如何选择二抗
羊抗兔：指把兔体内的抗原注射到羊的体内，而在羊的体内产生抗兔的抗体，即羊抗兔抗体，这个抗体来由羊产生的，是羊源的。
一抗如果是鼠抗人的抗体，二抗应该是另一种动物抗鼠的抗体，而且一抗和二抗的动物种属原则上不应该相同。一般来说，一抗多用兔抗和鼠抗，而二抗多用羊抗。二抗一般选择被碱性磷酸酶标记过的。
单克隆抗体是指针对于抗原标志物上某一种抗原决定簇产生的单一种类的抗体，一般通过杂交瘤细胞技术制得；
多克隆抗体是指针对抗原上多个抗原决定簇产生的抗体，一般将抗原免疫动物血清直接制得。对于一抗来说，现用单克隆抗体较多。
相对于一抗的特异性，二抗要求广谱抗体，最好能够一对多。现在购买的二抗多是二抗加亲和素生物素的复合物，与传统二抗有所不同。
In most cases, there are actually several antibody-conjugates that would work well in a particular application and finding the best one requires comparing them side-by-side.The following information is provided to help you decide which secondary antibody may be best for your particular application:
1. In what animal was the primary antibody developed?
For example if your primary antibody is raised in a mouse, you will need an anti-mouse secondary antibody. If it is raised in a rabbit, for example A2066 (rabbit anti-actin), you will need an anti-rabbit secondary antibody.
For example if your primary antibody is raised in a mouse, you will need an anti-mouse secondary antibody. If it is raised in a rabbit, for example A2066 (rabbit anti-actin), you will need an anti-rabbit secondary antibody.
2. What is the class and/or subclass of the primary antibody.
This is primarily important in the case of monoclonal antibodies. Polyclonal antibodies (generally developed in rabbit, goat, sheep or donkey) are typically IgG class immunoglobulins. For this reason, the secondary antibodies for these species will mainly be anti-IgG. Monoclonal antibodies (MAbs) are most commonly developed in mice and occasionally in rats. The class and subclass of our monoclonals is indicated in the product l
This is primarily important in the case of monoclonal antibodies. Polyclonal antibodies (generally developed in rabbit, goat, sheep or donkey) are typically IgG class immunoglobulins. For this reason, the secondary antibodies for these species will mainly be anti-IgG. Monoclonal antibodies (MAbs) are most commonly developed in mice and occasionally in rats. The class and subclass of our monoclonals is indicated in the product l
isting. For example, if the primary Mab is mouse IgM, one would want a secondary antibody that reacts with mouse IgM (anti-Mouse IgM or anti-Mouse IgG (Fab)).react to an emergency
If the primary monoclonal is one of the mouse IgG subclasses (IgG1, IgG2a, IgG2b, IgG3), almost any of our anti-mouse IgG secondary antibodies should bind to it. However, we offer different specificities to provide the end-user with options, especially for specific double labeling experiments. If the class and/or subclass of the primary antibody is not known, the anti-Mouse IgG (Fab) secondary antibodies may be used since they recognize most mouse immunoglobulin subtypes.
You may experience difficulty when choosing a the best secondary anti-mouse or anti-human IgG. This is because there are many specificities to IgG. The important thing to remember is that light chains (kappa and lambda) of immunoglobulins are shared among all the immunoglobulin classes. That is, IgG, IgM, IgA, IgD and IgE all have either kappa or lambda light chains. The heavy chain (lambda for IgG, µ for IgM and alpha for IgA), however, is class specific. The following summarizes the specificities we offer:
If the primary monoclonal is one of the mouse IgG subclasses (IgG1, IgG2a, IgG2b, IgG3), almost any of our anti-mouse IgG secondary antibodies should bind to it. However, we offer different specificities to provide the end-user with options, especially for specific double labeling experiments. If the class and/or subclass of the primary antibody is not known, the anti-Mouse IgG (Fab) secondary antibodies may be used since they recognize most mouse immunoglobulin subtypes.
You may experience difficulty when choosing a the best secondary anti-mouse or anti-human IgG. This is because there are many specificities to IgG. The important thing to remember is that light chains (kappa and lambda) of immunoglobulins are shared among all the immunoglobulin classes. That is, IgG, IgM, IgA, IgD and IgE all have either kappa or lambda light chains. The heavy chain (lambda for IgG, µ for IgM and alpha for IgA), however, is class specific. The following summarizes the specificities we offer:
o Polyvalent: react with all classes
o Fc and heavy-chain specific: react with heavy chains only, therefore, they are class specific (i.e. gamma chain specific reacts only with IgG; µ chain specific reacts only with IgM, etc.)
o Fab and whole molecule (wm) specific: react with heavy and light chains. Due to the light chain reactivity, they can react with all classes.
o Light chain (kappa, lambda) specific: react with all classes, since all classes use the same kappa or lambda light chains.
