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Introduction

The organic base melamine is used to make a number of products, including plastics, flame retardants, pigments, and fertilizers. The practice of adding melamine to animal feed and foods for human consumption in order to increase the apparent protein content has recently been reported. Because melamine contamination can cause serious illness or death in animals and people, there is increased interest in identifying methods for detecting melamine contamination in a variety of food products. In this application note, two melamine ELISA assay kits from Romer Labs are introduced and validated: the AgraQuant Melamine Assay and the AgraQuant Melamine Sensitive Assay.

The AgraQuant Melamine Assay is a direct competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of melamine and is intended for use with wheat gluten and pet food.¹ The AgraQuant Melamine Sensitive Assay is a more sensitive ELISA that has been validated for milk, milk powder, and other dairy products.² For both assays, test samples and enzyme-conjugated melamine are dispensed into microwells that have been pre-coated with melamine antibody. The test samples and standards compete with enzyme-conjugated melamine for binding to the antibody. Following incubation, the microwells are washed and substrate added, followed by a second incubation. After the second incubation, the reaction is quenched using the Stop Solution, and the absorbance of the mixture is measured at 450 nm using a microplate reader. The color intensity of the mixture is inversely proportional to the amount of melamine present in the standards or samples. The melamine concentrations of test samples are interpolated using a melamine standard curve.

Absorbance microplate readers from Molecular Devices are used to detect the absorbance in the melamine assays. Data collection and analysis are performed using a Melamine ELISA preconfigured protocol in SoftMax^® Pro GxP Software, an industry-standard analysis software for FDA 21 CFR Part 11 compliance.

Materials

• Molecular Devices microplate reader with absorbance detection mode, including:
  • SpectraMax ^® Plus 384 (cat. #PLUS 384)
  • SpectraMax 190 (cat. #190)
  • SpectraMax 340PC384 (cat. #340PC 384)
  • VersaMax™ (cat. #VERSAMAX)
  • SpectraMax M2/M2 ^e (cat. # M2 or M2E)
  • SpectraMax M5/M5 ^e (cat. #M5 or M5E)
  • FlexStation ^® 3 (cat. #FLEX3)
  • SpectraMax i3x (cat. #i3x)
  • SpectraMax iD3 (cat. #iD3)
• AgraQuant Melamine Test Kit (Romer Labs cat. #COKAQ9300)
• AgraQuant Melamine Sensitive Test Kit (Romer Labs cat. #COKAQ9400)

Methods

For both kits, the following procedure was used:

Reagents were equilibrated to room temperature prior to use.

Test wells were placed into a microwell strip holder. Unused wells were sealed in a plastic pouch with desiccant.

150 µL of melamine standards were added to test wells with a single-channel pipette, using a new tip for each dispense. The Melamine Assay kit includes 0, 20, 100, and 500 parts per billion (ppb) standards, while the Melamine Sensitive Assay kit includes 0, 20, 100, and 1000 ppb standards.

50 µL of conjugate were dispensed into each well using an 8-channel pipette.

The plate was gently rotated for 60 seconds and then incubated at room temperature for 30 minutes.

Contents of the wells were aspirated, and wells were washed with 400 µL of deionized water a total of four times.

Following the second wash, the plate was inverted onto absorbent paper to remove the remaining wash solution.

100 µL of Substrate was added to each well using a multi-channel pipette. The plate was incubated at room temperature for 20 minutes.

100 µL of Stop Solution was added to each well using a multi-channel pipette.

The absorbance of the wells was read on a VersaMax Microplate Reader at 450 nm using a pre-configured protocol in the SoftMax Pro Software.

Calibrators were plotted using a semi-log curve fit in the SoftMax Pro Software.

Results

Optical density (OD) values expressed as a percentage of the OD value of the zero ppb standard (B/B₀) were used to construct a semi-log response curve. Sample melamine content can be interpolated from the standard curve. If a sample contains greater than 500 ppb (regular kit) or 1000 ppb (sensitive kit), the sample should be further diluted and re-analyzed to obtain an accurate result. If a sample contains melamine below the 20 ppb standard, the result should be reported as “< (20 x dilution factor) ppb”.^{(1, 2)}

Figure 1 shows the standard curve for the Melamine Assay plotted as logit(B/B₀) vs. log (concentration), where B is the mean absorbance value for each standard and B₀ is the mean absorbance of the 0 ppb standard. Figure 2 shows the standard curve for the Melamine Sensitive Assay. The r² values were 0.999 for each standard curve, satisfying Romer Labs recommendation that the r² be no less than 0.985 for the Melamine Assay and no less than 0.990 for the Melamine Sensitive Assay. 50% inhibition was calculated as 124.2 for the Melamine Assay and 77.4 ppb for the Melamine Sensitive Assay; both values were comparable to those shown in the certificate of performance for each kit.

