On the other hand, TLC although cheaper and easy to perform, requires extensive sample preparation and is not a sensitive method. OTA by s-dot ELISA. The assay results were further co-evaluated with conventional analytical high-performance Budesonide liquid chromatography (HPLC) method. Results of the s-dot ELISA are in concordance with HPLC except for three samples that were unfavorable 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 g/kg) of OTA presence in cereals. Therefore, in conclusion, the developed s-dot ELISA is usually a better option 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. ((= 25)ITCC 2005A1ITCC?, IndiaITCC 1456A2ITCC, IndiaITCC 3167A3ITCC, IndiaITCC 2454A4ITCC, IndiaMTCC 1810A5MTCC, IndiaDFR_AO1-DFR_AO10A6CA15DFRL, IndiaDFR_AC1-DFR_AC10A16CA25DFRL, IndiaOTA unfavorable Aspergilli (= 15)ATCC 46283A26ATCC?, USANCIM 152A27NCIM?, IndiaNCIM 645A28NCIM, IndiaNCIM 650A29NCIM, IndiaMTCC 2798A30MTCC, IndiaMTCC 2797A31MTCC, IndiaDFR_AF1-DFR_AF9A32CA40DFRL, IndiaOTA positive Penicillia (= 32)ITCC 2156P1ITCC, IndiaITCC 2986P2ITCC, IndiaMTCC 1758P3MTCC, IndiaMTCC 2007P4MTCC, IndiaDFR_PVer1-DFR_PVer15P5CP19DFRL, IndiaDFR_PVir1-DFR_PVir13P20CP32DFRL, IndiaOTA unfavorable Penicillia (= 20)DFR_PCit1-DFR_PCit10P33CP42DFRL, IndiaDFR_PChr1-DFR_PChr5P43CP47DFRL, IndiaDFR_PH1-DFR_PH5P48CP52DFRL, IndiaOther OTA unfavorable fungi (= 8)MTCC 2089F1MTCC, IndiaMTCC 3693F2MTCC, IndiaMTCC 2081F3MTCC, IndiaMTCC 6479F4MTCC, IndiaMTCC 156F5MTCC, IndiaMTCC 286F6MTCC, IndiaITCC 149F7ITCC, IndiaITCC 3359F8ITCC, India Open in a separate windows ?Indian Type Culture Collection (ITCC); Microbial Type Culture Collection (MTCC); Defence Food Research Laboratory; ?American Type Culture Collection; ?National Collection of Industrial Microorganisms. Conjugation of OTA to BSA Ochratoxin A was coupled to BSA Rabbit polyclonal to DDX6 in the presence of EDC and NHS as described previously (Yu et al., 2005). Five milligrams of OTA was dissolved in 0.5 mL dimethyl sulfoxide (DMSO) that contained 6 mg NHS and 8 Budesonide mg EDC. The reaction was maintained at room heat for 2 h in the dark and then at 4C overnight. The solution was added to 10 mg imject BSA answer (Life Technologies, Bengaluru) drop-wise and then dissolved in 2 mL 0.1 M carbonate buffer (pH Budesonide 9.6), and maintained at room heat for 2 h. Following the reaction, the mixture was dialyzed against 10 mM PBS (pH 7.4), with the buffer replenished six occasions Budesonide over 72 h. Conjugation of OTA to OVA Ochratoxin-OVA conjugate was prepared by coupling OTA to OVA in the presence of a water-soluble carbodiimide (Chu et al., 1976) and used as solid-phase antigen for the indirect ELISA. In a typical reaction, Budesonide 0.5 mg of OTA in 0.2 mL of conjugation buffer was mixed with 2.5 mg of imject OVA (Life Technologies, Bengaluru) followed by 1 mg of EDC was added to the mixture with constant stirring. After the coupling reaction was carried out at 25C for 2 h, the mixture was dialyzed against PBS for 72 h and then lyophilized for storage. TNBS Assay Different ratios of proteinChapten conjugates were characterized by determining the available groups of surface lysine present in carrier proteins before and after conjugation. This was accomplished by using 2,4,6-trinitrobenzene-1-sulfonic acid (TNBS) reagent. The amount of amino groups present in the carrier protein before and after coupling with carboxylated hapten was directly quantitated with a UV/vis spectrophotometer at 335 nm (Sashidhar et al., 1994). Different conjugates were prepared at a concentration of 1 1 mg/mL and were reacted with 0.1% TNBS answer under alkaline conditions to determine the percentage of NH2 groups used during conjugation in different conjugates. An amount of 200 l of conjugate answer was taken and mixed with 200 l of 4% NaHCO3 answer. An amount of 200.