二抗的选择

最适合的二抗的选择方法

Secondary antibodies are a powerful research tool. High quality secondaries enable you to target, identify and purify proteins with greater accuracy across a range of detection techniques. Our secondaries can be supplied unconjugated or conjugated to enzymes and fluorophores to aid detection using techniques such as Western blots, Immunohistochemistry, immunofluorescence and ELISA.  

二抗是一个强大的研究工具。 高质量的二抗使您能够在各种检测技术中以更高的准确度定位，鉴定和纯化蛋白质。 我们可以提供非偶联或者偶联酶和荧光团的二抗，以帮助使用Western Blots，免疫组织化学，免疫荧光和ELISA等技术进行检测。

Advantages of using secondary antibodies   使用二抗的优势

1. Enhanced sensitivity提高灵敏度

2. Increased accuracy提高准确度

3. Greater flexibility of detection wavelength检测波长的灵活性更大

4. Signal amplification信号放大

5. No need to label your primary antibody无需标记一抗

What to consider 二抗选择需考虑以下5个因素：

1.Whole IgG or Fragment (F(ab')2, Fab, Ig)? 

全长IgG或者片段(F(ab')2, Fab, Ig)？

In general, a whole Immunoglobulin (e.g. IgG (H+L)) can be used as a secondary antibody. These will bind to both heavy and light chains of the Immunoglobulin (IgG, IgM, IgA etc) and will give the highest signal. Sometimes, it is preferable to use a fragment. Monovalent Fab fragments can effectively be used to block endogenous IgG in tissues and to 'change' the species of a primary antibody by blocking all binding sites, only allowing a secondary to bind to the single binding site of the monovalent Fab fragment. F(ab')2 fragments have been cleaved to remove the Fc domain of the IgG molecule. These are of particular use when Fc receptors are a problem on cell surfaces such as B cells. They are most commonly used in flow cytometry to ensure the secondary does not bind to the cell surface. 

通常，可以使用完整的免疫球蛋白（例如IgG（H + L））作为第二抗体。它们将与免疫球蛋白的重链和轻链（IgG，IgM，IgA等）结合，并产生最高信号。但有时候使用片段效果更好，单价Fab片段可以有效地用于阻断组织中的内源性IgG并通过阻断所有结合位点来“改变”一抗的种类，仅允许二级结合单价Fab片段的单一结合位点。 F（ab'）2片段已被切割以除去IgG分子的Fc结构域。当Fc受体在细胞表面例如B细胞上存在问题时，这些是特别有用的。 片段二抗最常用于流式细胞术，以确保二抗不会与细胞表面结合。 

2. The host species of your Primary (rabbit, chicken, goat, mouse, horse etc.) 

一抗的物种来源（兔，鸡，羊，鼠，马等）

The host species refers to the species of animal in which the antibody was raised. Your secondary antibody should be raised against the host species of your primary. For example, if your primary is raised in mouse, you need to select an anti-mouse secondary. It does not matter what species your secondary is raised in unless you are doing a multiple labelling experiment, where all secondaries should be raised in the same host species.

宿主物种是指抗体产生的动物物种，您的二抗选择应根据所使用一抗的宿主物种。 例如，如果您的一抗是鼠抗，则需要选择一个抗鼠的二抗。而产生二抗的物种没有要求，除非您需要进行多重标记实验，这就需要您的二抗物种来源都一致。

3.The class and subclass of the primary antibody (IgG/IgM, IgG1, IgG2a)

一抗的类别和亚型（IgG/IgM, IgG1, IgG2a）

Choose a secondary antibody that targets your primary antibody class and subclass. Primary monoclonal antibodies are usually generated in Mouse or Rabbit and have a specific subclass. Your secondary antibody can therefore be directed specifically against the subclass of your monoclonal primary e.g. IgG1. Having a subclass specific secondary can overcome problems of two primaries being raised in the same host when you're looking at multiple labelling experiments. An anti-IgG1 will not bind to an anti-IgG2a.