Figure 1. Melamine test kit standard curve. Melamine standards at 20, 100, and 500 ppb plotted as logit(B/B₀) vs. log (concentration). r² = 0.999. 50% inhibition = 124.2 ppb.

Figure 2. Melamine sensitive test kit standard curve. Melamine standards at 20, 100, and 1000 ppb plotted as logit(B/B₀) vs. log (concentration). r² = 0.999. 50% inhibition = 77.4 ppb..

According to data for test reproducibility presented in the Melamine Test Kit and Melamine Sensitive Test Kit certificates of performance, coefficients of variance (CVs)for standards are <9%. In this application note we demonstrate CVs of ≤5% for all standards. (See Table 1 and Table 2.).

Table 1. Melamine test kit. Average OD, standard deviation, average B/B₀, and CVs were calculated by SoftMax Pro Software. CVs ≤5% compare favorably to CVs <9% shown in the certificate of performance provided by Romer Labs for each test kit.

Table 2. Melamine sensitive test kit. Average OD, standard deviation, average B/B₀, and CVs were calculated by SoftMax Pro Software. CVs ≤5% compare favorably to CVs <9% shown in the certificate of performance provided by Romer Labs for each test kit.

Conclusion

The Melamine Test Kit and Melamine Sensitive Test Kit from Romer Labs, together with SpectraMax, VersaMax, VMax, and EMax microplate readers from Molecular Devices, offer a quality assurance screening solution for the detection of melamine contamination in food products such as milk, milk powder, yogurt, wheat gluten, and moist and dry pet food. Complete data analysis is provided by SoftMax Pro GxP Software, an industrystandard analysis software for FDA 21 CFR Part 11 compliance.

The AquaMax^® 4000 Microplate Washer from Molecular Devices enables automation of the ELISA wash steps and can be integrated with Molecular Devices StakMax^® Microplate Handling System for increased throughput. Users may also configure the StakMax system with Molecular Devices SpectraMax and VersaMax microplate readers. These combinations maximize throughput while minimizing manual plate handling.

References

1. AgraQuant Melamine Test Kit product insert and Certificate of Performance.
2. AgraQuant Melamine Sensitive Test Kit product insert and Certificate of Performance.

Learn more about VersaMax ELISA Microplate Reader >>

简介：

有机碱三聚氰胺可用于生产很多产品，如塑料、阻燃材料、颜料和肥料等。最近有报道称某些厂家在动物饲料和人类的食品中添加三聚氰胺用于增加其中蛋白质的含量。由于三聚氰胺潜在毒性可引起人和动物的一系列疾病，甚至死亡。所以在各种食品中检测三聚氰胺含量方法越来越受到大家重视。在此篇应用文章中介绍并验证了两种来自于罗默实验室的三聚氰胺ELISA检测试剂盒：AgraQuant三聚氰胺检测试剂盒和AgraQuant灵敏型三聚氰胺检测试剂盒。

AgraQuant三聚氰胺检测试剂盒采用的原理为直接竞争酶联免疫吸附法(ELISA)，主要用于麦麸和宠物食品中的三聚氰胺含量的检测。AgraQuant灵敏型三聚氰胺检测试剂盒适合于对灵敏度要求更高的ELISA实验，如对牛奶、奶粉和其它日常消费品中三聚氰胺的检测。两种试剂盒，都是将待检测的样品和酶标记三聚氰胺加入预先包被有三聚氰胺抗体的微孔板中，待检测样品和标准品与酶标记三聚氰胺相互竞争的与微孔板中已包被的抗体结合。孵育一段时间后，使用缓冲液清洗微孔板，随后加入底物后再孵育一段时间后显色。在第二次孵育后，加入反应终止液来终止反应，在酶标仪中检测其在450nm波长下的吸光度值。颜色深度与混合物中样品及标准品中三聚氰胺的含量成反比。样品中三聚氰胺的含量可由标准曲线拟合后算出。

Molecular Devices光吸收型读板机检测三 聚氰胺的吸光度值，针对三聚氰胺ELISA 检测使用SoftMax® Pro GxP 软件中预先 内置的模板进行数据的收集和分析，此软 件是工业标准的分析软件，具有美国 FDA21 CFR Part11认证。

材料：

• Molecular Devices具有吸收光检测模 式的微孔板读板机，包括：
  • SpectraMax ^® Plus 384 (cat. #PLUS 384)
  • SpectraMax 190 (cat. #190)
  • SpectraMax 340PC384 (cat. #340PC 384)
  • VersaMax™ (cat. #VERSAMAX)
  • SpectraMax M2/M2 ^e (cat. # M2 or M2E)
  • SpectraMax M5/M5 ^e (cat. #M5 or M5E)
  • FlexStation ^® 3 (cat. #FLEX3)
  • SpectraMax i3x (cat. #i3x)
  • SpectraMax iD3 (cat. #iD3)
• AgraQuant Melamine Test Kit (Romer Labs cat. #COKAQ9300)
• AgraQuant Melamine Sensitive Test Kit (Romer Labs cat. #COKAQ9400)

