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Cathy Olsen, PhD | Sr. Applications Scientist | Molecular Devices

Joyce Itatani | Applications Scientist | Molecular Devices

Introduction

Quantifying gluten in beer

With the recent rise in the prevalence of celiac disease, monitoring gluten levels in food and beverage has become increasingly important as more people strive to avoid gluten. FDA guidelines specify that foods labeled ‘gluten-free,’ ‘no gluten,’ ‘free of gluten,’ or ‘without gluten’ must contain less than 20 parts per million (ppm) of gluten. This is the limit of quantitation (LOQ) for currently accepted test methods.

Gluten occurs naturally in wheat, rye, barley, and crosses of these grains. It is a mixture of prolamin and glutelin proteins present in these grains. When foods are processed, and during digestion, intact prolamin is degraded into small peptide fragments which remain dangerous to celiac patients. These small peptide fragments cannot be detected by a sandwich ELISA due to that assay’s requirement for two epitopes. However, the RIDASCREEN^® Gliadin competitive ELISA can detect these small fragments using a new standard material, hydrolysate of wheat, rye, and barley (Figure 1). The R5 antibody used in this kit recognizes potentially toxic peptide sequences of gliadins from wheat and prolamins from rye and barley (Kahlenberg et al., 2005).

Figure 1. RIDASCREEN Gliadin competitive ELISA. Wells are precoated with gliadin. When sample and antibody conjugate are added, gluten in the sample competes for binding to the antibody, leading to a reduction in assay signal.

Many products are available for testing gluten content in a variety of sample types, but not all are quantitative, and not all are approved test methods. The RIDASCREEN^® Gliadin competitive ELISA is an AACC International approved method (38-55.01) and an AOAC-approved Official Method of Analysis (First Action OMA 2015.05) that has been evaluated in an international study by 18 labs for use with beer, starch syrup, and sourdough. In a second international collaborative study by the American Society of Brewing Chemists (ASBC), the RIDASCREEN^® Gliadin competitive ELISA was evaluated by 15 labs for five different beers, and this ELISA is now an ASBC international approved method (Beer-49).

Here we tested the gliadin levels in six commercially available beers to determine gluten levels with the RIDASCREEN Gliadin competitive ELISA. Beers tested included gluten-free, reduced gluten, weizenbier (wheat beer), and other varieties. The ELISA was read on the SpectraMax^® ABS Plus Microplate Reader and results were analyzed using SoftMax^® Pro Software. Quantitative results confirmed gluten levels below 20 ppm in the gluten-free and reduced-gluten beers.

Materials

• RIDASCREEN Gliadin Competitive (Art. No. R7021)
• Commercially available beers:\
  • Reduced gluten
  • Gluten-free
  • Weizenbier
  • Blond ale
  • American porter
  • India pale ale
• Cold water fish skin gelatin 40% (Electron Microscopy Sciences cat. #25560)
• Multi-Wash+ ^™ Microplate Washer (Molecular Devices cat. #MultiWash+)
• SpectraMax ABS Plus Microplate Reader (Molecular Devices cat. #ABS Plus)

Reagent preparation

All reagents in the kit were prepared as indicated in the product insert.

• Buffer (sample diluent) was diluted 1:5 with deionized water.
• Wash buffer was warmed in a 37°C water bath to dissolve any crystals, and then diluted 1:10 with deionized water.
• Antibody enzyme conjugate was shaken before pipetting, then diluted 1:11 with deionized water. Only the amount needed for assay was diluted.
• Substrate/chromogen (Red Chromogen Pro) and Stop solution were supplied as ready-to-use solutions.

Beer sample preparation

1 mL of each beer was combined with 9 mL 60% ethanol solution containing 10% cold water fish skin gelatin in a 15-mL conical tube. Samples were mixed thoroughly by vortexing and then placed on a rotator for 10 minutes. Samples were then centrifuged at 2500 rpm for 10 minutes. Supernatants were then ready for assay and could also be stored in closed containers in the dark at room temperature for up to four weeks. Supernatants were diluted 1:50 with diluted sample diluent (buffer) prior to assay.

ELISA

Three strip wells were placed into the microwell strip holder, and unused wells were stored in the foil pouch containing a desiccant packet.