二抗选择需要靶向您的一抗类别和亚类别，初级单克隆抗体通常在小鼠或兔子中产生并具有特定的亚类。 因此您的第二抗体可以特异性针对您的单克隆初级抗体亚类，如IgG1。当您在查看多个标记实验时，拥有一个特异性针对亚型类的二抗，可以在多重标记实验中特异识别在同宿主，不同亚型的一抗，克服交叉反应的问题，如抗-IgG1将不结合抗-IgG2a。

 Polyclonal antibodies may belong to more than one subclass. Our primary polyclonal antibodies are usually IgG (gamma chain globulin) isotype generated in Rabbit. Your secondary antibody would need to be anti-IgG and be able to recognise heavy and light chains, anti IgG H&L.

多克隆抗体可能属于多个亚类，我们的一级多克隆抗体通常是在兔子中产生的IgG（γ链球蛋白）同种型。 如果选用我们的一抗，则第二抗体需要是抗IgG，能够识别重链和轻链，抗IgG H＆L。

4. Other sources of immunoglobulin in your assay (block buffer, endogenous IgG)

您实验中其他来源的免疫球蛋白（封闭缓冲液，内源性IgG）

This is important to eliminate unwanted cross-reactivity with immunoglobulins therefore increasing specificity and reducing background signal. Some secondary antibodies are cross adsorbed so they don't recognise and react to other sources of IgG in the experimental system. These antibodies have been additionally purified to remove any immunoglobulins that bind to a specific species. This allows the production of highly specific antibodies that will not cross-react to certain specified species.

Example:

Rabbit anti-Mouse min x Rat

All IgG molecules that bind to Rat IgG have been removed, leaving only those that specifically bind mouse, but DO NOT bind rat.

消除与免疫球蛋白的交叉反应性，增加特异性并减少了背景信号是很重要的。一些二级抗体被交叉吸附，则它们不能识别实验系统中的其他IgG来源并对其起反应。这些抗体已被另外纯化以除去结合特定物种的任何免疫球蛋白。这可以获得不会与某些特定物种交叉反应的高度特异性抗体。

例如：

Rabbit anti-Mouse min x Rat

与大鼠IgG结合的所有IgG分子已被去除，只留下特异性结合小鼠的那些，但不结合大鼠。

5. The kind of label/conjugate needed

所需的标记物/偶联物的种类

Biorbyt's Range of Conjugates  Biorbyt抗体的偶联系列

Labels enable you to visualise your target protein. The detection method and application you are using dictate the conjugate options available to you. Biorbyt can provide both conjugated and unconjugated secondary antibodies. Our range of conjugates covers most wavelength of fluorophore and enzymes in an excellent range of target species.

标签使您能够可视化您的目标蛋白，您所使用的检测方法和应用程序决定了您可以使用的偶联标记物。 Biorbyt可以提供偶联和未偶联的二抗，我们的标记物系列覆盖了大多数荧光团波长和酶，并提供抗不同物种的二抗。

Conjugates are molecules that can be added to the secondary antibody to aid detection. Your choice is dependent on detection technique, laser availability if using Flow or immunofluorescence / fluorescent IHC and other labels within the experimental system. Immunofluorescence and flow cytometry need antibodies conjugated to fluorescent reporter labels e.g. Alexa Fluor, FITC, Qdot. ELISA and Western Blot are suited to chemiluminescent, colorimetric and fluorescence systems where enzyme linked secondaries are a good choice, e.g. horseradish peroxidase (HRP), alkaline phosphatase. Our selection tool will guide you to the best choice.

偶联物是可以添加到二抗中以辅助检测的分子。您的选择取决于检测技术，如在Flow或免疫荧光/荧光中仪器激光的可用性，和实验系统的其他标记物。免疫荧光和流式细胞术需要荧光标记的抗体，例如偶联 Alexa Fluor，FITC，Qdot。ELISA和蛋白质印迹适用于化学发光，比色和荧光系统，其中酶连接的二级体是一个很好的选择，例如辣根过氧化物酶（HRP），碱性磷酸酶。