Affinity Chromatography and its Applications in Diagnostic Virology

Pashu Sandesh, 06 March 2023

Dr Ragini Mishra1 and Dr Sonam Bhardwaj2


Affinity chromatography is a type of liquid chromatography for the separation, purification or specific analysis of sample components. Separation and purification of analytes by affinity chromatography are unlike most other forms of chromatography as it exploits the unique property of specific biological interactions to achieve separation and purification. Theoretically, it is capable of giving absolute purification, even from complex mixtures, in a single process. It was first developed by Arne Wilhelm Tiselius in the 1930s, and he won the Nobel Prize in 1948. This technique was originally developed for the purification of enzymes, but it has since been extended to nucleotides, nucleic acids, immunoglobulins, membrane receptors and even to whole cells and cell fragments.


The principle behind affinity chromatographic separation is based on a specific binding interaction between an immobilized ligand and its binding partner, e.g. antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions. It utilizes the reversible biological interaction or molecular recognition called affinity which refers to the attracting force exerted in different degrees between atoms which causes them to remain in combination. It offers high selectivity, resolution, and capacity in most protein purification schemes. It has the advantage of utilizing a protein's biological structure or function for purification. As a result, purifications can often be easily achieved with affinity chromatography. The technique requires that the material to be isolated is capable of binding reversibly to a specific ligand that is attached to an insoluble material. Under the correct experimental conditions, when a complex mixture containing the specific compound to be purified is added to the immobilized ligand (generally contained in a conventional chromatography column) only that compound will bind to the ligand.  All other compounds can therefore be washed away and the compound subsequently recovered by displacement from the ligand.

Components of affinity chromatography

1. Matrix

  • The matrix is an inert support to which a ligand can be directly or indirectly coupled.
  • In order for the matrix to be effective it must have certain characteristics.
  • Matrix should be chemically and physically inert.
  • It must be insoluble in solvents and buffers employed in the process
  • It must be chemically and mechanically stable.
  • It must be easily coupled to a ligand or spacer arm onto which the ligand can be attached.
  • It must exhibit good flow properties and have a relatively large surface area for attachment.
  • The most useful matrix materials are agarose and polyacrylamide.

2. Spacer arm

  • It is used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance.

3. Ligand

  • It refers to the molecule that binds reversibly to a specific target molecule.
  • The ligand can be selected only after the nature of the macromolecule to be isolated is known.
  • When a hormone receptor protein is to be purified by affinity chromatography, the hormone itself is an ideal candidate for the ligand.
  • For antibody isolation, an antigen or hapten may be used as a ligand.
  • If an enzyme is to be purified, a substrate analogue, inhibitor, cofactor, or effector may be used as an immobilized ligand.

Steps involved in affinity chromatography

Affinity medium is equilibrated in binding buffer. The sample is applied under conditions that favour specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes through the column. Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. The target protein is collected in a purified, concentrated form. The affinity medium is re-equilibrated with a binding buffer.

  These events can be summarized into the following three major steps:

1. Preparation of column

  • The column is loaded with a solid support such as sepharose, agarose, cellulose etc.
  • Ligand is selected according to the desired isolate.
  • The Spacer arm is attached between the ligand and solid support.

2. Loading of sample

  • A solution containing a mixture of substances is poured into the elution column and allowed to run at a controlled rate.

 3. Elution of ligand-molecule complex

  • The target substance is recovered by changing conditions to favour the elution of the bound molecules.        


Affinity chromatography is one of the most useful methods for separating and purifying specific products. It is essentially a sample purification technique, used primarily for biological molecules such as proteins.

 Its major application includes:

  • Separation of a mixture of compounds
  • Removal of impurities or in the purification process
  • In enzyme assays
  • Detection of substrates
  • Investigation of binding sites of enzymes
  • In in vitro antigen-antibody reactions
  • Detection of Single Nucleotide polymorphisms and mutations in nucleic acids

Applications in diagnostic virology

The specific use of immuno-affinity chromatography has proven to be an effective purification method, where an antibody directed towards a target protein is utilized. For example, an immuno-affinity chromatography technique for the purification of infective bluetongue virus (BTV) has been described using anti-core antibodies. BTV anti-core antibodies (prepared in guinea pig) were mixed with cell culture-grown BTV-1 and then the mixture was added to the cyanogens bromide-activated protein-A Sepharose column. Protein A binds to the antibody which in turn binds to the antigen (i.e. BTV). After thorough washing, antigen–antibody and antibody-protein A couplings were dissociated with 4M MgCl2, pH 6.5. Antibody molecules were removed by dialysis and virus particles were concentrated by spin column ultrafiltration. Dialyzed and concentrated material tested positive for BTV antigen by a sandwich ELISA and the infectivity of the chromatography-purified virus was demonstrated in cell culture. The main component of the envelope of most viruses is the glycoprotein. These glycoproteins can be purified or separated using affinity chromatography. Immunoaffinity chromatography is a powerful tool for the specific capturing of viral particles and virus-encoded membrane proteins directly from the cell culture broth. Dye-ligand columns have also been described for the purification and separation of virus particles which can be further utilized as purified antigens in diagnostic tools or for vaccine development. The dye-ligand column can be used to remove prion proteins, human immunodeficiency virus-1, and hepatitis B viral particles from biological fluids. Heparin affinity chromatography has been described to produce a differential vaccine without eliciting antibodies against the nonstructural proteins of the serotype O of the FMD virus. This method can then be used to produce a higher-quality vaccine compared with the PEG application in terms of the purity of the FMD vaccine. Affinity chromatography can obtain highly purified antigens from the equine infectious anaemia (EIA) virus. For that, the crude antigen is concentrated by poly ethylene glycol precipitation of culture fluids from persistently infected equine dermal cells with EIA virus, and the virus is disrupted with ether, and then added to a column of cyanogen bromide-activated Sepharose 4B to which EIA-specific antibody has been conjugated. The purified antigen is effectively released from the column with 5M MgCl2. This purified antigen can then be used to perform passive hemagglutination (HA) tests on sera from EAI infections. Thus affinity chromatography plays an important role in diagnostic virology and helps in the separation of various agents.

Dr Ragini Mishra1 (PhD Scholar- Division of Veterinary Microbiology, ICAR-IVRI, Izatnagar-243122, Bareilly)

Dr Sonam Bhardwaj2 (PhD Scholar- Division of Livestock Production and Management, ICAR-IVRI, Izatnagar-243122, Bareilly)