50 μL of each gliadin standard (0, 10, 30, 90 and 270 ng/mL) or prepared sample was added to duplicate wells. Duplicate wells were also set aside as a plate blank (no standard or sample added). 50 μL of diluted antibody enzyme conjugate was added to each well and mixed manually by gently shaking the plate. The plate was incubated for 30 minutes at room temperature. Next, the plate was washed three times with 250 μL of diluted wash buffer using the MultiWash+ Microplate Washer.

100 μL of substrate/chromogen was added to each well, including plate blank wells. The plate was mixed gently by shaking manually, and incubated 10 minutes at room temperature in the dark. 100 μL stop solution was then added to every well, and the plate was mixed gently by shaking manually. Within 10 minutes, absorbance was measured at 450 nm on the SpectraMax ABS Plus reader using an ELISA protocol in the protocol library of SoftMax Pro Software.

A standard curve was plotted using the semi-log curve fit in SoftMax Pro Software, and sample concentrations were calculated from the standard curve.

Results

From the gliadin standard curve (Figure 2), gliadin concentrations were interpolated automatically by the software. Concentrations (ng/mL) were adjusted to the values present in the original beer samples by multiplying by the dilution factor (500), and gliadin (ng/mL) was converted to gliadin (ppm) by dividing by 1000. To convert gliadin into gluten, a factor of two was used (definition Codex Alimentarius), with results ultimately expressed as gluten ppm (Figure 3). These calculations were added to the 'Unknowns' group table of the protocol in SoftMax Pro Software.

Figure 2. Gliadin ELISA standard curve. SoftMax Pro Software generated a standard curve including 10, 30, 90 and 270 ng/mL gliadin standards using a semi-log curve fit (R²=0.981).

R - Outside standard range Figure 3. Group table as seen in SoftMax Pro Software. Gliadin concentrations in beer samples were interpolated from the standard curve and multiplied by the dilution factor to obtain the adjusted concentration (ng/mL). Gliadin ppm and gluten ppm were calculated from the adjusted concentration.

Beers labeled reduced gluten or gluten-free had calculated gluten concentrations below 20 ppm, meeting FDA guidelines for these labels. For one of the gluten-free beer sample replicates, the OD value fell below that of the lowest gliadin standard, and this replicate was flagged as out of range, ‘R’ in the group table (Figure 3). Weizenbier, which was expected to have a higher gluten content, indeed showed gliadin values that exceeded levels present in the 270 ng/mL standard and was also flagged as out of range in the group table. To obtain an accurate quantitation of gluten, this beer would need to be further diluted. All the other beers tested ranged from 14 to 118 ppm gluten, with India pale ale falling beneath the 20 ppm ‘gluten-free’ level.

The blond ale, at 87 ppm gluten, could be labeled as ‘very low gluten’ by Codex Alimentarius standards.

Similar results are obtained using SpectraMax multi-mode readers (results not shown).

Conclusion

The RIDASCREEN Gliadin competitive ELISA provides a quantitative solution for gluten detection in beer. The hands-on time required to run this assay is reduced by using the MultiWash+ washer to automate wash steps. Detection of absorbance with the SpectraMax ABS Plus reader, and calculation of results using a SoftMax Pro Software protocol, further simplify the ELISA workflow and flag any result that warrants further attention. From sample to result, the time required to reliably determine gluten content is only about an hour.

Reference

Kahlenberg F., Sanchez D., Lachmann I., Tuckova L., Tlaskalova H., Mendez E., Mothes T. (2005). Monoclonal antibody R5 for detection of putatively coeliac-toxic gliadin peptides. European Food Research and Technology 222(1-2), 78-82.

