Iodination of Antibodies with Immobilized Iodogen.

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.

Purification of Labeled Antibodies Using Size-Exclusion Chromatography.

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.

Labeling Antibodies.

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.

Labeling Antibodies Using Europium.

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.

Labeling Antibodies Using Colloidal Gold.

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).

Labeling Antibodies with N-Hydroxysuccinimide-Long Chain (NHS-LC)-Biotin.

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.

Biotinylating Antibodies Using Biotin Polyethylene Oxide (PEO) Iodoacetamide.

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.

Biotinylating Antibodies Using Biotin-Long Chain (LC) Hydrazide.

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., ß-maleimidopropionic acid hydrazide [BMPH]) are also available to cross-link other targets to carbonyls.

Conjugation of Antibodies to Horseradish Peroxidase.

Antibodies conjugated with horseradish peroxidase (HRP) are one of the most widely used bioreagents in the biological sciences. This protocol is a basic method for adding HRP to a thiolated antibody and can be adapted for use with different cross-linkers. Conjugation methods usually focus on linking through the lysines on HRP because there are only six of them and their modification does not adversely affect enzyme activity.

Labeling Antibodies with Cy5-Phycoerythrin.

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.

Conjugation of Peptides to Thiol-Reactive Gel for Affinity Purification of Antibodies.

Thiol-reactive linkers, such as iodoacetyl or maleimide, bound to cross-linked agarose are used to attach cysteine-containing peptides covalently to this resin for use in affinity-purification protocols. It is critical to confirm that the peptide contains a reduced cysteine so that the thiol is available for conjugation to the thiol-reactive linker. The column should be sized appropriately for the amount of peptide to be used and the volume of serum to be processed. Excess binding sites on the column must be blocked with free cysteine before use.

Peptide Affinity Purification of Antibodies.

This protocol describes antibody purification using a peptide affinity column. Peptides can be designed that use naturally occurring cysteines within the protein target's primary sequence, or a cysteine can be added to either end of the peptide to provide free thiols for attachment. The peptides can then be covalently attached to resins bearing thiol-reactive linkers. The most commonly used thiol-reactive moieties are iodoacetyl and maleimide, both of which react selectively with peptides containing cysteine thiols. Although gravity can be used to cycle the antibody solution (e.g., serum) over the column (it is recommended that the antibody be cycled multiple times to obtain maximal yield), the use of a pump to apply the serum to the column in a continuous flow manner improves the yield of antibody. Similarly, washing the column after application of the antibody without and with 0.5 m NaCl should be performed with at least 20 column volumes.

Antibody Purification and Storage.

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.

Labeling Antibodies Using N-Hydroxysuccinimide (NHS)-Fluorescein.

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.

Labeling Antibodies Using a Maleimido Dye.

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.

Protein A and Protein G Purification of Antibodies.

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.

Purification of Antibodies: Diethylaminoethyl (DEAE) Chromatography.

Because IgG from most species (other than rodents) tends to have an isoelectric point around neutral, two approaches can be used when separating IgG using diethylaminoethyl (DEAE) resins. When serum containing antibodies is applied to DEAE at a slightly acidic pH, the IgG flows through the column while most other serum proteins bind to the DEAE. This method is best performed using a batch method. The DEAE beads can be kept in a disposable syringe containing a polypropylene frit, a glass reactor containing a coarse-sintered glass frit, or other suitable vessel. If the antibody solution is adjusted to a basic pH of 8-8.5, then IgG binds to the DEAE resin. After washing the column, the antibody is eluted by adding a buffer of increasing ionic strength to the column. Prepacked columns of many sizes are available for the isolation of antibodies by DEAE chromatography. Alternatively, DEAE medium can be swelled or prepped according to the manufacturer's instructions and a column can be poured when needed.

Ammonium Sulfate Fractionation of Antibodies.

Before the widespread availability and use of Protein A (rabbit) or Protein G (rodent) for the purification of IgG, the use of an ammonium sulfate "cut" was the standard method to isolate IgG and other serum proteins. The addition of ammonium sulfate reduces the effective solubility of proteins through direct competition for binding sites on the surface of the protein. The resulting precipitated proteins can be isolated by centrifugation. An ammonium sulfate concentration between 40% and 50% results in the precipitation of IgG from most species, and thus 50% is usually used. Because other proteins can be "trapped" within the aggregated protein, the use of ammonium sulfate does not result in a purified antibody fraction and as such should be considered a first step in a multistep antibody purification protocol.

Preparation of Antibody Using Caprylic Acid.

Caprylic acid has been used to enrich IgG from serum, ascites, and cell culture supernatants by precipitating the non-IgG serum proteins. By precipitating all of the unwanted serum proteins rather than the antibodies, the tendency of antibodies to aggregate when precipitated is avoided. This method should not be used with antibody sources that contain low concentrations of antibody, such as many cell culture supernatants, owing to the potential loss of high-affinity antibodies, which may be bound by the caprylic acid. After centrifugation, IgG remains in the supernatant, which can be used as the starting material for the next step in a multistep antibody purification protocol.

Isolation of IgY from Chicken Eggs.

Because IgY does not interact with either Protein A or Protein G, more traditional methods must be used for its isolation. To isolate chicken IgY from the yolk of chicken eggs, the yolks and the whites must be separated much in the way a cook does. Minimizing the egg white proteins is an important part of this process. Although eggs can be accumulated for short periods of time, IgY must be isolated from the yolk within 2-3 wk to avoid fouling of the material. In the first section of this protocol, sodium sulfate is used to precipitate protein fractions differentially from the chicken egg yolk. The second section describes an alternative differential precipitation technique that uses Polyethylene Glycol 6000 instead of sodium sulfate.