Covalent Immobilization of Affinity Ligands

Affinity purification and many assay techniques depend on the binding interactions of target biomolecules in liquid samples with specific chemical groups or macromolecules that are immobilized on a solid material. The latter class of molecules are called affinity ligands, and they can be attached (immobilized) to the solid material (stationary phase) in a variety of ways. Many assay methods, such as ELISA and western blotting, depend upon strong but noncovalent immobilization of antibodies or other ligands to the solid material (polystyrene microplates and nitrocellulose membrane, respectively).

By contrast, most affinity purification strategies – especially those involving antibodies and other proteins – depend upon covalent chemical conjugation of ligands to the solid support matrix. Affinity ligands that have broad applicability are commercially available in a variety of ready-to-use, pre-immobilized forms. Examples include Protein A agarose resin for general antibody purification and streptavidin magnetic beads for purifications involving biotinylated molecules.

Affinity chromatography utilizes the specific interactions between two molecules for the purification of a target molecule. A ligand having affinity for a target molecule is covalently attached to an insoluble support and functions as bait for capturing the target from complex solutions. The affinity ligand can be any molecule that will bind the target without also binding other molecules in the solution.

Small scale affinity purification using an antibody immobilized to a solid support. Chromatography has three main components: the mobile phase or solvent containing proteins, the stationary or solid phase also called the medium or resin (which may be agarose or other porous resin) and the chromatography column. Affinity chromatography is very selective and provides high resolution with an intermediate to high sample loading capacity. The protein of interest is tightly bound to the resin under conditions that favor specific binding to the ligand, and unbound contaminants are washed off. The bound protein is then recovered in a highly purified form by changing conditions to favor elution. Elution conditions may be specific, such as a competitive ligand, or nonspecific, such as changing pH, ionic strength, or polarity. The target protein is eluted in a purified and concentrated form.

• Small organic compounds that are able to dock into binding sites on proteins
• Inorganic metals that form coordination complexes with certain amino acids in proteins
• Hydrophobic molecules that can bind nonpolar pockets in biomolecules
• Proteins with specific binding regions that are able to interact with other proteins
• Antibodies, which can be designed to target any biomolecule through their antigen binding sites

The concept of using immobilized affinity ligands to target biomolecules has extended beyond just chromatographic applications with beaded agarose resins (still the most common). Affinity ligands are now coupled to magnetic particles, latex beads, nanoparticles, macro-beads, membranes, microplates, array surfaces, dipsticks and a host of other devices that facilitate the capture of specific biomolecules. Applications of affinity targeting include purification, scavenging (or removal of contaminants), catalysis (or modification of target molecules) and a broad range of analytical uses to quantify a target molecule in a sample solution.

Designing custom affinity supports that are able to target unique biomolecules requires methods to covalently link a ligand to an insoluble matrix. Regardless of the intended application, the chemical reactions that make possible ligand attachment are well characterized and facilitate the attachment of biomolecules through their common chemical groups. The types of functionalities generally used for attachment include easily reactive components such as primary amines, sulfhydryls, aldehydes, and carboxylic acids. Usually, the solid phase matrix first is activated with a compound that is reactive toward one or more of these functional groups. The activated complex then can generate a covalent linkage between the ligand and the support, resulting in ligand immobilization.

The type of linkage that is formed between the matrix and the immobilized ligand affects the performance of the affinity support in a number of ways. For example, if the linkage blocks or adversely affects the structure of the immobilized ligand, it will limit its effectiveness for affinity purification. A linkage that allows the coupled ligand to leach from the matrix will result in contamination of the purified protein and shorten the useful life of the affinity support. A conjugation chemistry that introduces a charged functional group into the support can cause nonspecific binding by promoting ion-exchange effects. A linkage that alters the structure of the matrix can change the flow and binding characteristics of the support.

Cyanogen bromide (CNBr)-activated supports are informative as an example of these adverse effects on affinity purification. This immobilization method results in a linkage that (1) constantly leaks ligand from the matrix, which then contaminants the purification, (2) includes a charged isourea group in the linkage, resulting in nonspecific binding, and (3) causes extensive crosslinking of the matrix, which reduces the ability of large molecules to penetrate into the interior of the resin.

Chemistry of CNBr resin (cyanogen bromide agarose).  This once-popular amine-reactive immobilization chemistry results in a charged isourea linkage that leaks and causes nonspecific binding.

