A Review of Gelatin Source Authentication Methods <s> Immunochemical Method (Enzyme-linked immunosorbent assay, ELISA) <s> This brief note addresses the historical background of the invention of the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA). These assays were developed independently and simultaneously by the research group of Peter Perlmann and Eva Engvall at Stockholm University in Sweden and by the research group of Anton Schuurs and Bauke van Weemen in The Netherlands. Today, fully automated instruments in medical laboratories around the world use the immunoassay principle with an enzyme as the reporter label for routine measurements of innumerable analytes in patient samples. The impact of EIA/ELISA is reflected in the overwhelmingly large number of times it has appeared as a keyword in the literature since the 1970s. Clinicians and their patients, medical laboratories, in vitro diagnostics manufacturers, and worldwide healthcare systems owe much to these four inventors. <s> BIB001 </s> A Review of Gelatin Source Authentication Methods <s> Immunochemical Method (Enzyme-linked immunosorbent assay, ELISA) <s> Gelatine is a collagen derivative obtained from bones and hides/skin mainly from bovine and pigs. As a consequence of the outbreak of bovine spongiform encephalopathy (BSE), the use of bovine gelatine in feed, food and pharmaceutical products has been restricted by regulatory authorities. However, no method was presently available for its specific detection. The large similarity in amino-acid sequences of collagens from different species make their immunochemical differentiation difficult when using polyclonal antibodies raised against the whole molecule [A. Venien, D. Levieux, J. Immunoassay Immunochem., in press]. To obtain bovine-specific antibodies, we immunized rabbits against putative species-specific sequences of the bovine collagen alpha 1(I) chain. Using these antibodies, an indirect ELISA was developed to allow a quick and easy differentiation between bovine and porcine gelatines. Moreover, a competitive indirect ELISA was found suitable to detect bovine gelatine in porcine gelatine purchased from laboratory chemicals suppliers down to a dilution of 2-4 parts per 1000 with CVs ranging from 5.7 to 7.7%. When testing mixtures of the largest possible range of industrial batches of bovine and porcine gelatines (skin/hides or bones origin, acid or alkaline processes, high or low Bloom) the detection limit was down to a dilution of 8 parts per 100 bovine gelatine in porcine gelatine. These ELISAs could be routinely used by pharmaceutical and food manufacturers to secure their supply chain. <s> BIB002 </s> A Review of Gelatin Source Authentication Methods <s> Immunochemical Method (Enzyme-linked immunosorbent assay, ELISA) <s> A real-time polymerase chain reaction assay using primers targeting the porcine-specific mitochondrial 12S rRNA gene and universal eukaryotic primers amplifying a conserved fragment of the nuclear 18S rRNA gene has been developed for the detection and quantification of porcine DNA in food and feedstuffs. The 18S rRNA primers were used as endogenous control for the total content of PCR-amplifiable DNA in the sample. The assay was tested on DNA extracted from raw and heat-treated binary mixtures of porcine tissues in a plant matrix, and on DNA extracted from reference feedstuff samples. Analysis of experimental mixtures demonstrated the suitability of the assay for the detection and quantification of porcine DNA in mixtures containing as little as 0.1%. <s> BIB003 </s> A Review of Gelatin Source Authentication Methods <s> Immunochemical Method (Enzyme-linked immunosorbent assay, ELISA) <s> In order to classify unknown gelatin into their species of origin, a simple and rapid method for the qualitative determination was developed using Fourier transform infrared (FTIR) in combination with attenuated total reflectance (ATR) and discriminant analysis. The spectra were analysed using a chemometric method, principal component analysis (PCA), to classify and characterise gelatin compounds using regions of the FTIR spectra in the range of 3290–3280 cm−1 and 1660–1200 cm−1 as calibration models. Results from PCA, which were subsequently represented by the Cooman’s plot showed a clear distinction between gelatin samples of bovine and porcine origins. This qualitative approach, besides providing a rapid determination of the source of gelatin, may also be established based on a second derivative study of the FTIR spectrum to alleviate any doubt of the gelatin source for applications in the food and pharmaceutical industries. <s> BIB004 </s> A Review of Gelatin Source Authentication Methods <s> Immunochemical Method (Enzyme-linked immunosorbent assay, ELISA) <s> Fucose-containing sulfated polysaccharides (FCSPs) extracted from seaweeds, especially brown macro-algae, are known to possess essential bioactive properties, notably growth inhibitory effects on tumor cells. In this work, we conducted a series of in vitro studies to examine the influence of FCSPs products from Sargassumhenslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively, on proliferation of melanoma B16 cells and to investigate the underlying apoptosis promoting mechanisms. Cell viability analysis showed that both FCSPs products, i.e., FSAR and FVES, decreased the proliferation of the melanoma cells in a dose-response fashion, with FSAR being more potent at lower dosages, and FVES being relatively more anti-proliferative than FSAR at higher dosages. Flow cytometric analysis by Annexin V staining of the melanoma cells exposed to the FCSPs products confirmed that both FSAR and