Antibody Labeling Methods

Conjugated Primary Antibodies

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Antibody Labeling Methods

Labeling chemistries often exploit primary amine groups to covalently attach labels to antibody molecules. The N-terminus of polypeptide chains and the side chain of lysine amino acid residues contain primary amines. It is possible to link labels to antibodies by using chemical groups that react with amines. The four main chemical approaches for antibody labeling are summarized below:


  1. NHS Esters: In the case of fluorescent dyes, it is typical to purchase an activated form of the dye with an inbuilt NHS ester (also called a ‘succinimidyl ester’). Linking the activated dye to antibody by chemical conjugation occurs under physiological or alkaline conditions to generate stable amide bonds. Excess reactive dye is removed by one of several possible methods, often column chromatography before the labeled antibody is used in an immunoassay.
  2. Heterobifunctional Reagents: If the label is a protein molecule (e.g. HRP, alkaline phosphatase, or phycoerythrin), then the antibody labeling procedure is complicated by the fact that both the antibody and label contain multiple amine groups. To restrict conjugation to the antibody, lysine residues on the antibody are modified to create a new reactive group (X), while lysine residues on the label are modified 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.
  3. Carbodiimides: Carbodiimides 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).These reagents 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).
  4. Sodium Periodate: Although this chemical is not compatible with the majority of labels, 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 lysine residues on antibody molecules. Since HRP itself has very few lysine residues, it is relatively easy to create antibody-HRP conjugates without significant HRP polymerization.


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