Several more effective beaded agarose, beaded acrylamide and magnetic bead affinity supports are commercially available in activated forms that are ready to use for coupling many different types of ligands. These activation chemistries and protocols have been optimized to assure excellent coupling yields and to generate stable covalent linkages that will not easily leach the immobilized ligand. The remainder of this article discusses these methods.

The most common functional target for immobilizing protein molecules is the amine group (–NH2): This group exists at the N-terminus of each polypeptide chain (called the alpha-amine) and in the side chain of lysine (Lys, K) residues (called the epsilon-amine). Because of its positive charge at physiologic conditions, primary amines are usually outward-facing (i.e., on the outer surface) of proteins; thus, they are usually accessible for conjugation without denaturing the protein structure.

NHS esters are reactive groups formed by EDC activation of carboxylate molecules. NHS ester-activated resins react with primary amines in slightly alkaline conditions (pH 7.2-8.5) to yield stable amide bonds. The immobilization reaction is usually performed in phosphate buffer at pH 7.2-8.0 for 0.5 to 4 hours at room temperature or 4°C. Primary amine buffers such as Tris-buffered saline (TBS) are not compatible because they compete for reaction; however, in some procedures, it is useful to add Tris or glycine buffer at the end of a conjugation procedure to quench (stop) the reaction.

A reliable and popular method for covalently attaching antibodies or other proteins to beaded agarose resin involves a chemistry called reductive amination. 

The immobilization reaction using reductive amination involves the formation of an initial Schiff base between the aldehyde and amine groups, which is then reduced to a secondary amine by the addition of sodium cyanoborohydride (NaCNBH3). The cyanoborohydride reducing agent used during the coupling process is mild enough not to cleave disulfides in most proteins, and also it will not reduce the aldehyde reactants–only the Schiff base intermediates. It is best to avoid stronger reducing agents such as sodium borohydride because of the potential for disulfide reduction of the protein and reduction of the aldehydes on the support to hydroxyls, effectively quenching the reaction. Depending on the type and amount of ligand present, a coupling reaction using reductive amination can achieve immobilization yields of greater than 85%.

Ligand immobilization on aldehyde-activated agarose resin. Each porous agarose bead contains trillions of available activation groups for conjugation. Thermo Scientific AminoLink Plus Coupling Resin is the basis for this reliable method of protein immobilization.

AminoLink Coupling Resin is crosslinked 4% beaded agarose that has been activated with aldehyde groups to enable covalent immobilization of antibodies and other proteins through primary amines. Coupling to AminoLink Resin can be accomplished in a single reaction at pH 7.2. Efficient formation of Schiff-base and reduction by sodium cyanoborohydride can be achieved. However, overall coupling efficiencies can be increased when the reaction is performed in two steps at different pH levels. At a pH of 9 to 10, Schiff-base formation is more efficient, but NaCNBH3 is only effective at near-neutral conditions. Thus, for proteins that are soluble at high pH, high coupling yields are possible when the reaction is performed at pH 10 for one hour, followed by neutralization and addition of NaCNBH3 for the remainder of the time.

Another amine-reactive strategy that can be used for immobilization is the azlactone ring present in Thermo Scientific UltraLink Biosupport. This is a unique, durable polyacrylamide-like resin formed by co-polymerization of acrylamide with azlactone.

A primary amine will react with an azlactone group in a ring-opening process that produces an amide bond at the end of a five-atom spacer. The group is spontaneously reactive with amines; no additives or catalysts are needed to drive the coupling process. The Thermo Scientific UltraLink Biosupport is a durable, azlactone-activated, beaded-polyacrylamide resin to covalently immobilize proteins and other primary amine biomolecules for preparation of affinity purification columns. It is supplied dry to assure stability of the azlactone groups until use. Adding a quantity of the support to a sample containing a protein or another amine-containing molecule causes immobilization to occur within about an hour. For most proteins, immobilization is most efficient when the coupling buffer contains a lyotropic salt, such as 0.6 M sodium citrate, which functions to drive the protein molecules toward the bead surface. This brings the hydrophilic amines close enough to the azlactone rings to react quickly.

Ligand immobilization on azlactone-activated resin. Each porous bead contains trillions of available activation groups for conjugation.

