What are antibody markers?
Antibody labeling refers to the use of chemical or biological methods to connect detectable markers (such as fluorescent dyes, enzymes, biotin, radioisotopes, etc.) to antibody molecules covalently or non-covalently, so that they can be specifically recognized and visualized in immunoassay. Antibody labeling is one of the core technologies of immunological experiments (such as ELISA, flow cytometry, immunofluorescence, Western blot, etc.).
First, the main types of antibody markers
Depending on the nature of the markers, antibody markers can be classified into the following categories:
1. Fluorescence labeling
- Markers: Fluorescent dyes (e.g. FITC, PE, Cy3, Cy5, Alexa Fluor series, DyLight, etc.).
- Application:
- Immunofluorescence (IF), flow cytometry (FACS), confocal microscopy.
- Multicolor detection (different fluorescent labeled antibodies can be used simultaneously).
2. Enzyme labeling
- Markers: horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase, etc.
- Application:
- ELISA, Western blot (by substrate color development or chemiluminescence detection).
Signal amplification (enzyme-catalyzed substrates generate strong signals).
3. Biotin labeling
Marker: Biotin, which amplifies the signal by streptavidin.
- Application:
- Combined with the avidin system (e.g. ABC method) to improve detection sensitivity.
For use in multi-stage amplification systems (e.g. mass spectrometry flow cytometry).
4. Radiolabeling
- Markers: radioactive isotopes (e.g., I ² ², I ³ ² P).
- Application:
Radioimmunoassay (RIA), autoradiography.
Due to safety concerns, it is gradually being replaced by non-radioactive methods.
5. Other markings
Metal tags (e.g. lanthanides): for mass spectrometry flow (CyTOF).
Gold nanoparticles: used in immunoelectron microscopy or lateral chromatography strips (e.g. colloidal gold strips).
DNA barcoding: for multiple detection (e.g. Olink technology).
Methods of antibody labeling
1. Chemical coupling method
- Amino coupling: Utilizes the free amino group of antibody lysine (Lys) to react with the active group of the marker, such as an NHS ester.
-Mercapto-coupling: Exposure of thiol (-SH) to react with maleimide groups by reducing disulfide bonds in the hinge region of the antibody.
Glycosylation modification: oxidation of the Fc segment of the antibody sugar chain, bound to the hydrazide group of the marker.
2. Genetic engineering law
Fusion protein tags: such as the expression of antibodies with fluorescent protein (eGFP) and enzymes (such as HRP) through gene recombination.
Click Chemistry: Introduce unnatural amino acids (e.g. Azide) to connect the markers through a click reaction.
3. Biotin-avidin system
- Label biotin first, followed by streptavidin (high affinity) ligase or fluorescently labeled avidin.
III. Application scenarios for antibody labeling
1. Immunohistochemistry (IHC): Fluorescence or enzyme-labeled antibodies target antigens in tissues.
2. Flow cytometry: multicolor fluorescence labeling to analyze cell surface/internal antigens.
3. Diagnostic reagents: such as colloidal gold-labeled rapid test strips (pregnancy test strips, infectious disease detection).
4. Drug conjugates (ADCs): Antibody-drug conjugates (e.g., targeted delivery of anticancer drugs).
IV. Precautions
1. Labeling efficiency: It is necessary to optimize the labeling ratio (e.g. fluorescent dye/antibody ratio, to avoid over-labeling leading to antibody inactivation).
2. Antibody activity protection: Avoid severe reaction conditions (e.g. pH, temperature).
3. Purification: removal of free markers (often gel filtration or dialysis).
4. Storage: Avoid light (fluorescent labeling) and avoid repeated freezing and thawing.
V. Frequently Asked Questions
Why is the antibody signal weak after labeling?
Possible causes: over-labeling (masking antigen binding check points), antibody denaturation, unpurified free markers.
How to choose a marking method?
According to the detection target (e.g., high sensitivity is required to select the enzyme label, multiple detection is required to select the fluorescence) and the experimental platform (e.g., the flow type requires dyes with different excitation/emission wavelengths).
The choice of antibody labeling technology directly affects the sensitivity and specificity of the experiment, and the optimization scheme needs to be combined with specific requirements.