方法：

针对两个检测试剂盒，都遵循以下检测步骤：

使用以前先将试剂放在室温环境下一段时间后再使用。

将检测板条放入板框适配器中，不使用的微孔板条放在具有干燥剂的塑料袋中。

使用单道枪将150μl三聚氰胺标准品加入检测孔中，每次分装后均换枪头，三聚氰胺检测试剂盒包含0,20,100和500ppb标准品，灵敏型三聚氰胺检测试剂盒包含0,20,100和1000ppb标准品。

使用8道排枪将50μl酶标记三聚氰胺分装入微孔板中。

将微孔板均匀震荡60秒后在室温条件下孵育30分钟。

将孔内的液体吸干后加入400μl去离子水后清洗4遍。

清洗后将微孔板倒扣在吸水纸表面，以便于去除残余洗液。

使用排枪将100μl底物溶液加入孔中。

将微孔板放在室温条件下孵育20分钟。

使用排枪将100μl终止反应液加入孔中。

使用VersaMax微孔板读板机，选择 SoftMax Pro软件内预置的模板检测 其在450nm波长下的光吸收值。

SoftMax Pro软件中选择Semi-log 曲线拟合方式。

结果

各标品的OD值与零标准品OD值的比值 (B/Bo)构建一条Semi-log方式拟合的 浓度效应学曲线。根据拟合生成的标准 曲线可计算出样品中三聚氰胺的浓度。 如果样品中三聚氰胺的含量高于 500ppb(常规试剂盒)或者1000ppb(灵 敏型试剂盒)，样品需要进一步的进行稀 释后重新分析以便获得精确的检测结 果。如果样品中三聚氰胺的含量低于 20ppb,结果中就会自动生成报告显示为 “< (20 x dilution factor) ppb” (1,2)。

图一为三聚氰胺检测试剂盒标准曲线logit (B/ Bo) vs. log(concentration)，B为每个 标准品的平均吸光度值，Bo为0ppb标准品 的平均吸光度值。图二为灵敏型三聚氰胺检 测试剂盒的标准曲线，每条标准曲线的r2值 均为0.999，罗默实验室推荐的r2值应不低 于0.985(常规试剂盒)和0.990(灵敏型试剂 盒)。通过标准曲线计算出50%抑制率的浓 度124.2ppb(常规试剂盒)和77.4ppb(灵敏 型试剂盒)；两个数值与试剂盒说明书中给 出的值相一致。

Figure 1. Melamine test kit standard curve. Melamine standards at 20, 100, and 500 ppb plotted as logit(B/B₀) vs. log (concentration). r² = 0.999. 50% inhibition = 124.2 ppb.

Figure 2. Melamine sensitive test kit standard curve. Melamine standards at 20, 100, and 1000 ppb plotted as logit(B/B₀) vs. log (concentration). r² = 0.999. 50% inhibition = 77.4 ppb..

According to data for test reproducibility presented in the Melamine Test Kit and Melamine Sensitive Test Kit certificates of performance, coefficients of variance (CVs)for standards are <9%. In this application note we demonstrate CVs of ≤5% for all standards. (See Table 1 and Table 2.).

Table 1. Melamine test kit. Average OD, standard deviation, average B/B₀, and CVs were calculated by SoftMax Pro Software. CVs ≤5% compare favorably to CVs <9% shown in the certificate of performance provided by Romer Labs for each test kit.

Table 2. Melamine sensitive test kit. Average OD, standard deviation, average B/B₀, and CVs were calculated by SoftMax Pro Software. CVs ≤5% compare favorably to CVs <9% shown in the certificate of performance provided by Romer Labs for each test kit.

结论：

罗默公司的三聚氰胺检测试剂盒和灵敏型三 聚 氰 胺 检 测 试 剂 盒 在 结 合 M o l e c u l a r Devices公司推出的SpectraMax, VersaMax, VMax, and EMax微孔板读板机，可 以针对各种奶制品（如鲜奶、奶粉、酸奶） 和面粉及各种宠物食品中三聚氰胺的含量获 得更加准确的检测结果。最后通过SoftMax Pro GxP软件获得全部数据的分析结果， 此款为工业标准分析软件并且符合FDA 21 CFR Part 11要求。

Molecular Devices公司的AquaMax® 4000 微孔板洗板机连接其StakMax堆板机可以 使得ELISA检测全自动化，增加检测通 量。用户也可以将StakMax ®堆板机与 Molecular Devices公司SpectraMax或 VersaMax微孔板读板机连接。减少手动操 作实现更高通量的检测。

References

1. AgraQuant Melamine Test Kit product insert and Certificate of Performance.
2. AgraQuant Melamine Sensitive Test Kit product insert and Certificate of Performance.

Learn more about VersaMax ELISA Microplate Reader >>

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