is a worldwide phenomenon affecting all major cereal crops
Analysis of OTA-Protein Conjugates Ochratoxin A-bovine serum albumin (BSA) conjugate was prepared for the immunization animals and OTA-OVA conjugate was made for the indirect enzyme-linked immunosorbent assay (ELISA) as the coating antigen both the conjugations were accomplished by a carbodiimide method
The strategy of utilizing distinctive conjugate proteins for the immunization of animals and the coating of ELISA plates was useful for successful screening for OTA specific monoclonal antibody (mAb)

Summary

Introduction

The incidence of microfungi and their secondary metabolites (mycotoxins) is a worldwide phenomenon affecting all major cereal crops. Though the presence and incidence of mycotoxins in cereals have been demonstrated since the early origins of organized crop cultivation and have. Dot-ELISA for detection of OTA from foods impacted mankind, their effects have largely been ignored until the recent past 40 years. The potency and sturdiness of mycotoxins and the significant losses annually to the health, trade, economy and the marketing of foods and feeds have attracted worldwide attention toward undertaking active research on mycotoxin detection and analysis (Priyanka et al, 2014; Ramana et al, 2014). Nephrotoxic, and teratogenic effects were well-documented (Mor et al, 2014; Mantle et al, 2015) and it is involved in the Balkan endemic nephropathy (BEN) and in the Chronic Interstitial Nephropathy (Bayman and Baker, 2006)

Methods

Results

Conclusion