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Introduction

Mycoplasma, the smallest and simplest of the prokaryotes, are common contaminants of cell cultures. Symptoms of mycoplasma contamination include a reduction in the rate of proliferation and changes in cellular responses, including gene expression. Because mycoplasma cannot be detected by simply examining cell cultures under a microscope, a sensitive and reliable assay is needed to determine whether contamination is present. Traditional mycoplasma detection methods involve time-consuming staining or PCR protocols, and the results can be difficult to interpret.

The MycoAlert^™ Assay and MycoAlert PLUS Assay from Lonza provide a rapid and convenient way to detect viable mycoplasma in cell cultures using a luminescence microplate reader. The assays take advantage of mycoplasmaspecific enzymes found in major mycoplasma species to screen for contamination. The MycoAlert PLUS Assay is the next-generation kit, which provides higher light output than the standard MycoAlert Kit, allowing for the use of a wide range of luminescence readers as well as testing of unused media, media supplements, or water. The setup procedure is the same for both kits.

In the first step of the assay, MycoAlert Reagent is added to cell culture supernatant, lysing viable mycoplasma and initiating a bioluminescent luciferase reaction (Figure 1) that generates light from background ATP. In the second step, MycoAlert Substrate is added and reacts with the released mycoplasmal enzymes to convert ADP to additional ATP. Bioluminescence is then detected on a plate reader to measure the level of ATP present in the sample before and after the addition of the MycoAlert substrate.

Figure 1: Bioluminescent reaction to detect ATP.

Readings taken on a luminescence plate reader before and after substrate addition are used to obtain a ratio that indicates whether mycoplasma is present. If no mycoplasmal enzymes are present, the second reading shows no increase over the first (ratio < 0.9 with the MycoAlert Assay and < 1.0 with MycoAlert PLUS), but if the sample is positive for mycoplasma, the second reading is higher than the first (ratio > 1.2). Figure 2 outlines the assay workflow.

Figure 2. MycoAlert assay workflow. The luminescence measurement for Reading B is divided by Reading A to obtain a ratio indicative of the presence (ratio > 1.2) or absence (ratio < 0.9 with the MycoAlert Assay and < 1.0 with MycoAlert PLUS) of mycoplasma.

In this application note, we demonstrate how Molecular Devices microplate readers with luminescence detection mode provide superior sensitivity and ease of use for reliable mycoplasma detection using MycoAlert assays. Both the original MycoAlert Assay Kit as well as MycoAlert PLUS, a newer version with increased sensitivity, were tested and shown to be compatible with Molecular Devices microplate readers.

Materials

• MycoAlert Mycoplasma Detection Kit (Lonza cat. #LT07-318)
• MycoAlert PLUS Mycoplasma Detection Kit (Lonza cat. #LT07-710)
• MycoAlert Assay Control Set (Lonza cat. #LT07-518)
• 96-well polystyrene solid white plates (Greiner cat. #655075)
• Microplate readers
  • SpectraMax ^® i3 Multi-Mode Microplate Reader
  • SpectraMax i3x Multi-Mode Microplate Reader
  • SpectraMax Paradigm ^® Multi-Mode Microplate Reader
  • SpectraMax M5 Multi-Mode Microplate Reader
  • FilterMax ^™ F5 Multi-Mode Microplate Reader
  • SpectraMax L Microplate Luminometer

Methods

Methods for MycoAlert and MycoAlert PLUS Assays are basically the same, except for the concentration range of assay control used, and are detailed below. Each kit was used on the same day that its reagents were reconstituted.

Dilution series of the MycoAlert Positive Assay Control for each assay format were prepared as indicated in Table 1. Dilutions were made in MycoAlert Assay Buffer. 100 µL of each sample was pipetted into a solid, white, flat-bottom 96-well plate. Samples were run in triplicate. MycoAlert Reagent was added to each well and incubated at room temperature for 5 minutes. The plate was then read on Molecular Devices microplate readers with luminescence detection mode (Read A). MycoAlert Substrate was then added to the wells, and the plate was incubated for 10 minutes at room temperature. The plate was then re-read on the plate reader (Read B). Ratios for each sample were calculated as Read B/Read A. Both reads were performed with the instrument settings shown in Table 2. Data were generated and analyzed in SoftMax^® Pro Software. Final results were displayed using the bar graph feature available in SoftMax Pro 6.5 or higher.

