Antibodies are largely used in immunoassays to identify and measure the presence of antigens. The antibody that recognizes the antigen is referred to as the ‘primary’ antibody and gives specificity to the assay. A ‘label or tag’ is additionally integrated in to the assay utilizing one of the two approaches: direct or indirect detection methods to provide measurability. Typically used immunoassay strategies are provided in Table 1 together with instances of the kinds of labels that might be used.
Table 1: Types of Immunoassay Experiment and Associated Labels
| Immunoassay |
Labels |
| Western Blotting |
Enzymes (typically HRP, or AP) |
| ELISA |
Enzymes, Biotin/Streptavidin |
| Immunoflouorescence |
Fluorescent dyes |
| Immunohistochemistry |
Enyzme, Biotin/Streptavidin |
| Flow Cytometry |
Fluorescent proteins or dyes, tandem dyes |
Bio-Synthesis are capable of using wide varieties of conjugation chemistry. Commonly used antibody labeling methods are outline below:
Antibody Labeling and Conjugation Methods
Antibodies contain large numbers of surface amine groups (Lys or N-terminal amines, a primary amine). Those surface amine groups can easily react with an acylating reagent, such as NHS-ester or isothiocyanate activated small molecule, to form a stable amide bond or equivalent. The products obtained usually contain multiple labels per molecule.
The four main chemical approaches for antibody labeling are followed:
- NHS Esters: In the case of fluorescent dye labels it is usual to purchase an activated form of the label with an inbuilt NHS ester (also called a ‘succinimidyl ester’). The activated dye can be reacted under appropriate conditions with antibodies (all of which have multiple lysine groups). Excess reactive dye is removed by one of several possible methods (often column chromatography) before the labeled antibody can be used in an immunoassay.
- Heterobifunctional Reagents: If the label is a protein molecule (e.g. HRP, alkaline phosphatase, or phycoerythrin) the antibody labeling procedure is complicated by the fact that the antibody and label have multiple amines. In this situation it is usual to modify some of the lysines on one molecule (e.g. the antibody) to create a new reactive group (X) and lysines on the label to create another reactive group (Y). A ‘heterobifunctional reagent’ is used to introduce the Y groups, which subsequently react with X groups when the antibody and label are mixed, thus creating heterodimeric conjugates. There are many variations on this theme and you will find hundreds of examples in the literature on the use of heterobifunctional reagents to create labeled antibodies and other labeled biomolecules.
- Carbodiimides: These reagents (EDC is one very common example) are used to create covalent links between amine- and carboxyl-containing molecules. Carbodiimides activate carboxyl groups, and the activated intermediate is then attacked by an amine (e.g. provided by a lysine residue on an antibody). Carbodimides are commonly used to conjugate antibodies to carboxylated particles (e.g. latex particles, magnetic beads), and to other carboxylated surfaces, such as microwell plates or chip surfaces. Carbodiimides are rarely used to attach dyes or protein labels to antibodies, although they are important in the production of NHS-activated dyes (see above).
- Sodium Periodate: This chemical cannot be employed with the vast majority of labels but is quite an important reagent in that it is applicable to HRP, the most popular diagnostic enzyme. Periodate activates carbohydrate chains on the HRP molecule to create aldehyde groups, which are capable of reacting with lysines on antibody molecules. Since HRP itself has very few lysines it is relatively easy to create antibody-HRP conjugates without significant HRP polymerization.
- Click Chemistry: Click chemistry-mediated antibody labeling methods that result in site-specific labeling of antibody heavy chain glycans using mild conditions. The methods involve the enzymatic incorporation of azide-modified sugars into antibody heavy chain glycans away from the antibody binding domain. Then azide modified antiobdies are reacted with dibenzocyclooctyne (DBCO)-functionalized probe of choice (e.g., Alexa Fluor® 488 DBCO alkyne, FITC alkyne, oligo). The average degree of labeling is 3–3.5 labels per antibody.
Key Consideration for Antibody Labeling
It is critical to remember that your antibody will contain substances other than antibody; minimally a buffer and/or salts, and possibly other proteins and additives. Compatibility of the mixture with labeling methods may not have been a key consideration when the antibody was initially purified and formulated; occasionally it will be necessary to re-purify the antibody prior to carrying out the labeling reaction. Purification may involve the removal of stabilizing proteins (e.g. BSA) or the removal of low molecular weight substances, such as sodium azide, tris buffer or glycine. The different labeling methods may be negatively impacted to different extents by the various additives but, as mentioned earlier, the majority of labeling methods exploit lysine residues on antibodies, thus substances with primary amines should generally be avoided in antibody labeling procedures.
Antibody Concentration and Purity
For optimal labeling efficiency, the antibody is required to be reasonably pure (e.g. >90%, preferably >95%) and at a concentration of >0.5mg/ml. Many commercially available antibodies are provided in a form suitable for labeling, but you should be aware that this is not always so. Before labeling, Bio-Synthesis will conducts antibody quality check with an additional fee if antibody quality is not specified. For example, antibodies may be sold in the form of hybridoma tissue culture supernatant (TCS), ascites fluid or crude serum. TCS often contains many other proteins and culture nutrients (e.g. amino acids) which are particularly problematic. Purification (e.g. on protein A columns) is more or less obligatory if TCS is your starting point. Ascites fluids and crude serum have higher concentrations of antibody than TCS, but these materials are impure and contain high concentrations of amino acids; further purification of the antibody will generally be required.
Contact Bio-Synthesis for custom antibody labeling.