最適合的二抗的選擇方法

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.  

二抗是一個(gè)強(qiáng)大的研究工具。 高質(zhì)量的二抗使您能夠在各種檢測(cè)技術(shù)中以更高的準(zhǔn)確度定位，鑒定和純化蛋白質(zhì)。 我們可以提供非偶聯(lián)或者偶聯(lián)酶和熒光團(tuán)的二抗，以幫助使用Western Blots，免疫組織化學(xué)，免疫熒光和ELISA等技術(shù)進(jìn)行檢測(cè)。

Advantages of using secondary antibodies   使用二抗的優(yōu)勢(shì)

1. Enhanced sensitivity提高靈敏度

2. Increased accuracy提高準(zhǔn)確度

3. Greater flexibility of detection wavelength檢測(cè)波長(zhǎng)的靈活性更大

4. Signal amplification信號(hào)放大

5. No need to label your primary antibody無(wú)需標(biāo)記一抗

What to consider 二抗選擇需考慮以下5個(gè)因素：

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

全長(zhǎng)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））作為第二抗體。它們將與免疫球蛋白的重鏈和輕鏈（IgG，IgM，IgA等）結(jié)合，并產(chǎn)生最高信號(hào)。但有時(shí)候使用片段效果更好，單價(jià)Fab片段可以有效地用于阻斷組織中的內(nèi)源性IgG并通過(guò)阻斷所有結(jié)合位點(diǎn)來(lái)“改變”一抗的種類(lèi)，僅允許二級(jí)結(jié)合單價(jià)Fab片段的單一結(jié)合位點(diǎn)。 F（ab'）2片段已被切割以除去IgG分子的Fc結(jié)構(gòu)域。當(dāng)Fc受體在細(xì)胞表面例如B細(xì)胞上存在問(wèn)題時(shí)，這些是特別有用的。 片段二抗最常用于流式細(xì)胞術(shù)，以確保二抗不會(huì)與細(xì)胞表面結(jié)合。 

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

一抗的物種來(lái)源（兔，雞，羊，鼠，馬等）

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.

宿主物種是指抗體產(chǎn)生的動(dòng)物物種，您的二抗選擇應(yīng)根據(jù)所使用一抗的宿主物種。 例如，如果您的一抗是鼠抗，則需要選擇一個(gè)抗鼠的二抗。而產(chǎn)生二抗的物種沒(méi)有要求，除非您需要進(jìn)行多重標(biāo)記實(shí)驗(yàn)，這就需要您的二抗物種來(lái)源都一致。

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

一抗的類(lèi)別和亞型（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.

二抗選擇需要靶向您的一抗類(lèi)別和亞類(lèi)別，初級(jí)單克隆抗體通常在小鼠或兔子中產(chǎn)生并具有特定的亞類(lèi)。 因此您的第二抗體可以特異性針對(duì)您的單克隆初級(jí)抗體亞類(lèi)，如IgG1。當(dāng)您在查看多個(gè)標(biāo)記實(shí)驗(yàn)時(shí)，擁有一個(gè)特異性針對(duì)亞型類(lèi)的二抗，可以在多重標(biāo)記實(shí)驗(yàn)中特異識(shí)別在同宿主，不同亞型的一抗，克服交叉反應(yīng)的問(wèn)題，如抗-IgG1將不結(jié)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.

多克隆抗體可能屬于多個(gè)亞類(lèi)，我們的一級(jí)多克隆抗體通常是在兔子中產(chǎn)生的IgG（γ鏈球蛋白）同種型。 如果選用我們的一抗，則第二抗體需要是抗IgG，能夠識(shí)別重鏈和輕鏈，抗IgG H＆L。

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

您實(shí)驗(yàn)中其他來(lái)源的免疫球蛋白（封閉緩沖液，內(nèi)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.

消除與免疫球蛋白的交叉反應(yīng)性，增加特異性并減少了背景信號(hào)是很重要的。一些二級(jí)抗體被交叉吸附，則它們不能識(shí)別實(shí)驗(yàn)系統(tǒng)中的其他IgG來(lái)源并對(duì)其起反應(yīng)。這些抗體已被另外純化以除去結(jié)合特定物種的任何免疫球蛋白。這可以獲得不會(huì)與某些特定物種交叉反應(yīng)的高度特異性抗體。

例如：

Rabbit anti-Mouse min x Rat

與大鼠IgG結(jié)合的所有IgG分子已被去除，只留下特異性結(jié)合小鼠的那些，但不結(jié)合大鼠。

5. The kind of label/conjugate needed

所需的標(biāo)記物/偶聯(lián)物的種類(lèi)

Biorbyt's Range of Conjugates  Biorbyt抗體的偶聯(lián)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.

標(biāo)簽使您能夠可視化您的目標(biāo)蛋白，您所使用的檢測(cè)方法和應(yīng)用程序決定了您可以使用的偶聯(lián)標(biāo)記物。 Biorbyt可以提供偶聯(lián)和未偶聯(lián)的二抗，我們的標(biāo)記物系列覆蓋了大多數(shù)熒光團(tuán)波長(zhǎng)和酶，并提供抗不同物種的二抗。

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.

偶聯(lián)物是可以添加到二抗中以輔助檢測(cè)的分子。您的選擇取決于檢測(cè)技術(shù)，如在Flow或免疫熒光/熒光中儀器激光的可用性，和實(shí)驗(yàn)系統(tǒng)的其他標(biāo)記物。免疫熒光和流式細(xì)胞術(shù)需要熒光標(biāo)記的抗體，例如偶聯(lián) Alexa Fluor，F(xiàn)ITC，Qdot。ELISA和蛋白質(zhì)印跡適用于化學(xué)發(fā)光，比色和熒光系統(tǒng)，其中酶連接的二級(jí)體是一個(gè)很好的選擇，例如辣根過(guò)氧化物酶（HRP），堿性磷酸酶。