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There are many uses for antibodies labeled with metal ions. Most of these methods involve first attaching a metal chelator to the antibody molecule. This is achieved using standard cross-linking chemistry and then adding the desired metal at appropriate concentration and pH. The method described here outlines a basic procedure for creating a lanthanide conjugate. Lanthanide conjugates are used for proximity assays, as MRI contrast agents, or for mass cytometry experiments. Different metals and chelators can be substituted, but the basic procedures are similar.

There are several techniques for biotinylating antibodies, from the most basic (using NHS-ester biotin to label primary amines) to more complex experiments (modifying sulfhydryls and carbohydrates). Biotinylation of free sulfhydryls, described here, can be effectively mediated using haloacetyl biotin derivatives. To modify an antibody using this reagent, sulfhydryls must be available. Digestion of antibodies by the enzyme pepsin produces F(ab')2 fragments, which can be separated by mild reduction into two sulfhydryl-containing, univalent Fab' fragments. Alternatively, thiol groups can be added by modifying amines with an appropriate cross-linker.

This introduction outlines general strategies for labeling proteins, with an emphasis on methods that are used primarily for labeling antibodies. It covers the specific site of modification, cross-linker options, types of labels, and postlabeling cleanup methodology, along with the advantages and disadvantages of each method. In general, polyclonal antibodies are more versatile and resistant to activity loss than are monoclonal antibodies. Greater care must be taken when labeling monoclonal antibodies to ensure a quality conjugate. The methods outlined here can be adapted for a variety of labels including multiple labels on the same immunoglobulin. The most important consideration when undertaking an antibody labeling experiment is to maintain the activity of the antibody. This is an empirical process and will often require additional experiments to optimize the label of a particular antibody. When successful, these reagents are very useful and adaptable biomolecules. This introduction provides the reader with methods and options for producing a variety of labeled immunological tools.

Hydrazide derivatives are useful for biotinylating antibodies at oxidized carbohydrate groups. This protocol uses oxidation conditions that will convert most if not all possible hydroxyl sites to aldehydes. Each antibody may require different oxidation conditions to optimize labeling. Some monoclonal antibodies may be deficient in glycosylation, making this method suboptimal. Other labeling reagents (fluorophores) are also available as hydrazides. Hetero-bifunctional cross-linkers (e.g., β-maleimidopropionic acid hydrazide [BMPH]) are also available to cross-link other targets to carbonyls.

Many antibody labeling procedures call for a desalting or purification step requiring size-exclusion chromatography (SEC). The method outlined here contains information needed to desalt an antibody conjugate. Similar procedures would be used for ion-exchange chromatography using a gradient of increasing ionic strength. Resins can be purchased in bulk (as in this protocol), or commercially available columns are available.

Colloidal gold–antibody conjugates are easy to prepare and are an excellent choice for microscopic applications. Colloidal gold is an aqueous suspension of nanometer-sized particles of gold. Typically, chloroauric acid, HAuCl4, is reduced with dilute solutions of sodium citrate, as described here. This will cause the gold to form small aggregates that will associate with proteins. Gold particles of specific sizes can be isolated and differentiated microscopically, allowing these particles to be used for multiple-label experiments. Colloidal gold-labeled antibodies are widely used in electron microscopy (EM), and can be used for light microscopy but require additional steps (silver enhancement).

Iodination, a chemical or enzymatic incorporation of 125I to specific amino acid side chains, is a commonly used method for labeling antibodies with radioisotopes. Commercially available products make iodination of antibodies a simple and quick process. One example, used here and available at Pierce, is the "Iodination bead," or N-chloro-benzenesulfonamide immobilized on nonporous, polystyrene beads.

Labeling antibodies with biotin (biotinylation) is a useful and simple technique. Biotin's small size (244 Da) usually has little effect on the biological activity of the protein target. The most common way to biotinylate an antibody is to cross-link a biotin succinimidyl ester to a primary amine. There are many commercially available types of biotin analogs that can be used for labeling. They vary in reactive group chemistry as well as spacer length. For example, a common analog used for biotinylation is the succinimidyl ester of biotin with an aminohexanoic acid spacer (Long Chain or LC-Biotin), utilized here. A PEG spacer of varying length can also be used.

Fluorophore-maleimide derivatives are effective for labeling sulfhydryl-containing molecules. Maleimide groups react with free thiols at pH 6.5-7.5 forming a covalent bond. Reducing agents should be avoided during the conjugation step. This protocol uses the cross-linker N-succinimidyl S-acetylthioacetate (SATA) to introduce thiol groups on the antibody while maintaining the divalent nature of the antibody. Alternatively, the antibody can be digested and reduced to monovalent Fab fragments, which can then be labeled directly with maleimido derivatives.

N-Hydroxysuccinimide (NHS)-ester derivatives are among the most commonly used reagents for labeling proteins. The method described here can be adapted to use practically any NHS fluorophore. Generally, a fluorophore is covalently bound to a macromolecule such as an antibody and acts as a reporter molecule used to measure the presence of the macromolecule. These fluorescently labeled bioactive reagents are suitable for use in immunofluorescence, flow cytometry, and numerous other biological applications. There are several widely used dyes available in convenient formats. This protocol can be used with any amine-reactive (e.g., PFP, isothiocyanate) fluorophore derivative.

Protein A and Protein G are immunoglobulin-binding proteins expressed in Staphylococcus aureus and Streptococcus sp., respectively, that have been adapted for use in purifying large amounts of IgG. They are available covalently attached to affinity resins such as 4% cross-linked agarose, making them suitable for low-pressure antibody isolation. Protein A is not recommended for the isolation of mouse mAbs because it lacks affinity for mouse IgG1, or for the isolation of antibodies from sheep, goat, chicken, hamster, or rat. IgGs from most species bind to Protein G at near physiological pH and ionic strength with a higher affinity than IgG binding to Protein A. Therefore, the pH required to dissociate bound IgG is lower, resulting in the loss of activity for some antibodies. If this is observed, Protein A may be an alternative if the IgG from the species being isolated can be purified using Protein A. Neither Protein A nor Protein G can be used for the isolation of chicken antibodies.

Antibodies have become a common and necessary tool in biochemistry, cell biology, and immunology laboratories. There are many different types of antibodies and antibody fragments being used for a myriad of applications. As a result, many different purification protocols have been developed to obtain antibodies of the desired specificity and sensitivity. Here, we introduce the options for small- to large-scale antibody purification and isolation of polyclonal and monoclonal antibodies (and fragments generated from these) that target-specific proteins, as well as methods to properly purify antibodies that recognize posttranslational modifications. Optimal conditions for the long-term storage of antibodies are also discussed.

Conjugates of the FRET dye Cy5-phycoerythrin (Cy5PE) with antibodies are relatively straightforward to make. The protocol does require synthesis of the Cy5PE tandem dye. Phycoerythrin (PE) can be purchased from multiple vendors. This type of conjugate is useful for immunofluorescence studies involving protein targets with low expression levels. Although the entire conjugation can be performed in a single day, there is an overnight stopping point. When initially making Cy5PE derivatives, several different conjugates with varying ratios of Cy5 to PE should be made. These should be tested by conjugating to a well-characterized antibody. Absorbance spectra readings are a very worthwhile step to determine the quality of the Cy5PE label.