Making Immunospecific AC Media with Custom Ligands

Extracted from Affinity Chromatography Vol. 1: Antibodies, GE Healthcare, 2016

If an AC medium is not available, a ligand (such as a pure antigen or an antibody) can be covalently coupled to a suitable matrix to create an immunospecific affinity medium for purification. Although this process requires careful development and optimization, it is often worthwhile, for example when a specific protein needs to be prepared on a regular basis. Immunospecific purification is particularly useful if the target molecules bind weakly or not at all to protein A or protein G and can also be used to remove key contaminants.

This section describes the simplest coupling method, that is, when a ligand is coupled

via its primary amine group to a pre-activated medium. GE offers two pre-activated AC media for coupling of antigen or antibody ligands: NHS-activated Sepharose® 4 Fast Flow, which is available in bulk packs for packing columns, and NHS-activated Sepharose® High Performance, which is available in convenient, prepacked HiTrap™ NHS-activated HP columns.

The excellent hydrophilic properties of the base matrices of NHS-activated Sepharose® chromatography media minimize nonspecific adsorption of proteins that can reduce the binding capacity of the target protein. The pH range for coupling is well suited to the stability characteristics of many immunoglobulins. Furthermore, the chromatography media are stable at  high pH to allow stringent washing procedures (subject to the pH stability of the coupled ligand).

NHS-activated Sepharose® chromatography media are used for the initial capture step; a size exclusion chromatography step is commonly used after immunocapture to ensure a highly pure and homogenous target protein (removal of monomers, dimers, and any leached ligand).

If no primary amine group on the ligand to be coupled is available, ligand attachment via carboxyl, thiol, or hydroxyl groups can be considered. These procedures are described in the handbook Affinity Chromatography: Principles and Methods (18102229).

A pure ligand is required that has a proven reversible high affinity for the target molecule. Using an antigen or an anti-antibody as a ligand will give a high degree of purification. If possible, test the affinity of the interaction.

Immunospecific interactions often require harsh elution conditions. Collect fractions into a neutralizing buffer, such as 60 to 200 µl 1 M Tris-HCl, pH 9.0 per milliliter fraction.


Figure 3.35 shows the partial purification of an IgE-stimulating factor from a human T-cell line, using IgE as the specific affinity ligand coupled to HiTrap™ NHS-activated HP 1 ml column. Figure 3.36 shows purification of anti-mouse Fc-IgG from sheep serum using mouse IgG1 coupled to HiTrap™ NHS-activated HP 1 ml column.

Purification of an IgE-stimulating factor from a human T-cell line.

Fig 3.35. Purification of an IgE-stimulating factor from a human T-cell line.


Purification of anti-mouse Fc-IgG from sheep antiserum.

Fig 3.36. Purification of anti-mouse Fc-IgG from sheep antiserum.

Coupling ligands to HiTrap™ NHS-activated HP columns

The protocol below describes the preparation of a prepacked HiTrap™ NHS-activated HP column and a recommendation for a preliminary purification protocol. Many of these details are generally applicable to NHS-activated Sepharose® chromatography media. Coupling can take place within the pH range of 6.5 to 9.0 with a maximum yield achieved at around pH 8.0.

A general column packing procedure is described in Appendix 5 (Column packing and preparation).

Buffer preparation
Acidification solution
: 1 mM HCl (kept on ice)

Coupling buffer: 200 mM sodium hydrogen carbonate, 500 mM sodium chloride, pH 8.3

Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.45 µm filter before use.

The activated product is supplied in 100% isopropanol to preserve the stability prior to coupling. Do not replace the isopropanol until it is time to couple the ligand.


Ligand and HiTrap™ column preparation

  1. Dissolve the desired ligand in the coupling buffer to a final concentration of 0.5 to 10 mg/ml (for protein ligands) or perform a buffer exchange using a desalting column (see Chapter 2). The optimal concentration depends on the ligand. Dissolve the ligand in one column volume of coupling buffer.
  2. Remove the top cap and apply a drop of acidification solution to the top of the column to avoid air bubbles.
  3. Connect the Luer adapter (or tubing if using a pump or system) to the top of the column.
  4. Remove the snap-off end at the column outlet.


Ligand coupling

  1. Wash out the isopropanol with acidification solution. Use 3 × 2 ml for HiTrap™ 1 ml and 3 × 10 ml for HiTrap™ 5 ml.

Do not exceed flow rates of 1 ml/min for HiTrap™ 1 ml columns and 5 ml/min for HiTrap™ 5 ml columns at this stage to avoid irreversible compression of the prepacked medium.

  1. Immediately inject 1 ml (HiTrap™ 1 ml) or 5 ml (HiTrap™ 5 ml) of the ligand solution onto the column.
  2. Seal the column and leave for 15 to 30 min at 25°C or 4 h at 4°C*.

* Coupling efficiency can be measured after this step. Procedures are supplied with each HiTrap™ NHS-activated HP column.

If larger volumes of ligand solution are used, recirculate the solution by connecting a second syringe to the outlet of the column and gently pump the solution back and forth for 15 to 30 min. Recirculation can also be performed by connecting a peristaltic pump, for example, Pump P-1.