FVES induced apoptosis. The FCSPs-induced apoptosis was evidenced by loss of plasma membrane asymmetry and translocation of the cell membrane phospholipids and was accompanied by the activation of caspase-3. The FCSPs bioactivity is proposed to be attributable to distinct structural features of the FCSPs, particularly the presence of sulfated galactofucans (notably in S.henslowianum) and sulfated fucans (notably in F. vesiculosus). This study thus indicates that unfractionated FCSPs may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3. <s> BIB005 </s> A Review of Gelatin Source Authentication Methods <s> Immunochemical Method (Enzyme-linked immunosorbent assay, ELISA) <s> We presented a novel nanoUPLC-MS(E) workflow method that has potential to identify origin of gelatin in some dairy products; yoghurt, cheese and ice cream. In this study, the method was performed in two steps. In the first step, gelatin was extracted from these products before the MS-sample preparation. In the second step, tryptic gelatin peptides were separated and analyzed with ultra-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (nanoUPLC-ESI-q-TOF-MS(E)). The novelty of this setup was that it functioned in a data independent acquisition mode and that alternate low and elevated collision energy was applied to acquire precursor and product ion information. This enabled accurate mass acquisition on the peptide level to identify the gelatin peptides. The marker peptides specific for porcine and bovine could be successfully detected in the gelatin added to the dairy products analyzed, revealing that the detection of marker peptides in the digested gelatin samples using nanoUPLC-ESI-q-TOF-MS(E) could be an effective method to differentiate porcine and bovine gelatin in the dairy products. <s> BIB006 </s> A Review of Gelatin Source Authentication Methods <s> Immunochemical Method (Enzyme-linked immunosorbent assay, ELISA) <s> Porcine gelatine is a common adulterant found in edible bird’s nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g–1, respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg–1 (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry. <s> BIB007 </s> A Review of Gelatin Source Authentication Methods <s> Immunochemical Method (Enzyme-linked immunosorbent assay, ELISA) <s> Competitive 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 α2 (I) chain (pAb1 and pAb2) and α1 (I) chain (pAb3). The limit of detection (IC15) of the three ELISAs was 0.033, 0.082 and 0.052 μg/mL respectively. The median inhibitory concentration (IC50) of pAb1, pAb2 and pAb3 was 0.265, 0.394 and 0.228 μg/mL respectively, as well as able to recognise porcine and bovine gelatins. pAb1 showed 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 62.8–125.4% with intra- and inter-day coefficient of variants (CVs) of 2.9–5.4% and 4.7–9.6% respectively when using pAb3. Taking into account all abovementioned factors, pAb3 appeared sufficient for EBN authentication. <s> BIB008
The principle of enzyme-linked immunosorbent assay (ELISA) is based on the binding effect of the antibody with specific macromolecular analytes -antigens -such as protein/peptide BIB005 . Alternatively, the antibody might be the analytes rather than an antigen. In addition, a measurable signal is produced through the introduction of chemically linked antibodies or antigen with a detectable label in order to allow for measurement  . The reporter labels that have been utilised in ELISA are basically enzymes such as horseradish peroxidase, alkaline phosphatase and glucose oxidase BIB001 . ELISA method is advantageous because of structural specificity, detectability, sensitivity and high sample throughput capacity  BIB003 ). It equally offers simplicity in term of sample preparation as high purity is not necessary. Thus, immunochemical differentiation is a valuable alternative method for authentication of gelatin source  . Recent research efforts on the development of ELISA technique for gelatin source authentication have been reported. Briefly, the experimental procedure involves (i) identification of suitable specie-specific sequence (from gelatin peptides) (ii) production of synthetic peptides (iii) crosslinking of peptides with keyhole limpet hemocyanin (iv) immunisation of rabbit (iv) recovery of produced of polyclonal antibodies (v) development of an indirect and/or competitive indirect ELISA. However, there might be slight variation in the literature. BIB002 developed indirect and competitive indirect ELISA using polyclonal anti-peptide antibodies for differentiation of bovine from porcine gelatins. It was reported that competitive indirect ELISA was more appropriate to detect bovine gelatin in porcine gelatin to a dilution of 2-4 parts per 1000. BIB007 also developed indirect ELISA using anti-peptide polyclonal antibodies for porcine gelatin detection in dibble bird's nests. The result showed that the developed ELISA was able to detect at least 0.5 ng/μg porcine gelatin in spiked samples. In another study,  developed indirect ELISA using anti-peptide (collagen α2) polyclonal antibodies for authentication of gelatin source in confectionery products. The developed ELISA showed low cross-reactivity to fish and chicken gelatin and the detection limit was 0.05 μg/ mL. Furthermore, BIB008 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. The challenge of immunochemical method is lack of antibodies stability in alkaline or acidic process which is commonly used for gelatin production BIB006 . Also, ELISA method lack enough species-specific detection of mixture containing porcine gelatin and low concentration of bovine gelatin BIB004 .