Development of a Highly Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detection of Sudan I in Food Samples

Abstract

A highly selective and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) for Sudan I was developed. Two hapten derivatives with different lengths of carboxylic spacer at the azo-bound para-position were synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugates were used as immunogens, while the hapten-ovalbumin (OA) conjugates were applied as coating antigens. The antisera which were obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. At optimal experimental conditions it was found that IC50 and LOD values of seven pairs based on four antisera and two coating antigens were in the range of 0.3-2 ng/mL and 0.02-0.1 ng/mL, respectively. The most sensitive ELISA could be established with Sudan I-propionic acid-OA coating antigen and the antiserum which was obtained with the corresponding immunogen. The cross-reactivity values of the four antisera with Sudan II, III, and IV was estimated with 0.1-14.3%. No cross-reactivity was found with six edible colorants Sunset yellow, Amarant, Kermes, Indigotin, Bright blue and Lemon yellow, indicating high specificity for Sudan I. Six food samples were fortified with Sudan I and extracted by simple sample preparation. The methanolic extracts after dilution with methanol:water (5:95, v/v) were analyzed by the developed ELISA. Assay precision and accuracy was estimated by determination of three replicates. Acceptable recovery rates of 92.5-114% and intra-assay coefficients of variation of 5.9-24.8% were obtained. The data were validated by conventional HPLC method. As revealed, both methods were highly correlated (r = 0.9851, n = 7), demonstrating the applicability of the developed ELISA for Sudan I analysis in food samples.

Ultrasensitive and Specific Detection of Salbutamol in Swine Feed, Meat, and Urine Samples by a Competitive Immunochromatographic Test Integrated with Surface-Enhanced Raman Scattering

“…The carboxylic acid in SAL derivative was activated by DCC/NHS ester method and linked to BSA and OA to form SAL-BSA and SAL-OVA SAL conjugates (Han et al 2007). The prepared SAL-OVA conjugate was used as a coating antigen, while SAL-BSA conjugate was used as an immunogen.…”

“…The prepared SAL-OVA conjugate was used as a coating antigen, while SAL-BSA conjugate was used as an immunogen. The polyclonal Abs against SAL were produced by immunizing rabbits with immunogen as the similar processes described in our previous literature (Han et al 2007). Briefly, two adult New Zealand rabbits were immunized with SAL-BSA.…”

Direct detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in food samples without derivatisation step by a sensitive and specific monoclonal antibody based ELISA

“…Via the carboxylic acid group, the AMOZ derivatives were conjugated to carrier proteins by the DCC/NHS activation method as described in the literature (Franek et al, 2001;Han et al, 2007). Briefly, equimolar amounts (0.15 mmol) of CPAMOZ, NHS, and DCC were dissolved in 300 lL of DMF and the mixture was incubated overnight at 25°C.…”

“…The most used immunoassays are enzyme-linked immunosorbent assays (ELISAs) which exhibit significant advantages over traditional chromatographic methods, such as high sensitivity and specificity, simple sample preparation, high throughput, and therefore, low cost per sample. Many ELISAs have been widely used for the determination of low molecular contaminants in the food and environmental analysis (Deng et al, 2003;Franek, Deng, Kolar, & Socha, 2001;Han et al, 2007;Wang et al, 2007). In the last decade, there have been a few ELISAs for the determination of nitrofurans and their derivatized metabolites (Chang, Peng, Wu, Wang, & Yuan, 2008;Cheng et al, 2009;Cooper, Caddell, Elliott, & Kennedy, 2004;Cooper, Samsonova, Plumpton, Elliott, & Kennedy, 2007;Gao, Chen, Cheng, Lei, & Zeng, 2007;Kotkova et al, 2005;Li, Liu, & Wang, 2009;Li, Liu, Zhang, Li, & Wang, 2010;Liu et al, 2007), and one ELISA using monoclonal antibody for the detection of NPAMOZ in spiked shrimp samples was reported (Umaporn et al, 2009).…”

Electrochemical Detection of Sudan I Using a Multi-Walled Carbon Nanotube/Chitosan Composite Modified Glassy Carbon Electrode

“…To date, many methods have been investigated for the determination of Sudan I, such as high-performance liquid chromatography (HPLC) [3][4][5][6][7][8][9], gas chromatography (GC) [10], capillary electrophoresis [11], immunoanalysis [12,13], chemiluminescence flow injection analysis [14], and plasmon resonance light scattering (PRLS) [15]. Most of these techniques exhibit high sensitivity and excellent selectivity, but some are also time-consuming, expensive, and unsuitable for in situ assays.…”