In hydrolysates obtained by one- and two-step hydrolysis, gliadin content was assessed by immunoenzymatic assay with G12 (AgraQuant® Gluten G12®; Romer Labs GmbH, Tulln an der Donau, Austria) and R5 (RIDASCREEN® Gliadin; R-Biopharm Inc., Pfungstadt, Germany) antibodies according to the manufacturer’s instructions and previous report [5 (link)]. The G12 monoclonal antibody recognizes an immunoreactive 33-mer peptide, while the R5 monoclonal antibody recognizes a potentially immunogenetic QQPFP sequence that occurs multiple times in prolamine molecules. Assays were performed in triplicate, and gliadin content was calculated based on the standard curve. The results are presented as relative residual immunoreactivity [%] in respect to the untreated gliadin sample or prehydrolyzed gliadin by commercial digestive enzymes in the case of two-step hydrolysis.
Agraquant gluten g12
Manufactured by Romer Labs
Sourced in Austria
The AgraQuant Gluten G12 is a quantitative ELISA test kit designed for the detection and measurement of gluten in various food and feed samples. The kit utilizes the G12 monoclonal antibody, which is specific for the detection of the immunodominant 33-mer peptide from gluten proteins.
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5 protocols using agraquant gluten g12
1
Gliadin Content Assessment in Hydrolysates
2
Quantifying Gluten with Multi-epitope ELISA
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The Enzyme‐Linked Immunosorbent Assay (ELISA) was conducted using the G12 mAb kit (AgraQuant® Gluten G12®, 10 001 994; Romer Labs, Newark, DE), the R5 mAb kit (RIDASCREEN® Gliadin, Art. No. R7001; R‐Biopharm, Darmstadt, Germany) and the R‐BioPharm Total Gluten kit (RIDASCREEN® Art. No. R7401). This assay contains a mixture of four mAbs: mAb R5 as well as mAbs to the HMW‐GS, LMW GSs and rye seaclins (Lacorn et al., 2019 (link)). The sample preparation and measurements were conducted following the manufacturer's instructions for the kits.
3
Gluten-Free Bread Gluten Analysis
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To ensure the gluten content remained below the required threshold (<3 ppm), gluten analysis was conducted on the prepared bread. To accurately measure gluten levels, specialized testing methods such as Ridascreen Gliadin (R-Biopharm AG, Darmstadt, Germany); Veratox for Gliadin R5 (Neogen, Lansing, Michigan, USA); and AgraQuant Gluten G12 (Romer Labs, Getzersdorf, Austria) were used. All values were designated as mean ± standard deviation (n = 3).
The gluten content in the gluten-free bread product was analyzed using the gliadin test Ridascreen (R-Biopharm AG, Darmstadt, Germany), which was also used by Ja Myung Yu [64 (link)].
The gluten content in the gluten-free bread product was analyzed using the gliadin test Ridascreen (R-Biopharm AG, Darmstadt, Germany), which was also used by Ja Myung Yu [64 (link)].
4
Quantifying Wheat Toxic Epitopes
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The R5 Ridascreen Gliadin (R-Biopharm) sandwich enzyme immunoassay and the AgraQuant Gluten G12 (Romer Labs) sandwich enzyme assay were used to measure differences in the toxic epitope content in the various treatments and cultivars. Prolamin extracts were prepared in four replicates using the methodology provided by the kit suppliers. To measure the gliadin content of wheat flour samples, extracts were diluted by 1:5000 in the case of R5 and 1:10,000 in the case of G12 because of the different detection limits. ELISAs were performed as outlined in the manuals of the assays provided by the manufacturers. Results were interpreted by interpolating optical density (OD) values from the standard values, corrected by the dilution factor used for the flour samples. Calculated gliadin contents determined by the ELISAs were normalized by the protein content and gliadin/gluten ratio of each sample.
5
Comparative Evaluation of ELISA Kits for Gluten Detection
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The four spiked samples
were analyzed using four ELISA systems, including Ridascreen Gliadin
(“R5 ”, R7001, R-Biopharm), AgraQuant gluten G12 (Romer-Labs, Austria), Ridascreen Total Gluten (R7041, R-Biopharm, Germany), and Wheat/Gluten ELISA kit II (Morinaga , Japan). Table 1 summaries the details of each ELISA kit. Total hordein
isolate served as a common external hordein standard for all kits.
Three individual extractions of each spiking level were conducted,
and three measurements of each extraction were performed. Two sets
of kit standards and two sets of hordein external standards were also
served. The recovery was calculated by dividing the calibrated gluten
content results with the theoretical spiking hordein content.
were analyzed using four ELISA systems, including Ridascreen Gliadin
(“
isolate served as a common external hordein standard for all kits.
Three individual extractions of each spiking level were conducted,
and three measurements of each extraction were performed. Two sets
of kit standards and two sets of hordein external standards were also
served. The recovery was calculated by dividing the calibrated gluten
content results with the theoretical spiking hordein content.
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