Table 1. Preparation of standard dilution series for MycoAlert and MycoAlert PLUS Assays. MycoAlert Positive Assay Control was diluted into MycoAlert Assay Buffer at the ratios indicated.

Table 2. General instrument settings for the MycoAlert assays. *The SpectraMax Paradigm reader uses a Luminescence Detection Cartridge to detect signal for this assay. All other readers tested use an integrated luminescence detection mode.

Results

Sensitivity criteria for luminescence readers have been defined by Lonza for each kit. For the MycoAlert Kit, assay control samples should have a ratio of > 1.2 for dilutions of at least 1:8. For the MycoAlert PLUS Kit, a ratio > 1.2 should be produced by dilutions of at least 1:1000.

All Molecular Devices microplate readers with luminescence detection mode exceeded the sensitivity requirements as specified by Lonza for both MycoAlert and MycoAlert PLUS Kits. Representative data from the SpectraMax i3x Multi-Mode Microplate Reader for both MycoAlert and MycoAlert PLUS assays are shown in Figure 3. Table 3 shows the ratios obtained on all Molecular Devices plate readers tested for the minimum required assay control dilutions for each assay.

Figure 3: Sensitivity of the SpectraMax i3x reader in detecting increasing dilutions of the MycoAlert Assay Control. Negative samples contained only MycoAlert Assay Buffer. For each kit, the reader was able to detect the lowest dilution as positive. Asterisks indicate the dilution of assay control that should be minimally detectable for each kit. Left: MycoAlert Assay Kit; Right: MycoAlert PLUS Kit.

Table 3. Ratios obtained for assay control at minimum required dilutions. All ratios exceeded the required threshold of 1.2.

Conclusion

The results presented here confirm that all Molecular Devices microplate readers with luminescence detection mode exceed the sensitivity requirements set by Lonza for detecting mycoplasma with the MycoAlert and MycoAlert PLUS Assays. With both kits, minimal increases in assay signal indicative of mycoplasma contamination can easily be detected.

Molecular Devices microplate readers, in combination with MycoAlert Assays, enable sensitive and rapid detection of mycoplasma, ensuring that contamination is readily detected and saving valuable time in the effort to monitor cell culture contamination.

Learn more about SpectraMax i3x Multi-Mode Detection Platform

简介

支原体是目前已知最小的原核生物，是一种常见于哺乳动物细胞培养中污染源。支原体污染让细胞培养人员付出很大代价，因为它们改变了受污染细胞的形态、活力和代谢特征。通过显微镜很难在培养基中发现支原体。目前针对这种情况的污染需要一种灵敏可靠的检测方法，传统的支原体污染物检测方法如耗时标记法和繁琐的PCR等，检测结果也很难得到准确分析。

MycoAlert支原体检测试剂盒和MycoAert PLUS检测试剂盒均来自Lonza Walkersville 有限公司，此试剂基于生化反应配合具有 发光功能微孔板读板机来检测某种支原体 酶活力。这些酶的存在提供了一种快速筛 选过程，可以非常灵敏的对测试样品进行 支原体污染检测。MycoAert PLUS作为下 一代的检测试剂盒，较上一代标准版试剂 盒具有更高光量子输出效率，化学发光读 板机获得更宽的动态学范围，无论是培养 基、细胞培养基上清、水均能进行相应检 测。两种试剂盒的程序步骤一致。

生物化学发光反应检测ATP含量

第一步首先将MycoAlert试剂加入细胞培 养上清液中，裂解支原体触发化学发光反 应，如图一所示；第二步，有活力的支原 体被裂解并释放出能与MycoAlert底物反 应的酶，催化ADP 转化为ATP，通过检测 样品中加入MycoAlert底物前后ATP的水 平，可以得到其比值并指明支原体是否存 在。如果这些酶不存在，那么第二次的读 数将不会增加。(MycoAlert的Ratio＜0.9 和MycoAert PLUS的Ratio＜1.0)，如果样 品阳性的话，其第二次检测值高于第一 次，Ratio＞1.2，如图二所示为检测流 程。

