Labels for detecting antibodies
Antibodies are labeled with various substances for detection.
When using a commercial kit to label antibodies, care must be taken not to lose the activity of the antibody.
Common types of labels
・Fluoresecent labeling
・Enzyme labeling
・Biotin
・Magnetic beads, agarose beads, magnetic agarose beads
・Colloidal gold
Fluoresecent labeling
Fluorescent labeled antibodies are used for flow cytometry and immunofluorescence staining. Multiple antibodies with different fluorescent labels can be used for double or triple staining.
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Common fluorophores |
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Type of fluorophore |
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| Fluorescent dye (low molecular weight fluorescent dye) | FITC (fluorescein isothiocyanate), Alexa Fluor® dye Cy dyes, etc. |
| Fluorescent protein | PE (phycoerythrin), APC (allophycocyanin), etc. |
Application example of fluorescent-labeled antibody: Immunofluorescence staining
Immunofluorescence staining with an antibody to an organelle marker
HeLa cells were stained with an antibody to the Golgi protein GM130.
Antibody：Anti-GM130 mAb-Alexa Fluor® 488（Code No. M179-A48)
Application example of fluorescent-labeled antibody: Flow cytometry
T cells are separated by type using fluorescent-labeled antibodies to cell surface markers.
T cells are separated into helper T cells, cytotoxic T cells, etc., by flow cytometry using antibodies labeled with different fluorophores (CD4-FITC, CD8-PE).
PE-labeled anti-CD8 antibody (Clone: KT15, Code No. D271-5)
FITC-labeled anti-CD4 antibody (Clone: GK1.5, Code No. D341-4)
Enzyme labeling
Enzyme-labeled antibodies are used for ELISA, Western blotting, and immunostaining.
Labeled antibodies are detected by reaction with a substrate that emits light or changes color.
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Commonly used enzymes |
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| Enzyme name | Chromogenic substrate | Chemiluminescence substrate |
| HRP（Horseradish peroxidase） | DAB and TMB | Luminol-based (ECL) |
| AP（Alkaline phosphatase） | BCIP/NBT and pPNPP | Dioxetane-based (CDP-star®) |
Application example of enzyme-labeled antibodies: Immunohistochemical staining
The result of immunohistochemical staining indicates that autophagy was induced in the liver tissue.
Application example of enzyme-labeled antibodies: Western blotting
Primary antibody: Mouse anti-GFP antibody (Clone: 1E4, Code No. M048-3)
Secondary antibody: Anti-mouse IgG antibody (HRP-labeled, Code No. 330)
Biotin
Biotin-labeled antibodies are used in flow cytometry, immunofluorescence staining, and immunohistochemical staining. Biotin-labeled antibodies are detected by reaction with dye-, enzyme-, or fluorophore-labeled avidin.
Antibody: Biotin-labeled anti-GP2 (Glycoprotein 2) antibody
Agarose, magnetic beads, magnetic agarose beads
Antibody-conjugated resins, such as magnetic beads and agarose, are used for immunoprecipitation.
>The principle and method of immunoprecipitation (IP)
Colloidal gold
Gold-labeled antibodies are used in immunoelectron microscopy. This method is highly sensitive, and the signal does not fade as in fluorescent-labeling and enzyme-labeling.
Immunoelectron micrograph of autophagosomes detected using the marker LC3
Primary antibody: Anti-LC3 monoclonal antibody
| [Related] Links to the list of direct-labeled antibody products | |
| Fluorescent | FITC-labeled antibodies PE-labeled antibodies Alexa Fluor® 488-labeled antibodies Alexa Fluor® 594-labeled antibodies Alexa Fluor® 647-labeled antibodies |
| Enzyme | HRP-labeled antibodies AP-labeled antibodies |
| Other | Biotin-labeled antibodies Antibody-conjugated magnetic beads Antibody-conjugated magnetic agarose Primary antibody-conjugated agarose |
Main causes of non-specific reactions
Various non-specific reactions of antibodies
Non-specific reactions should always be considered even if the data seem clear.
Non-specific reactions can make truly negative samples look positive (false positives) and truly positive samples look negative (false negatives).
Main causes of non-specific reactions of antibodies
・Fc receptors
・Non-specific reactions of a secondary antibody
・Heterophilic antibodies
・Rheumatoid factors
Fc receptors
False positive signals may occur when samples (cells and tissues) express receptors for the Fc portion of the antibody molecule (Fc receptors).