Cathy Olsen, PhD | Sr. Applications Scientist | Molecular Devices

Joyce Itatani | Applications Scientist | Molecular Devices

简介

定量啤酒中谷蛋白

由于最近乳糜泻患病率呈上升趋势，对于 越来越多的需要避免谷蛋白摄入的人们来 说，监测食品和饮料中的谷蛋白水平变得 尤为重要。FDA 指南中明确指出食品标签 上标明“gluten-free”、“no gluten”、 “free of gluten”或“without gluten” 的，谷蛋白含量必须小于 20 ppm。这是 目前检测方法能够接受的定量限。

谷蛋白天然存在于小麦、黑麦、大麦和这 类谷物中。在这些谷物中，它是存在于这 些谷物中的丙氨酸和谷氨酸蛋白的混合 物。在食品生产和食物消化工程中，完整 的醇溶谷蛋白分解成小肽片段，这些小肽 对乳糜泻患者仍然是有危害的。这些小肽 片段无法用三明治 ELISA 检测，因为这种 分析方法需要 2 个抗原表位。然而， RIDASCREEN 麦醇溶蛋白竞争 ELISA 可以 检测这些小肽片段，它是通过一个新的标 准品，小麦，黑麦和大麦的水解产物来检 测的 ( 图 1 )。这个试剂盒中的 R5 抗体用 来识别来自小麦的醇溶蛋白和来自黑麦和 大麦的醇溶谷蛋白这些可能有害的多肽片 段 (Kahlenberg et al., 2005) 。

***图 1 RIDASCREEN 麦醇溶蛋白竞争 ELISA。*微孔板的孔中用麦醇溶蛋白预包被。当加入 样品和抗体结合物后，样品中的谷蛋白和抗体结合，导致检测信号降低

很多产品能够检测不同类型样品中的谷蛋 白含量，但是不是所有能够定量检测，不 是所有都通过了认证检测。RIDASCREEN 麦醇溶蛋白竞争 ELISA 试剂盒是 AACC 国 际认证 (38-55.01) 和 AOAC 官方认证的分 析方法 ( 第一行动 OMA 2015.05 ) ，经过 国际上 18 个实验室用啤酒，淀粉糖浆和 酵母评估。在第二国际协作研究中，美国 优势 • 采用经过认证的方法定量检测 啤酒和其他食品中的谷蛋白含 量 • MultiWash+ 微孔板洗板机自动 进行 ELISA 洗板，节省时间 • 使用 SoftMax Pro 软件快速获 得分析结果 酿酒化学家协会 (ASBC) 选出 15 个实验 室，通过 5 种不同啤酒对 RIDASCREEN 麦 醇溶蛋白竞争 ELISA 试剂盒进行评估。这 种 ELISA 方法现在是 ASBC 国际认证的方 法 (Beer-49) 。

本文我们利用 RIDASCREEN 麦醇溶蛋白竞 争 ELISA 试剂盒检测了 6 种市场上买到的 啤酒中的麦醇溶蛋白水平。检测的啤酒包 括无谷蛋白，低谷蛋白，小麦啤酒和其他 种类的啤酒。ELISA 通过 SpectraMax ABS Plus 微孔板读板机读板，数据结果通过 SoftMax Pro 软件分析。定量分析结果证 实，无谷蛋白和低谷蛋白啤酒的谷蛋白含 量低于 20 ppm。

材料

• RIDASCREEN 麦醇溶蛋白竞争试剂盒 (Art. No. R7021)
• 市面上买到的啤酒：\
  • 低谷蛋白
  • 无谷蛋白
  • 小麦啤酒
  • 金色麦芽酒
  • 美式波特
  • 印度麦酒
• 40%冷水鱼皮明胶 (Electron Microscopy Sciences cat. #25560)
• Multi-Wash+ 微孔板洗板机 (Molecular Devices cat. #MultiWash+)
• SpectraMax ABS Plus 微孔板读板机 (Molecular Devices cat. #ABS Plus)

试剂制备

试剂盒中所有试剂都按照产品说明书制备。

• 缓冲液 ( 样品稀释液 ) 去离子水 1：5 稀释
• 洗涤缓冲液 37℃ 温育去除结晶， 去离子水 1：10 稀释
• 抗体酶结合物混匀，去离子水 1：11 稀释。现用现稀释
• 底物和终止液即开即用

啤酒样品制备

1 mL 啤酒和 9 mL 含有 10% 冷水鱼皮明胶 的 60% 乙醇混合于 15 mL 离心管中，涡 旋混合均匀。2500 rpm 离心 10 分钟。上 清即可用于分析检测，也可存储在密闭容 器中黑暗下置于室温 4 周。上清用稀释好 的样品稀释液 1：50 稀释后再分析检测。