One other option for immobilizing amine-containing affinity ligands is the use of carbonyl diimidazole (CDI) to activate hydroxyls on agarose supports to form reactive imidazole carbamates. This reactive group is formed on the support in organic solvent and stored as a suspension in acetone to prevent hydrolysis. Reaction of the support in an aqueous coupling buffer with primary amine-containing ligands causes loss of the imidazole groups and formation of carbamate linkages. The coupling process occurs at basic pH (8.5-10), but it is a slower reaction with proteins than reductive amination or azlactone coupling. CDI-activated resins are particularly adept at immobilizing peptides and small organic molecules. The reaction also can be done in organic solvent to permit coupling of water-insoluble ligands. Thermo Scientific Pierce CDI-Activated Agarose is carbonyldiimidazole affinity chromatography resin, activated for covalent immobilization of N-nucleophiles and primary amine ligands in aqueous or organic solvent conditions.

Ligand immobilization on CDI-activated agarose resin. Each porous bead contains trillions of available activation groups for conjugation.

A form of amine-based immobilization is possible using an amine-to-amine crosslinker when a specific binding interaction can be used to bind and orient a ligand to an already-immobilized molecule. For example, Protein G will affinity-bind to antibodies (IgG), and Protein G Agarose is readily available. If a crosslinker, such as DSS, is added to Protein G Agarose that has been incubated with purified IgG, covalent crosslinks will form between primary amines on the various polypeptide components represented. Some of these will effectively crosslink the IgG to the Protein G (and the Protein G is already covalently immobilized on the agarose bead).

This antibody immobilization method is called "IgG orientation." When the method is used to immobilize antibodies for immunoprecipitation, it is called "Crosslink IP.

The method can be used for other kinds of affinity pairs besides antibodies with Protein A or Protein G. For example, glutathione agarose can be used for orientation-crosslinking of GST-tagged fusion proteins.

Ligand immobilization by orientation and crosslinking. After an antibody is bound to Protein G agarose, DSS is added to covalently crosslink the proteins.

Often it is advantageous to immobilize affinity ligands through functional groups other than just amines. In particular, the thiol group can be used to direct coupling reactions away from active centers or binding sites on certain protein molecules.

Sulfhydryls (–SH) exists in the side chain of cysteine (Cys, C). Often, as part of a protein's secondary or tertiary structure, cysteines are joined together between their side chains via disulfide bonds (–S–S–). These must be reduced to sulfhydryls to make them available for immobilization. 

Because amines occur at many positions on a protein’s surface, it is usually difficult to predict where a coupling reaction will occur. Sulfhydryl groups typically are present in fewer numbers than primary amines and, therefore, enable more selective immobilization of proteins and peptides. Sulfhydryls for conjugation can be added to peptide ligands at the time of peptide synthesis by adding a cysteine residue at one end of the molecule. This ensures that every peptide molecule will be oriented on the support in the same way after immobilization. Thiol groups (sulfhydryls) can be indigenous within a protein molecule or they may be added through the reduction of disulfides or through the use of various thiolation reagents. Thermo Scientific SulfoLink Coupling Resin is porous, crosslinked, 6% beaded agarose that has been activated with iodoacetyl groups for covalent immobilization of cysteine-peptides and other sulfhydryl molecules.

Maleimide-activated reagents react specifically with sulfhydryl groups (–SH) at near neutral conditions (pH 6.5-7.5) to form stable thioether linkages. The maleimide chemistry is the basis for most crosslinkers and labeling reagents designed for conjugation of sulfhydryl groups. However, the method is seldom used to immobilize ligands onto agarose beads for use in affinity purification. One exception is maleimide-activated polystyrene microplates; these provide a method to bind sulfhydryl-peptides that otherwise would not coat effectively onto the plate surface.

Iodoacetyl-activated supports react with sulfhydryl groups at physiologic to alkaline conditions (pH 7.2 to 9), resulting in stable thioether linkages. To limit free iodine generation, which has the potential to react with tyrosine, histidine and tryptophan residues, these reactions are usually performed in the dark.

Thermo Scientific SulfoLink Coupling Resin is beaded agarose with an iodoacetyl group at the end of a long spacer arm. Immobilization of sulfhydryls occurs through displacement of the iodine atom. UltraLink Iodoacetyl Resin uses the same chemistry but is built on the acrylamide-based UltraLink Biosupport (see above).

Pyridyl disulfides react with sulfhydryl groups over a broad pH range to form disulfide bonds. As such, conjugates prepared using this chemistry are cleavable with typical disulfide reducing agents, such as dithiothreitol (DTT).

