A Reliable Enzyme Linked Immunosorbent Assay for the Determination of Bovine and Porcine Gelatin in Processed Foods

Abstract

Since gelatin-containing foods pose a risk for eliciting allergic reactions in sensitized individuals, a novel sandwich enzyme linked-immunosorbent assay (ELISA) for the detection and quantification of bovine and porcine gelatin in processed foods was developed. Rabbits and goats were immunized with bovine gelatin, and three antisera (pAb1 and pAb2 from rabbits, and pAb3 from goats) were obtained. We established a sandwich ELISA method based on a combination of these antibodies. In this study, two sandwich ELISA methods, rabbit pAb2-pAb1 and goat pAb3-pAb3, were evaluated for sensitivity, specificity, cross-reactivity, and applicability. Both ELISA methods were highly specific for bovine and porcine gelatin but had little reactivity with fish gelatin. The detection and quantification limits for porcine gelatin were found to be 0.78 ng/mL and 1.56 ng/mL, respectively. The established sandwich ELISA methods produced no false-positives, except for heated meat products or false negatives when various commercial foods were analyzed for their gelatin content. The rabbit pAb2-pAb1 ELISA cross-reacted with boiled squid, while the goat pAb3-pAb3 ELISA did not. Thus, the proposed goat pAb3-pAb3 ELISA method is a reliable tool for the detection of gelatin contaminants present in processed foods.

An evaluation of Fourier transforms infrared spectroscopy method for the classification and discrimination of bovine, porcine and fish gelatins

“…In these studies, discrimination of both gelatins is accomplished by using analytical methods including the applications of spectroscopic, chemical, liquid chromatography and immunochemical techniques (Nhari et al, 2012). Doi, Watanabe, Shibata, and Tanabe (2009) developed a sandwich ELISA method that can be used for detecting bovine and porcine gelatin in processed foods for the patients who have gelatin allergy (Doi et al, 2009). Nemati, Oveisi, Abdollahi, andSabzevari (2004) have differentiated raw bovine and porcine gelatins by principal component analysis (PCA) based on amino acid analysis obtained from RP-HPLC analysis (Nemati et al, 2004).…”

Real-Time Polymerase Chain Reaction for Halal Authentication of Gelatin in Soft Candy

“…Such methods are infrared spectroscopy coupled with chemometrics of Principal Component Analysis (PCA) for differentiation of porcine and bovine gelatins (Hashim et al, 2010) and those with fish gelatine (Cebi et al, 2016), high performance liquid chromatography coupled with fluorescence detector and chemometrics of PCA (Nemati et al, 2004;Raraswati et al, 2013) and with some types of mass-spectrometer detectors (Zhang et al, 2009;Yilmaz et al, 2013), electrophoretic analysis (Hermanto et al, 2013), Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) combined with PCA (Azira et al, 2014), Enzyme-Linked Immuno-Sorbent Assay (ELISA) (Doi et al, 2009;Venien and Levieux, 2005), conventional method using calcium phosphate precipitation test (Hidaka and Liu, 2003) and Polymerase Chain Reaction (PCR) (Demirhan et al, 2012;Cai et al, 2012). The PCR is an ideal technique to be used for fat and sensitive detection of porcine DNA in gelatin due to the higher stability of DNA compared to protein (Aida et al, 2007).…”

Determination of porcine gelatin in edible bird's nest by competitive indirect ELISA based on anti-peptide polyclonal antibody

“…These methods demonstrate successful establishment of adulterants detection, but due to the requirement of expensive instrument, long time as well as highly skilled operators, development of enzyme-linked immunosorbent assay (ELISA) would be a favourable alternative. Additionally, this assay has high sensitivity and throughput as reported by Venien and Levieux (2005b) and Doi, Watenabe, Shibata, and Tanabe (2009) in detecting gelatin in raw and processed products, respectively. Therefore, the aim of this study was to evaluate the efficiency of polyclonal antibodies (pAbs) against peptide immunogens in detecting porcine gelatin in EBN by competitive indirect ELISA.…”

“…Due to low immunogenicity of gelatin (Venien & Levieux, 2005a), the anti-peptide antibodies against porcine species-specific amino acid sequence of collagen a2 (I) chain (pAb1 and pAb2) and collagen a1 (I) chain (pAb3) were used. This approach has been done by Doi et al (2009) by applying anti-peptide antibodies as capture antibodies in the sandwich ELISA for mammalian gelatin quantification. In addition, it also showed good reactivity toward bovine gelatin in the competitive indirect ELISA (Venien & Levieux, 2005b).…”