ELISA

取出 3 列复孔板条放置在微孔板条支架 上，其余存放在有干燥剂的铝箔小袋中。 将 50 µL 麦醇溶蛋白标准品 ( 0、10、30、 90 和 270 ng/mL ) 或制备好的样品加入复 孔中。同时留有复孔 ( 没加标准品或样品 ) 作为板空白。每孔中加入 50 µL 稀释好的 抗体酶结合物并轻摇板子混匀。板子室温 孵育 30 分钟。然后，用 MultiWash+ 微孔 板洗板机 250 µL 稀释的洗涤缓冲液洗板3 次。

每孔加入 100µL 底物，包括板空白。轻摇 板混匀，黑暗下室温放置 10 分钟。每孔 加入 100 µL 终止液，轻摇板混匀。10 分 钟后，用 SpectraMax ABS Plus 调用 SoftMax Pro 软件中预存的 ELISA 方法在 450 nm 处检测光吸收值。 在 SoftMax Pro 软件中用 semi-log 拟合出 标准曲线，样品浓度通过代入标准曲线中 计算得出。

结果

通过麦醇溶蛋白标准曲线 ( 图 2 ) ，软件自 动 计 算 出 麦 醇 溶 蛋 白 浓 度 。 最 终 浓 度 (ng/mL) 用计算出的浓度乘以原始啤酒样 品的稀释因子 (500) 得出，麦醇溶蛋白 (ng/mL) 除以 1000 转变成麦醇溶蛋白 (ppm)。为了将麦醇溶蛋白转变成谷蛋 白，使用因子 2 ( 国际食品药典定义 ) ，最 终结果用谷蛋白 ppm 表示 ( 图 3 ) 。这些 计算都已添加在 SoftMax Pro 软件 ‘Unknow’样品组的图表中。

***图 2 麦醇溶蛋白 ELISA 标准曲线。*SoftMax Pro 软件把 10，30，90 和 270 ng/mL 麦醇溶蛋白标准 品用 semi-log 方式进行曲线拟合 (R2 = 0.981)

R - Outside standard range ***图 3 SoftMax Pro 软件中显示的各组信息。*啤酒样品中的麦醇溶蛋白含量代入标准曲线中计算得 到，乘以稀释倍数计算出最终含量 (ng/mL)。麦醇溶蛋白 ppm 和谷蛋白 ppm 由最终浓度计算得出

标有低谷蛋白和无谷蛋白的啤酒检测出的 谷蛋白含量均低于 20 ppm，符合 FDA 指 南要求。一个无谷蛋白啤酒样品的重复， 检测出的 OD 值低于麦醇溶蛋白标准品的最 低值，这个重复标记为超出检测范围，在 分组图表中表示为‘R’( 图 3 )。谷蛋白含 量高的小麦啤酒，实际结果显示麦醇溶蛋 白检测值超过标准品上限 270 ng/ mL，在 分组图中也显示超出检测范围。为了得到 更准确的定量谷蛋白，应该继续稀释这款 啤酒。其他啤酒检测范围在 14-118 ppm 谷 蛋白，印度麦酒的谷蛋白含量低于 20 ppm ‘无谷蛋白’水平。

金色麦芽酒 87 ppm 谷蛋白，按照国际食 品法典标准可以贴上‘谷蛋白含量极低’ 的标签。

在 SpectraMa x 多功能微孔板读板机上获 得了同样的检测结果 (结果未显示) 。

结论

RIDASCREEN 麦醇溶蛋白竞争 ELISA 试剂 盒能够定量啤酒中的谷蛋白含量。使用 MultiWash+ 洗板机自动洗板可以大大减 少手动操作时间。采用 SpectraMax ABS Plus 微孔板读板机检测光吸收值，采用 SoftMax Pro 软件中的方法计算检测结果， 以后还可以进一步简化 ELISA 工作流程并 标记所有结果。从样品到结果，检测得到 可靠的谷蛋白含量仅需 1 小时。

参考文献

Kahlenberg F., Sanchez D., Lachmann I., Tuckova L., Tlaskalova H., Mendez E., Mothes T. (2005). Monoclonal antibody R5 for detection of putatively coeliac-toxic gliadin peptides. European Food Research and Technology 222(1-2), 78-82.

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