1. What form of antibody should be used?
Most people prefer affinity isolated antibodies. These products are the most purified, and therefore, give the lowest amount of non-specific binding. However, in certain cases, IgG fractions may be considered. IgG fractions may have the benefit of containing very high affinity antibodies. Affinity isolation may remove some very high affinity antibodies because they bind so tightly to the affinity matrix that they are not eluted. Therefore, IgG fractions may be useful in situations where very high affinity is required, most commonly when the a
Most people prefer affinity isolated antibodies. These products are the most purified, and therefore, give the lowest amount of non-specific binding. However, in certain cases, IgG fractions may be considered. IgG fractions may have the benefit of containing very high affinity antibodies. Affinity isolation may remove some very high affinity antibodies because they bind so tightly to the affinity matrix that they are not eluted. Therefore, IgG fractions may be useful in situations where very high affinity is required, most commonly when the a
ntigen of interest is rare or present in low abundance.
2. What kind of label?
The label is very application dependent. For immunoblotting and ELISA, enzyme-labeled secondary antibodies are the most popular. Peroxidase is economical, rapid and a more stable enzyme, in general, than alkaline phosphatase. Also, It has become very popular for use in chemiluminescent detection systems. Alkaline phosphatase on the other hand is considered more sensitive than peroxidase particularly when colorimetric detection is used. For cell or tissue staining, alkaline phosphatase, peroxidase or fluorescent secondary antibodies may be used for cytochemistry or histochemistry. Secondary antibodies labeled with a fluorochrome (FITC, phycoerythrin, and Quantum Red) may be used for flow cytometry.
To generate increased amplification, a two-step biotin/avidin system may be used. Biotin binds with very high affinity to avidin resulting in an essentially irreversible interaction. A biotinylated secondary antibody is applied first, then avidin, ExtrAvidin. or streptavidin conjugated to an enzyme or fluorochrome binds to multiple sites on the biotinylated second
The label is very application dependent. For immunoblotting and ELISA, enzyme-labeled secondary antibodies are the most popular. Peroxidase is economical, rapid and a more stable enzyme, in general, than alkaline phosphatase. Also, It has become very popular for use in chemiluminescent detection systems. Alkaline phosphatase on the other hand is considered more sensitive than peroxidase particularly when colorimetric detection is used. For cell or tissue staining, alkaline phosphatase, peroxidase or fluorescent secondary antibodies may be used for cytochemistry or histochemistry. Secondary antibodies labeled with a fluorochrome (FITC, phycoerythrin, and Quantum Red) may be used for flow cytometry.
To generate increased amplification, a two-step biotin/avidin system may be used. Biotin binds with very high affinity to avidin resulting in an essentially irreversible interaction. A biotinylated secondary antibody is applied first, then avidin, ExtrAvidin. or streptavidin conjugated to an enzyme or fluorochrome binds to multiple sites on the biotinylated second
ary antibody, thus amplifying the signal and resulting in greater sensitivity than that achieved with an antibody-enzyme or antibody-fluorochrome conjugate alone.
3. Is there an appropriate adsorbed secondary antibody available?
If working with human tissues, cells or proteins, choose a secondary antibody that has been adsorbed with human serum or human IgG. If working with bovine tissues, cells or proteins, choose a product that has been adsorbed with bovine serum or bovine IgG, etc. Using these products will reduce non-specific background
If working with human tissues, cells or proteins, choose a secondary antibody that has been adsorbed with human serum or human IgG. If working with bovine tissues, cells or proteins, choose a product that has been adsorbed with bovine serum or bovine IgG, etc. Using these products will reduce non-specific background
4. Is a F(ab )2 fragment product necessary?
If working with tissues or cells that have Fc receptors (thymus, spleen, blood, leukocytes, B cells etc.), choose an F(ab )2 fragment when possible to eliminate non-specific binding through Fc receptors present on cells. Alternatively, Fc receptors can be blocked. Perform an absorption step, using purified IgG from the host species of the your secondary antibody. In that case an F(ab)2 secondary antibody may not be necessary.
If working with tissues or cells that have Fc receptors (thymus, spleen, blood, leukocytes, B cells etc.), choose an F(ab )2 fragment when possible to eliminate non-specific binding through Fc receptors present on cells. Alternatively, Fc receptors can be blocked. Perform an absorption step, using purified IgG from the host species of the your secondary antibody. In that case an F(ab)2 secondary antibody may not be necessary.
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