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查看更多产品信息 99-1000 - FAQs (99-1000)
7 个常见问题解答
不适用。一位客户在2014年初通知了我们货号99-1000未检测到食蟹猴的IgG亚类。就其他非人类灵长类动物而言，我们不知道该试剂盒是否适用于这些动物的样品。
在人类IgG亚类ELISA试剂盒（货号99-1000）中，人类IgG亚类标准品是部件号50287HK。每批50287HK根据名为67/97的WHO参考标准进行校准。关于该标准的更多细节见Klein F等人（1985）Clin Chem Acta 150（2）：119-127的摘要。
在人类IgG亚类ELISA试剂盒（货号99-1000）中，所提供的8联管的所有孔都包被山羊多克隆抗FITC抗体，用作一般捕获试剂。当您将单独的FITC标记的亚类特异性单克隆抗体添加到孔中时（手册（https://tools.thermofisher.com/content/sfs/manuals/PI99-1000_Human%20IgG%20ELISA%20Kit%20Rev%201208.pdf）第2页的检测程序中的步骤2），它们会与山羊捕获抗体结合。ELISA夹心的第二层会执行亚类特异性捕获功能。当您向孔中添加样品（即血清、标准品和对照品）时，样品中存在的任何人类IgG将与借由标记的FITC被捕获至板子上的亚类特异性抗体结合。通过试剂盒中提供的这些亚类特异性的小鼠抗人类IgG抗体的单克隆抗体实现亚类特异性检测。在将HRP标记的抗人类IgG抗体和TMB显色剂添加到孔中后，会发生真正的亚类检测。每个IgG亚类的量在其自己的孔中单独测定。
这些亚类特异性组的一个示例见手册（https://tools.thermofisher.com/content/sfs/manuals/PI99-1000_Human%20IgG%20ELISA%20Kit%20Rev%201208.pdf）第2页的检测程序的步骤1。在这个例子中，所示的设置是针对IgG1的，但假如说您只想测量IgG4，您也可以遵循相同的设置。不同的是，不要将小鼠抗人类IgG1抗体上样到这些孔，而是使用小鼠抗人类IgG4抗体代替。如果您只想检测IgG4，则板中的其余孔可用于其他样品，而不是用于其他亚类的标准曲线和各自的样品。但是，我们建议您每次进行检测时，在您准备的每个微孔板上运行目标IgG亚类的标准曲线。
试剂盒中的对照品由冻干的人血清组成，并且所提供的人类IgG标准品包含所有四个亚类，其浓度在批次特定的手册中显示。即使您可能只想测量1个或2个亚类，您也将使用包含所有亚类的标准品。如果您只检测1个亚类如 IgG3，其他3个亚类也不会干扰IgG3的检测，因为捕获抗体具有IgG3特异性。试剂盒中的检测抗体是抗人类IgG的HRP偶联物，其同样有效地检测所有亚类并检测孔中捕获的所有人类IgG。
请注意，试剂盒中的抗体包被板是可以分拆为8孔联管使用的。您不必一次运行整个微孔板或两块板。将任何未使用的8孔联管储存于2-8°C并使其保持干燥。应牢固密封单个联管中未使用的孔，以防止水分在检测运行时进入。
To estimate the contribution of the non-specific interaction and Fc receptor binding, staining protocols using an antibody directed to an irrelevant antigen (for example, DNP) having the same isotype as the antibody of interest may be analyzed in parallel with the antibody of interest. The antibody directed to the irrelevant antigen is known as the isotype control.
Antibodies are useful for identifying and localizing proteins and other antigens both on the surface of cells as wells as inside cells. The utility of antibodies lies in their specificity and avidity for their antigens, an interaction which is mediated by the antigen binding sites. Antibodies can also react with components other than their antigens in several ways. They can associate with cellular components by non-specific protein-protein interactions. They can associate with fatty components by hydrophobic interaction. Antibodies can also bind to antibody receptors expressed on the surface of some cell types. For example, antibodies of the IgG class can bind to the cell surface Fc receptors known as CD16, CD32, and CD64. Under ideal conditions, various types of non-specific binding will be prevented by including blocking proteins (BSA, milk, or animal serum products) in antibody incubation reaction mixtures. Antibody binding to Fc receptors can be prevented by preincubation of cells with serum preparations containing immunoglobulins.
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No. A customer notified us early in 2014 that Cat. No. 99-1000 did not detect IgG subclasses from cynomolgus monkeys. As far as other nonhuman primates are concerned, we do not know if this kit will work with samples from these animals.
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In the IgG Subclass Human ELISA Kit (Cat. No. 99-1000), the human IgG Subclass Standard is Part Number 50287HK. Each batch of 50287HK is calibrated against a WHO reference standard designated 67/97. More details about this standard are in the abstract from Klein F et al. (1985) Clin Chem Acta 150 (2):119-127.
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In the IgG Subclass Human ELISA Kit (Cat. No. 99-1000), all of the wells of the 8-well strips provided are coated with a goat polyclonal anti-FITC antibody, which serves as a general capture reagent. When you add the individual FITC-labeled, subclass-specific monoclonals to the wells (step 2 in the assay procedure on page 2 of the manual - https://tools.thermofisher.com/content/sfs/manuals/PI99-1000_Human%20IgG%20ELISA%20Kit%20Rev%201208.pdf), they bind to the goat capture antibody. This second layer of the ELISA sandwich now performs the subclass-specific capture function. When you add samples (i.e., serum, standards, and controls) to the wells, any human IgG present in the samples will bind to the subclass-specific antibodies that are captured on the plate via their FITC labels. The subclass-specific detection is enabled by each of these subclass-specific mouse monoclonal anti-human IgG antibodies provided in the kit. The actual subclass detection occurs after you add the HRP-labeled anti-human IgG antibody and the TMB chromogen solution to the wells. The amount of each IgG subclass is determined separately in its own set of wells.
An example of one of these subclass-specific sets is shown under step 1 in the assay procedure on page 2 of the manual - https://tools.thermofisher.com/content/sfs/manuals/PI99-1000_Human%20IgG%20ELISA%20Kit%20Rev%201208.pdf. In this case, the setup shown is for IgG1, but if you wanted to measure only IgG4, for example, you would follow the same setup. However, instead of loading these wells with mouse anti-human IgG1, you would use the mouse anti-human IgG4 instead. If all you want to detect is IgG4, the rest of the wells in the plate can be used for other samples instead of additional standard curves for the other subclasses and their respective samples. However, we suggest running a standard curve for the IgG subclass of interest on each plate that you prepare, and each time you run the assay.
The control in the kit consists of lyophilized human serum, and the human IgG standard provided contains all four subclasses at the concentrations indicated on the lot-specific manual. Even though you may want to measure only 1 or 2 subclasses, you'll be using a standard that contains all of them. The other 3 subclasses don't interfere with detection of IgG3, for example, because the capture antibody is IgG3-specific. The detection antibody in the kit is an HRP conjugate of an anti-human IgG that detects all of the subclasses equally effectively and detects all of the human IgG captured in the wells.
Note that the antibody-coated plates in the kit come as 8-well strips that you snap into the frame provided. You do not have to run an entire plate or both plates at one time. Store any unused 8-well strips at 2-8 degrees C and keep them dry. Unused wells in individual strips should be sealed securely to prevent the entry of moisture while running the assay.
Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.