Pre-activated pyridyldithiol resins are not commonly available as ready-to-use supports. However, the mention of this reaction chemistry provides the opportunity to illustrate how many different variations of affinity resins can be prepared by individual researchers using commonly available reagents. In this case, an amine-activated resin, Thermo Scientific CarboxyLink, can be modified with a crosslinker, such as Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'-(2-pyridyldithio) propionamido) hexanoate) to make an activated resin for reversible sulfhydryl immobilization.

Most biological molecules do not contain carbonyl ketones or aldehydes in their native state. However, it might be useful to create such groups on proteins in order to form a site for immobilization that directs covalent coupling away from active centers or binding sites. Glycoconjugates, such as glycoproteins or glycolipids, usually contain sugar residues that have hydroxyls on adjacent carbon atoms; these cis-diols can be oxidized with sodium periodate to create aldehydes as sites for covalent immobilization

Hydrazide-activated resins and compounds will conjugate with carbonyls of oxidized carbohydrates (sugars) at pH 5 to 7, resulting in formation of hydrazone bonds.

Hydrazide chemistry is useful for labeling, immobilizing or conjugating glycoproteins through glycosylation sites, which are often (as with most polyclonal antibodies) located at domains removed from the key binding sites whose function one wishes to preserve. Coupling antibodies in this manner specifically targets heavy chains in the Fc portion of the molecule, helping to ensure the best possible retention of antigen binding activity by the ends of the Fv regions.

Activated hydrazide agarose support was formerly available as Pierce CarboLink Coupling Gel. That older product has been replaced by Thermo Scientific GlycoLink Resin, which is hydrazide-activated UltraLink Biosupport (a durable acrylamide resin; see discussion above). The coupling procedure has been improved by incorporating the use of aniline as a catalyst.

Ligand immobilization on hydrazide-activated resin. Each porous bead contains trillions of available activation groups for conjugation.

The carboxyl group is a frequent constituent of many biological molecules. Peptides and proteins contain carboxyls (–COOH) at the C-terminus of each polypeptide chain and in the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E). Like primary amines, carboxyls are usually on the surface of protein structure.

Carboxylic acids may be used to immobilize biological molecules through the use of a carbodiimide-mediated reaction. Although no activated support contains a reactive group that is spontaneously reactive with carboxylates, chromatography supports containing amines (or hydrazides) can be used to form amide bonds with carboxylates that have been activated with the water-soluble carbodiimide crosslinker EDC (Part No. 22980).

Because peptides and proteins contain multiple carboxyls and amines, direct EDC-mediated crosslinking usually causes random polymerization of polypeptides. Nevertheless, this reaction chemistry is used widely in immobilization procedures (e.g., attaching proteins to a carboxylated surface) and in immunogen preparation (e.g., attaching a small peptide to a large carrier protein). In fact, some degree of peptide polymerization is beneficial in antibody production and purification procedures because it increases overall peptide conjugation (loading) and ensures that the peptide ligands are presented in multiple orientations.

Ligand immobilization using EDC crosslinker. Each porous agarose bead contains trillions of available activation groups for conjugation.

For molecules devoid of easily reactive functional groups, immobilization may be difficult or even impossible using the methods discussed above. In particular, certain drugs, steroids, dyes or other small organic molecules often have structures that contain no available “handles” for immobilization. Other molecules have functional groups that have low reactivity or are sterically hindered. However, some of these compounds have active (or replaceable) hydrogens that can be condensed with formaldehyde and an amine using the Mannich reaction.

Formally, the Mannich reaction consists of the condensation of formaldehyde (or another aldehyde) with ammonia and another compound containing an active hydrogen. Instead of using ammonia, this reaction can be done with primary or secondary amines or even with amides. Ligand immobilization occurs when diaminodipropylamine (DADPA) agarose resin is used as the primary amine for this reaction. The Pierce PharmaLink Immobilization Kit was a complete set of reagents with DADPA resin for this method, but that particular product is no longer offered.

Immobilization via the Mannich reaction. 

Susan R. Mikkelsen, Eduardo CortÓn. Bioanalytical Chemistry. Chapter 16. John Wiley and Sons, Inc. Hoboken, New Jersey. 2016

Greg T. Jermanson. Bioconjugate Techniques (Third Edition). Elsevier Inc. 2013

Cummings TF, Shelton R. Mannich Reaction Mechanisms. (1990) J. Org Chem. 25(3): 419—423.