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

Abstract

a b s t r a c tCompetitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for rapid detection of porcine gelatin in edible bird's nest (EBN). Three ELISAs were developed by using polyclonal rabbit antibodies against porcine species-specific amino acid sequences of collagen a2 (I) chain (pAb1 and pAb2) and a1 (I) chain (pAb3). The limit of detection (IC 15 ) of the three ELISAs was 0.033, 0.082 and 0.052 mg/mL respectively. The median inhibitory concentration (IC 50 ) of pAb1, pAb2 and pAb3 was 0.265, 0.394 and 0.228 mg/mL respectively, as well as able to recognise porcine and bovine gelatins. pAb1showed slight cross-reactivity with cave nest and egg white, while pAb2 exhibited slight cross-reactivity with blood cave nest and egg white. No cross-reactivity was observed with EBNs and egg white for pAb3. The recoveries of porcine gelatin spiked EBNs were in the range of 62.8e125.4% with intra-and inter-day coefficient of variants (CVs) of 2.9e5.4% and 4.7e9.6% respectively when using pAb3. Taking into account all abovementioned factors, pAb3 appeared sufficient for EBN authentication.

Origin Determination and Differentiation of Gelatin Species of Bovine, Porcine, and Piscine through Analytical Methods

“…However, the porcine limed bone has not been detected by competitive indirect ELISA (Venien and Levieux, 2005). Tukiran et al (2016a) investigated limit of detection of 0.05 mg/mL which is satisfactory for the scope of adulteration lower than this would not be economically profitable and prone to cross contamination problem. Weber et al (2009) developed a competitive indirect ELISA which was applied to several fish gelatins and isinglass samples used in food production for the determination of parvalbumins from various fish species.…”

“…Detection and quantitation of gelatin in food and pharmaceutical products have also been achieved by immunochemical methods which are less expensive, can be completed in shorter time and do not require highly skilled staff (Tukiran et al, 2016a;Venien and Levieux, 2005). ELISA is among the types of immunochemical analytical methods.…”

A Review of Gelatin Source Authentication Methods

“…The developed ELISA showed low cross-reactivity to fish and chicken gelatin and the detection limit was 0.05 μg/mL. Furthermore, Tukiran et al (2016a) also developed competitive indirect ELISA based on the anti-peptide polyclonal antibody for determination of porcine gelatin in edible bird’s nest. Among the three porcine species-specific peptides, the sequence that used collagen α1 was found to be sufficient for authentication of edible bird’s nest with a detection limit of 0052 μg/mL.…”

“…However, large similarities between structures of gelatin from different sources make their differentiation difficult ( Nemati et al 2004 ). Many gelatin source authentication methods have been developed including: spectroscopic method ( Hashim et al 2010 ; Hermanto & Fatimah 2013 ), immunochemical method ( Tukiran et al 2015 , 2016a ), nucleic acid based method ( Cai et al 2012 ; Demirhan et al 2012 ; Malik et al 2016 ; Mutalib et al 2015 ; Shabani et al 2015 ; Sudjadi et al 2015 ), mass spectrometric method HPLC/MS ( Yilmaz et al 2013 ; G.-F. Zhang et al 2008 ; G. Zhang et al 2009 ), chromatographic-chemometric method ( Azilawati et al 2015 ), electrophoretic analysis ( Azira et al 2014 ) and chemisorption ( Hidaka & Liu 2003 ).…”

Near Infrared Spectroscopy Based on Supervised Pattern Recognition Methods for Rapid Identification of Adulterated Edible Gelatin

“…Currently, some analytical methods for the identification of gelatin in products include the electrophoretic method [4,5], enzyme linked immune sorbent assay (ELISA) [6][7][8], high performance liquid chromatography (HPLC) [9][10][11], and polymerase chain reaction (PCR) [12][13][14][15]. However, most of these methods are expensive and time consuming although the sensitivity and accuracy are high.…”