Washing and deactivation

Deactivate any excess active groups that have not coupled to the ligand, and wash out the non-specifically bound ligands, by following the procedure below:

Buffer A: 500 mM ethanolamine, 500 mM sodium chloride, pH 8.3
Buffer B
: 100 mM acetate, 500 mM sodium chloride, pH 4.0

  1. Inject 3 × 2 ml (HiTrap™ 1 ml) or 3 × 10 ml (HiTrap™ 5 ml) of Buffer A.
  2. Inject 3 × 2 ml (HiTrap™ 1 ml) or 3 × 10 ml (HiTrap™ 5 ml) of Buffer B.
  3. Inject 3 × 2 ml (HiTrap™ 1 ml) or 3 × 10 ml (HiTrap™ 5 ml) of Buffer A.
  4. Leave the column for 15 to 30 min at room temperature or approximately 4 h at 4 °C.
  5. Inject 3 × 2 ml (HiTrap™ 1 ml) or 3 × 10 ml (HiTrap™ 5 ml) of Buffer B.
  6. Inject 3 × 2 ml (HiTrap™ 1 ml) or 3 × 10 ml (HiTrap™ 5 ml) of Buffer A.
  7. Inject 3 × 2 ml (HiTrap™ 1 ml) or 3 × 10 ml (HiTrap™ 5 ml) of Buffer B.
  8. Finally, inject 2 ml (HiTrap™ 1 ml) or 10 ml (HiTrap™ 5 ml) of a buffer with neutral pH to adjust the pH.


Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, for example phosphate-buffered saline (PBS), 0.05% sodium azide, pH 7.2.

pH stability of the chromatography medium when coupled to the selected ligand depends on the stability of the ligand. Sodium azide can interfere with many coupling methods and some biological assays. It can be removed using a desalting column.

Sodium azide is carcinogenic, handle with care.

Performing a purification on a coupled HiTrap™ NHS-activated column

Use high quality water and chemicals. Filtration through 0.45 µm filters is recommended. Optimal binding and elution conditions for purification of the target protein must be determined separately for each ligand (see below for suggested elution buffers). The general protocol given here can be used for preliminary purification.

For the first run, perform a blank run to ensure that any loosely bound ligand is removed. Samples should be centrifuged immediately before use and/or filtered through a 0.45 µm filter. If the sample is too viscous, dilute with binding buffer.

Sample binding properties can be improved by adjusting the sample to the composition of the binding buffer. Perform a buffer exchange using a desalting column (see Chapter 2) or dilute the sample in binding buffer.

  1. Prepare the column by washing with:
    (i). 3 ml (HiTrap™ 1 ml) or 15 ml (HiTrap™ 5 ml) binding buffer.
    (ii). 3 ml (HiTrap™ 1 ml) or 15 ml (HiTrap™ 5 ml) elution buffer (see below for advice on elution buffers).
  2. Equilibrate the column with 10 column volumes of binding buffer.
  3. Sample preparation. The sample should be adjusted to the composition of the binding buffer. This can be done by either diluting the sample with binding buffer or by buffer exchange or desalting (see Desalting and buffer exchange). The sample should be filtered through a 0.45 µm filter or centrifuged immediately before it is applied to the column.
  4. Apply the sample, using a syringe fitted to the Luer adapter or by pumping it onto the column. Recommended flow rates: 0.2 to 1 ml/min (HiTrap™ 1 ml) or 1 to 5 ml/min (HiTrap™ 5 ml)*. The optimal flow rate is dependent on the binding constant of the ligand.
  5. Wash with binding buffer, 5 to 10 column volumes or until no material appears in the effluent. Excessive washing should be avoided if the interaction between the protein of interest and the ligand is weak, since this can decrease the yield.
  6. Elute with elution buffer; 1 to 3 column volumes is usually sufficient but larger volumes might be necessary.
  7. The purified fractions can be desalted (see Desalting and buffer exchange).
  8. Re-equilibrate the column by washing with 5 to 10 column volumes of binding buffer. The columns is now ready for a new purification of the same kind of sample.

* 1 ml/min corresponds to approximately 30 drops/min when using a syringe with a 1 ml HiTrap™ column; 5 ml/min corresponds to approximately 120 drops/min when using a syringe with a 5 ml HiTrap™ column.

To preserve the activity of acid-labile IgG, we recommend adding 60 to 200 µl of  1 M Tris-HCl pH 9.0 to collection tubes, which ensures that the final pH of the sample will be approximately neutral.


Elution buffers

Immunospecific interactions can be very strong and sometimes difficult to reverse. The specific nature of the interaction determines the elution conditions. Always check the reversibility of the interaction before coupling a ligand to an affinity matrix. If standard elution buffers do not reverse the interaction, alternative elution buffers that can be considered are listed below:

  • Low pH (below pH 2.5)
  • High pH (up to pH 11.0)
  • Substances that reduce the polarity of a buffer can facilitate elution without affecting protein activity: dioxane (up to 10%), ethylene glycol (up to 50%).


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