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查看更多产品信息 IL-1 alpha Mouse ELISA Kit - FAQs (EMIL1ALPHA)
8 个常见问题解答
所有ELISA试剂盒均以夹心ELISA形式提供，其中捕获抗体已经包被在96孔板上。典型的检测使用生物素化的检测抗体，接着使用链霉亲和素-HRP和HRP底物。大多数试剂盒以单个96孔板的形式提供，有些试剂盒提供2个或5个96孔板。试剂盒通常包含：
•包被捕获抗体的96孔联排板
•蛋白质标准品
•洗涤缓冲液，10x
•ELISA 缓冲液/稀释液，10x
•检测抗体（大多数试剂盒中，生物素化）
•HRP试剂（HRP偶联的二抗或链霉亲和素）（100x）
•底物（通常为TMB）
•终止液
•粘性封板膜
运行内部对照以及空白对照和标准曲线是监测任何ELISA试剂盒性能的绝佳方法。内部对照是用试剂盒测量的含有已知量的分析物的样品。内部对照的一个关键属性，以及它们与标准曲线样品的区别在于，它们会尽可能地复制您实际样品的组分。这一点很重要，因为您的样品可能含有影响分析物检测的成分，而这种影响方式与试剂盒中提供的标准稀释液的影响方式不同。换句话说，内部对照可以让您确定在标准稀释液（标准曲线）和样品基质（内部对照）中检测等量的分析物是否会给出相同的结果。同样重要的是要记住，内部对照可以让您对结果的准确度充满信心。内部对照有助于确保不同检测在每次运行时都能始终如一。
内部对照的来源之一是含有已知量的待测分析物的自然存在的样品（如血清或血浆）。这种阳性对照样品可以原样运行，或者也可以稀释至各种浓度，最好用自然存在的相同类型的阴性样品稀释。如果可以的话，最好运行至少2个内部对照。其中一个分析物的浓度通常在标准曲线的最低点和曲线的中点之间，而另一个的浓度在中点和最高点之间。有些研究人员还会运行一个浓度接近曲线中点的内部对照。
如果您无法获得自然存在的对照品，您可以自己制备。假设您要使用ELISA试剂盒（货号KHC0061）测量人血清样品中的白细胞介素-6（IL-6）。首先，您需要一些合并的正常人类血清作为内部对照的样品基质。该血清的IL-6含量最好是微乎其微（但是已知存在），或者低到无法测量。接下来，您应该使用试剂盒中提供的人类IL-6标准，将已知量的人类IL-6添加到该血清中以建立内部对照。按照产品手册中的说明制备标准曲线后，将一些剩余的浓缩IL-6标准品添加到血清基质中。如上所述，如果您的ELISA微孔板中有足够的孔，您可以制备具有高和低IL-6水平或高、中和低水平的对照。
即使您的样品类型不是血清并且您也不是测量IL-6，也可按照上述通用程序使用您的目标蛋白质和样品基质制备内部对照。 点击此处（https://tools.thermofisher.com/content/sfs/brochures/TR0058-Spike-and-Recovery.pdf）查找加标回收率和稀释度线性分析的有价值的资源。如果您需要更多帮助，请联系技术支持LifeScience-CNTS@thermofisher.com。
我们的ELISA试剂盒可分为多个不同的组（参见蛋白质分析手册（https://www.thermofisher.com/us/en/home/global/forms/protein-handbook-registration.html）第35页的表2），考虑的许多因素包括：靶标蛋白质分类、灵敏度、读出方法或检测靶标蛋白的特异性磷酸化状态的能力。
•标准的比色法ELISA试剂盒使用标准比色读出，具有出色的灵敏度和检测范围。典型灵敏度<10 pg / mL，标准曲线范围约为10-250 pg / mL，但有些试剂盒的范围更广。
•超灵敏ELISA试剂盒使用标准比色读出，但能够检测和分析低至0.5 pg/mL的蛋白质。基于0.5-20 pg/mL的测量范围，这些试剂盒特别适用于高度稀释的样品。
•Chemi ELISA试剂盒使用化学发光检测技术获得高灵敏度（<1 pg/mL），并且灵活性高，测量范围可达0.5至2000 pg / mL。
•磷酸化ELISA试剂盒能够特异性检测关键信号蛋白的磷酸化，且特异性极高，其通常用于补充免疫印迹的结果并提供量化数据。
我们提供完整的ELISA试剂盒、试剂组和抗体对。ELISA试剂盒是完整的即用型试剂盒，包含预包被的检测板、所有缓冲液、捕获和检测抗体。大多数试剂盒以单块板子的格式提供，但有些试剂盒提供2或5块板子。试剂组适用于需要核心试剂盒组件但更喜欢自己制备缓冲液并自己包被板子的研究人员。抗体对是配对的检测和捕获抗体，适用于需要处理大量样品的研究人员。
All ELISA kits are provided in the sandwich ELISA format with capture antibody already coated onto a 96 well plate. Typical detection uses a biotinylated detection antibody followed by Streptavidin-HRP and HRP substrate. Most kits are available as single 96-well plate kits, some are available as 2- and 5-plate kits. Kits typically contain:
96 Well Strip Plate coated with capture antibody
Standard protein
Wash buffer, 10x
ELISA buffer/Diluent, 10x
Detection antibody (in most kits, biotinylated)
HRP Reagent (either secondary antibody or streptavidin conjugated to HRP)(100x)
Substrate (usually TMB)
Stop solution
Adhesive plate covers
Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.
