How are the labels conjugatedto antibodies in practice?
Two types of labeling methods are commonly used depending on what part of the antibody is labeled. The first is to label the amino groups (NH2 groups) of the antibody (the NH2 type), and the second is to label the thiol groups (SH groups) (the SH type). Each method has advantages and disadvantages. The NH2 type and SH type methods are suitable for low and high molecular weight labels, respectively.
Labels are linked to the amino groups (NH2 groups) of antibodies.
Among the four amino acids with side-chain amino groups (glutamine, lysine, arginine, asparagine), only lysine can be labeled. In addition, the N-terminal amino group can be labeled. Thus, there are many sites that can be labeled as shown in the diagram on the right.
The procedure is relatively simple as shown in the diagram below.
Antibodies are labeled by incubating with an active ester derivative of a fluorophore or enzyme. A number of active ester derivatives of fluorophores and enzymes are commercially available.
・Simple procedure. Commercial kits are available.
・The addition of low molecular weight labels, such as FITC and biotin, to multiple sites enhances the sensitivity.
・Excessive labeling can impair the activity of the antibody.
・High molecular weight labels, such as enzymes and PE, may cause steric hindrance and alter antibody activity.
The SH type method that labels amino acids with an SH group (cysteine) is known as the maleimide method. The labeling sites are limited, and most are clustered at the hinge region. There are few cysteines in the variable region (antigen-binding site).
Antibodies are reduced by 2-mercaptoethanol (2-ME), which splits the molecule at the hinge region. The SH groups of the reduced antibodies are reacted with a maleimide-activated fluorophore or enzyme.
・High molecular weight labels, such as enzymes and PE, can be added without causing steric hindrance at the antigen-recognition site.
・Multiple fluorophores and enzymes cannot be conjugated
Primary antibodies are pre-labeled with a fluorescent dye or an enzyme. Processing time can be reduced by the use of direct-labeled primary antibodies.
Primary antibodies are detected using a secondary antibody labeled with a fluorophore or an enzyme.
Primary antibodies are pre-labeled with biotin and detected by labeled avidin (the biotin-avidin complex).
|Direct method||Indirect method|
|Commercial products||A limited number of direct-labeled primary antibodies are commercially available.
If labeled antibodies are not commercially available, you will need to label them yourself.
|Labeled secondary antibodies are readily available commercially. Only in rare cases, are they not commercially available.|
|Processing time, complexity||Processing time can be reduced because the reaction step with a secondary antibody is eliminated.
It could be more complicated if you need to label the antibodies.
|It requires the time and additional steps for the reaction with a secondary antibody.
A labeled secondary antibody can be used with various primary antibodies.
|Multiple staining||Multiple staining can be performed using antibodies derived from the same animal species.||Multiple staining requires primary antibodies derived from different animals species.|
|Non-specific reaction||Non-specific reaction of a secondary antibody is eliminated.||Non-specific reaction of a secondary antibody may occur.|
|Other issues||Direct labeling should be performed carefully to avoid inactivation of the antibody.||Signals are stronger if the secondary antibody is polyclonal.|
Alexa Fluor®488-labeled antibodies
Alexa Fluor®594-labeled antibodies
Alexa Fluor®647-labeled antibodies
Primary antibody-coated agarose