The Protein Man's Blog | A Discussion of Protein Research

Immunoaffinity purification utilizes the unique high specificity of purified antibodies (polyclonal and monoclonal) immobilized onto a solid matrix (porous agarose beads) to rapidly and selectively purify target analytes from a complex mixture. Optimal elution conditions allow for the efficient purification of an active analyte while still allowing later regeneration of the immobilized antibodies.

This starts with the process of adsorption, where the target antigen binds to the immobilized antibody by its specific affinity for that antibody. Adsorption is most efficient in aqueous buffers under physiological conditions (PBS, neutral pH 7.0-7.4). Following antigen capture, the unbound proteins and proteins bound by nonspecific interactions are washed away.

The elution of the target antigen from the immobilized antibody (or desorption) is the most delicate step of immunoaffinity purification. The target antigen can be desorbed from the immobilized antibody by counteracting the four forces that stabilize the antigen-antibody complex (ionic, hydrogen bonding, van der Waals interactions and hydrophobic bonds) which are all reversible.  This can be accomplished with different elution conditions that weaken the antibody-antigen affinity interactions, but some are harsh and must be carefully chosen to maintain antigen activity.

Common Immunoaffinity elution strategies:

• Altering the pH
• Altering the ionic state
• Using a denaturant
• Using chaotropic agents

The appropriate elution strategy varies depending on the type of target molecule and the composition of its chemical binding properties. The most common elution method is to lower the pH with 0.1 M glycine HCl, pH 2.5-3.0, which disrupts the ionic and hydrogen bonds between the antigen and antibody. However, some antibodies and proteins are damaged by low pH, but can be neutralized by adding 1/10^th volume of alkaline buffer, 1M Tris HCl pH 8.5, immediately after recovery. If lowering the pH is not effective, then the next best option is to use a commercially available elution buffer such as G-Biosciences IgG Elution Buffer or Gentle IgG Elution Buffer, which is designed to destabilize the antigen-antibody complex without denaturation or inactivation.

Some less-common and harsher elution strategies for dissociating high affinity antibody-antigen complexes include the use of chaotropic agents, denaturants, competing agents, organic modifiers, or altering temperature. These methods must be considered cautiously because they can damage the proteins by disrupting structure, resulting in low yields and activity of the purified protein. For example, chaotropic salts denature and/or alter a protein’s ionic strength by disrupting the hydrogen bonding network between macromolecules in a solution. Some typical chaotropic agents are thiocyanate (SCN^-), trifluoroacetate (CF₃COO^-), perchlorate (ClO₄ ^-), iodide (I^-) or chloride (Cl^-) (listed in order from strongest to mildest). Denaturing agents, like urea and guanidinium hydrochloride, affect elution by promoting protein unfolding. Organic modifiers can be used, but they can permanently denature antibodies when used at high concentrations. The most extreme eluent is the detergent sodium dodecyl sulfate (SDS) which denatures and inactivates the antibody-antigen complex. It may be worthwhile to sacrifice the activity of the target antigen and antibody by using SDS, because it prepares the antigen for SDS-PAGE and mass spectrometric analysis which can be used to identify the target molecule with certainty.

G-Biosciences Elution Buffers for Immunoaffinity Purification

• IgG Elution Buffer (Cat# 786-205, 786-206, 786-545): Amine based, acidic (pH2.8) buffer that effectively dissociates most protein-protein and antibody-antigen binding interactions without permanently affecting protein structure suitable for most immunoaffinity purification systems.
  • Note: Some antibodies can be damaged by low pH
• Gentle IgG Elution Buffer (Cat# 786-200, 786-201, 786-202): High salt, near neutral, phosphate free buffer. Elutes pH sensitive antibodies and antigens without denaturation or inactivation.

Table 1. Summary of commonly used elution conditions

 Here's some more topics that you may find interesting:

• How to generate an antibody affinity column for protein purification?
• CNBr-activated resin to immobilize ligands for affinity chromatography
• Epoxy-activated resin: a versatile affinity chromatography support