Running internal controls along with the blanks and the standard curve is an excellent way to monitor the performance of any ELISA kit. Internal controls are samples containing known amounts of the analyte being measured with the kit. A key attribute of internal controls, and what differentiates them from the samples of the standard curve, is that they duplicate the composition of your actual samples as closely as possible. This is important because your samples may contain components that affect detection of the analyte differently than does the Standard Diluent provided in the kit. In other words, internal controls allow you to determine if assaying the same amount of analyte in the Standard Diluent (the standard curve) and in the sample matrix (internal controls) gives the same results. It's also important to remember that internal controls give you confidence in the accuracy of your results. Internal controls help ensure that the assay is performing consistently from assay to assay, every time you run it.
One source of internal controls is naturally occurring samples (such as serum or plasma) containing known amounts of the analyte you want to measure. Such positive control samples can be run as-is, or they can be diluted to various known concentrations, preferably with a naturally occurring negative sample of the same type. It's a good idea to run at least 2 internal controls, if you can. One usually has an analyte concentration between the lowest point on the standard curve and the curve's midpoint, while the other has a concentration between the midpoint and the highest concentration. Some researchers also run an internal control that falls close to the midpoint of the curve.
If you don't have access to naturally occurring controls, you can prepare them yourself. Let's say that you will be measuring interleukin-6 (IL-6) in human serum samples with an ELISA kit, Cat. No. KHC0061. First you will need to obtain some pooled normal human sera, which will be used as the sample matrix for your internal controls. Preferably, the IL-6 content of this serum should be negligible (but known) or too low to measure. Next you should add known amounts of human IL-6 to this serum to create the internal controls, using the human IL-6 standard provided in the kit. After you prepare the standard curve as described in the product manual, add some of the leftover concentrated IL-6 standard to the serum matrix. As described above, you can prepare controls with high and low IL-6 levels, or high, medium, and low, if you have enough wells in your ELISA plate.
Even if your sample type is not serum and you're not measuring IL-6, follow the general procedure outlined above to prepare your internal controls with your protein and sample matrix of interest. A valuable resource describing Spike and Recovery and Linearity of Dilution assays can be found here (http://tools.thermofisher.com/content/sfs/brochures/TR0058-Spike-and-Recovery.pdf). If you need more help, please contact Technical Support at techsupport@thermofisher.com.
Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.
Our ELISA kits can be categorized into several different groups (see Table 2, Page 35 of the Protein Analysis Handbook - http://www.thermofisher.com/us/en/home/global/forms/protein-handbook-registration.html), based on a number of factors: target protein class, sensitivity, readout method, or ability to detect specific phosphorylation states of the target protein.
Standard colorimetric ELISA kits use a standard colorimetric readout and allow excellent sensitivity and detection range. Typical sensitivity is less than 10 pg/mL and standard curve range is around 10-250 pg/mL, although there are some kits with wider ranges.
Ultrasensitive ELISA kits use a standard colorimetric readout but enable detection and analysis of proteins to levels as low as 0.5 pg/mL. With measurement range of 0.5-20 pg/mL, these kits are especially useful with highly diluted samples.
Chemi ELISA kits use chemiluminescence detection for high sensitivity (less than 1 pg/mL) and are highly flexible with a wide measurement range from 0.5 to 2000 pg/mL.
Phospho ELISA kits enable the specific detection of phosphorylation of key signaling proteins with high specificity, and are often used to supplement western blot results and provide quantitative data.
Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.
We offer full ELISA kits, Reagent Sets and Antibody Pairs. ELISA kits are complete, ready-to-use kits with pre-coated plates, all buffers, capture, and detection antibodies included. Most kits are single plate format, but some are available in 2- or 5-plate formats. Reagent Sets are for researchers who need the core kit components but prefer to make their own buffers and coat their own plates. Antibody Pairs are matched pairs of detection and capture antibodies for researchers who need to process large numbers of samples.
Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.