Non-specific reactions of a secondary antibody
Secondary antibodies are selected based on the species of animal used to generate the primary antibody. When using a polyclonal secondary antibody, a portion of the secondary antibody may react with proteins of other animal species.
Heterophilic antibodies
Human blood sometimes contains antibodies that bind to mouse antibodies. These antibodies are called human anti-mouse antibody (HAMA). Antibodies that react with antibodies of other animal species include goat (HAGA), sheep (HASA), and rabbit (HARA) antibodies.
These antibodies are collectively called “heterophilic antibodies”.
The exact reason for the presence of heterophilic antibodies is unknown. HAMA can be found in a significant proportion of healthy individuals who have never been exposed to mice.
Rheumatoid factors
Human blood may contain antibodies that react with human antibodies. A well-known example is autoantibodies commonly found in patients with rheumatoid arthritis. They are typically IgM-type immunoglobulins specific for the Fc fragment of human IgG and called rheumatoid factors (RFs).
Although RFs are directed against the Fc fragment of human antibodies, they also react with antibodies of other species, causing false positives/negatives in tests that use mouse antibodies.
In antibody-based clinical tests, interference from RFs in the samples should not be ignored, because RF levels are high in a few percent of rheumatoid arthritis patients and healthy individuals, and in about ten percent of the elderly. False reactions do not only reduce the reliability of the test, but also endanger the lives of misdiagnosed patients.
How to reduce non-specific reactions
Four ways to reduce non-specific reactions
・Remove the Fc region of the antibody.
・Include antibodies against heterophilic antibodies (HAMA Blocker, etc.).
・Pre-mix the sample with IgG from the animal species in which the secondary antibody is produced.
・Use a polyclonal secondary antibody preabsorbed with serum of the same animal.
Methods to remove the Fc region before labeling
Below are the methods to remove the Fc region of antibodies before labeling. Non-specific reactions caused by Fc receptors are eliminated by using a labeled secondary antibody without the Fc region. One of the following two methods can be used, depending on the molecular weight of the label.
・Labeling SH groups after pepsin digestion
・Labeling NH2 groups after papain digestion
Method 1. Pepsin digestion (for labeling SH groups)
In this method the pFc′ fragment is removed by pepsin digestion, followed by labeling
the SH groups in the F(ab’) fragment. This method is commonly used because the
antibody-binding site will not be blocked by the label.
First, the Fc fragment of IgG is removed by pepsin digestion. The resulting F(ab′)2 fragment is isolated by gel filtration chromatography.
Next, the isolated F(ab′)2 fragment is reduced by 2-mercaptoethylamine (2MEA) treatment to cleave the F(ab′)2 fragment at the hinge region into two F(ab’) fragments. An enzyme label is added to the F(ab’) fragment (the SH type modification), and gel filtration chromatography is used to separate enzyme-labeled F(ab’) from unlabeled F(ab’) and the unreacted enzyme.
Method 2. Papain digestion (for labeling NH2 groups)
In this method the Fc region is removed by papain digestion, followed by labeling
NH2 groups.
First, IgG is digested by papain into Fab and Fc fragments, and then the Fab fragments are isolated by affinity chromatography using one of the following two methods. The first is to use antigen-coated beads to capture the Fab fragments. The second is to use Protein A (or Protein G) to remove the Fc fragment.
Subsequently, a fluorophore is added to the isolated Fab (NH2 type modification), and the reaction results in fluorescent-labeled Fab.
Examples for reduction of non-specific reactions
Example 1 for reduction of non-specific reactions
Removal of the Fc region of a secondary antibody (to produce Fab fragments) reduces false positive results caused by Fc receptor binding to the Fc region.
Removal of the Fc region of a secondary antibody (to produce Fab fragments) reduces false negative results caused by Fc-specific antibodies.
Example 2 for reduction of non-specific reactions
False positive reactions caused by heterophilic antibodies (human anti-mouse antibody, or HAMA) are reduced by adding anti-human IgG (in the example below) specific for HAMA. This type of blocking agent (known as HAMA blockers) is commercially available.
False positive results caused by Fc receptors are also reduced by adding IgG from the animal species in which the secondary antibody is raised.
As illustrated above, more accurate results are obtained by reducing non-specific reactions.