What is antibody conjugation? Antibody conjugation, also known as antibody labeling, is a technique for modification of antibodies which involves with the attachment of a specific tag to an antibody. These labeled antibodies can be used to isolate and purify a protein of interest from a complex mixture, usually cells, tissues or whole organisms.
Horseradish peroxidase (HRP) is a commonly used enzyme for conjugation of antibodies. There are only six lysines on HRP and their modification does not adversely affect enzyme activity. Fluorescent molecules such as FITC, PE, APC and PerCP are also available for labeling antibodies.
After generating antibodies for customers, Sino Biological also provides antibody conjugation service, which can custom conjugation of an antibody to HRP, biotin or a fluorophore.
Table 1. Fluorescent labeling for antibodies
|Conjugate||Ex (nm)||Em (nm)||Fluorescence Color||Relative Brightness|
|PerCP (peridinin chlorophyll protein)||490||675||2|
|FITC (fluorescein isothiocyanate)||490||520||3|
A variety of colorimetric and fluorescent labels are linked to primary antibodies for use in detection, sorting, and microscopy applications. Sino Biological has manufactured 1,000+ conjugated antibodies which allow for direct detection of a target antigen without a secondary antibody.
|Flow cytometric analysis of human PDL1 (CD274) expression on HCC827 cells with PE-conjugated anti-PDL1 antibody (Cat: 10084-R611-P).
|Immunofluorescence staining of human NFKBIA in HeLa cells with FITC-conjugated anti-NFKBIA antibody (Cat: 12045-MM02-F). The image showing nucleus staining of HeLa cells.|
Like all proteins, antibodies are composed of amino acids. In theory, bioconjugation is possible via most amino acids. Several useful techniques are available for lysine-based conjugation of antibodies described as below.
This method is useful for the conjugation of antibodies with widely available fluorescent dyes such as rhodamine derivatives. It is typically performed in a phosphate buffer with subsequent on-column separation from the unlabeled dye. The main disadvantage is that the esters are unstable because they are moisture-sensitive. The labeled antibody should be used immediately after the end of the reaction.
Gemtuzumab ozogamicin (Mylotarg®) was the first antibody-drug conjugate (ADC) on the market. A semi-synthetic calicheamicin derivative was equipped with an NHS ester in order to conjugate the toxin to lysines of a humanized IgG4. However, due to a lack of improvement in clinical benefit to patients, Mylotarg® was withdrawn from the market in 2010.
This method may be used to make fluorescein isothiocyanate (FITC), which is very popular in the preparation of fluorescent proteins and antibodies. Isothiocyanate is more stable than NHS but it is harder to make and your labeling reaction will likely be less efficient with this method. As with NHS, the excess dye should be removed after the reaction by chromatography.
Carbodiimide-derived compounds convert carboxyl groups on proteins into reactive intermediates that can react with lysines. The high reactivity of carbodiimides means that they can be used to label antibodies with relatively inert materials such as magnetic or gold particles. The most commonly used carbodiimide is EDC. NHS is sometimes added to the reaction to aid the formation of relatively stable intermediates.
The method is simple, but similarly to NHS, EDS is hygroscopic, so you will need to use your antibody immediately after labeling.
This method is useful for generation specific HRP-antibody conjugates. Periodate activates HRP by creating aldehyde molecules that interact with lysine residues. The HRP itself has only a few lysine residues, so enzyme polymerization is not a significant concern. The bonds between HRP and the antibody are reversible unless stabilized by adding sodium cyanohydride.
1. Cal, P. M., Bernardes, G. J., & Gois, P. M. (2014). Cysteine‐Selective Reactions for Antibody Conjugation. Angewandte Chemie International Edition, 53(40), 10585-10587.
2. Dennler, P., Fischer, E., & Schibli, R. (2015). Antibody conjugates: from heterogeneous populations to defined reagents. Antibodies, 4(3), 197-224.