图二：MycoAlert试剂盒检测流程， ，化学发光信号检测流程A次读值和B次读值，其 中B比上A的比率值，（Ratio＞1.2）代表有污染，或者（MycoAlert的Ratio＜0.9和 MycoAlert PLUS的Ratio＜1.0）代表无污染。

这篇应用文章介绍了如何利用Molecular Devices公司的具有化学发光的微孔板读 板机进行相应检测，仪器和试剂盒组合简 单易操作，进行相应检测时表现出很高的 灵敏度。两种类型的检测试剂盒均可在 Molecular Devices公司推出的具有化学发 光的微孔板读板机进行相应的检测实验， 获得较优的结果。

材料：

• MycoAlert Mycoplasma Detection Kit (Lonza cat. #LT07-318)
• MycoAlert PLUS Mycoplasma Detection Kit (Lonza cat. #LT07-710)
• MycoAlert Assay Control Set (Lonza cat. #LT07-518)
• 96-well polystyrene solid white plates (Greiner cat. #655075)
• 微孔板读板机
  • SpectraMax i3 多功能微孔板读板机
  • SpectraMax i3x 多功能微孔板读板机
  • SpectraMax Paradigm 多功能微孔板读板机
  • SpectraMax M5 多功能微孔板读板机
  • FilterMax F5 多功能微孔板读板机
  • SpectraMax L 微孔板读板机

方法

MycoAlert和MycoAlert PLUS试剂盒基本 原理一致，但是其两种试剂盒检测浓度动 态学范围不同，详细信息如下，每种试剂 盒在使用当天进行试剂配置。

MycoAlert阳性质控样品稀释关系如表一 所示，100 μL系列稀释的MycoAlert阳性 质控样品加于白色96-孔发光多孔板中做3 个重复。100 μL MycoAlert试剂(溶于 MycoAlert缓冲液中)加于每个孔中并在室 温下孵育5分钟。孔板置于发光型微孔板 读板机中，使用SoftMax® Pro 软件(读数A) 的读取所选孔的读数。100 μL的MycoAlert 底物(溶于MycoAlert缓冲液)立即加入到每 个孔中并在室温下孵育10分钟。孔板置于 发光型微孔板读板机中进行检测，所选孔 的读数(读数 B)。仪器设置见表二。p>

表一：准备MycoAlert和MycoAlert PLUS试剂标准品进行梯度稀释， 阳性质控试剂梯度稀释与MycoAlert缓冲液中。

表二：MycoAlert检测时仪器设置方式。

结果

我们利用Lonzede公司的MycoAlert试剂 盒针对所有发光型微孔板读板机进行灵敏 度评价，针对于MycoAlert试剂盒质控样 品在1:8稀释倍数关系时获得Ratio＞1.2， 而针对于MycoAlert PLUS试剂盒质控样品 在1:1000稀释倍数关系时可获得Ratio＞ 1.2。

所有的Molecular Devices公司推出的发光 功能的微孔板读板机均能获得超过预期的 灵敏度，我们列举了利用SpectraMax i3x 多功能微孔板读板机和两种试剂盒检测数 据值，如图三所示；而表三显示所有 Molecular Devices的微孔板读板机对阳性 样品稀释至最低比率关系也可检测到 Ratio＞1.2的信息情况。

图三：使用SpectraMax i3x 对阳性质控样品梯度稀释后获得的检测结果， 其中阴性样品仅包括 MycoAlert缓冲液，每种试剂盒，微孔板读板机均能检测阳性质控样品最低稀释度信号。 左侧图为MycoAlert试剂盒，右侧图为MycoAlert PLUS试剂盒。

表三： 不同读板机在阳性质控最低稀释关系可获得Ratio 1.2情况。

结论

结果显示所有Molecular Devices公司具有 发光功能的微孔板读板机均能够满足此试 剂盒的检测要求，并获得灵敏的检测结 果；Molecular Devices微孔板读板机结合 MycoAlert试剂盒，能够灵敏、迅速的检 测支原体含量，确保及时检测到污染物， 节省后期实验的宝贵时间。

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