Affinity Chromatography

There are several bacterially derived proteins that have a strong affinity for the constant regions of immunoglobulins that can form the basis of affinity purification schemes (see Table 2). Of these, protein A and G have found the widest application. Protein A binds well to IgG from humans (apart from IgG3), rabbit, and guinea pig (1). It is less useful for the purification of mouse IgG as it has a lower affinity for IgG1 and IgG3 from that species (see Table 2). Protein G is produced by some strains of streptococci and this protein will bind to all the human IgG subclasses and also has a stronger affinity for mouse IgG (see Note 20 and ref. 8). Both protein A and G bind predominantly to the Fc region of IgG, but they do also bind to sites in the variable domain of the heavy chain and so will also have some affinity for antibody fragments. However protein L, produced by Peptostreptococcus magnus, binds exclusively to the kappa light chain (9) and may be more useful in specific cases and particularly for the purification of antibody fragments (see Subheading 3.3.). Jacalin, a lectin isolated from the seeds of the jack fruit (Artocarpus heterophyllus), has an affinity for D-galactose and has been shown to bind to IgA1. Although this has been used to develop an affinity purification scheme for IgA1 (10), jacalin will also bind to other serum proteins, such as haemopexin and C1-inhibitor (11).

Agarose-based chromatography media provide a suitable basis for the preparation of affinity chromatography supports and of the many choices available Sepharose 4B is suitably versatile. Protein ligands can be readily attached to form suitable matrices for affinity chromatography by activation of the agarose with cyanogen bromide producing a derivative, which will couple to the amino groups of proteins at basic pH. The activation process is not difficult but requires access to an efficient fume-hood as cyanogen bromide is toxic, forming hydrogen cyanide at acid pH, and thus it may be prudent to purchase pre-activated resin. Protein A agarose and protein G agarose can also be purchased. The protocol (see Subheading 3.2.3.1.) is suitable for protein A, G, or L. Purification of antibodies by affinity chromatography has been reviewed recently by Huse et al. (12).

Table 2

Binding of Protein A, G, and L to the Immunoglobulin Classes and Subclasses of Different Species

Table 2

Binding of Protein A, G, and L to the Immunoglobulin Classes and Subclasses of Different Species

Protein A

Protein G

Protein La

Human

IgG1

+++++

+++++

+++

IgG2

+++++

+++++

+++

IgG3

++

+++++

+++

IgG4

+++++

+++++

+++

IgM

+

-

+++

IgE

++

-

+++

IgA

+

-

+++

IgD

-

-

+++

Mouse

IgG1

++

++++

++++

IgG2a

+++++

+++++

++++

IgG2b

+++++

+++++

++++

IgG3

+++++

+++++

++++

Rabbit

IgG

+++++

++++

+

Goat

IgG

+

+++

-

Sheep

IgG

++

++++

-

Dog

IgG

+++++

++

+

Pig

IgG

+++++

++++

++++

Rat

IgG

+

++

++++

Cow

IgG

++

++++

-

Horse

IgG

++

++++

+/-

Guinea pig

IgG

+++++

++++

+++

Hamster

IgG

+++

++++

++++

Donkey

IgG

+++

+++++

-

Chicken

IgY

-

-

-/+

Strong binding, +++++; weak binding +; no binding -. "Protein L binds to the k light chain and thus will not bind to all classes of immunoglobulin.

Strong binding, +++++; weak binding +; no binding -. "Protein L binds to the k light chain and thus will not bind to all classes of immunoglobulin.

3.3.3.1. Preparation of Cyanogen Bromide-Activated Agarose

It should be stressed that cyanogen bromide is very toxic and thus it is particularly important to consult and adhere to local safety regulation regarding its use, the decontamination of containers and equipment and the disposal of solutions used. Purchase the mini mum amount of cyanogen bromide needed for the procedure. The procedure should be conducted in an efficient fume cupboard. The activated agarose prepared by this method is not stable and the ligand coupling should be conducted immediately.

1. Prepare the ligand solution in advance by dissolving the protein A/ G/L at a concentration of 2-5 mg/mL (using 5 mg per mL of agarose) in coupling buffer (0.1 M bicarbonate buffer, 0.5 M NaCl, pH 8.3; see Note 21).

2. Wash Sepharose 4B with a 100-fold excess of water, resuspend in water, and allow to settle for 30 min. Note the settled volume and remove the excess water.

3. Add an equal volume of 0.1 M sodium carbonate, pH 10.5, and mix gently while monitoring the pH with an electrode.

4. In a fume cupboard, rapidly weigh out 0.5 g of cyanogen bromide per 5 mL of settled volume of Sepharose.

5. Add the cyanogen bromide to the stirring Sepharose and maintain the pH between 10.5 and 11.0 by the addition of 5 M NaOH (see Note 22).

6. Once the pH has stabilized, filter the reaction mixture using a Buchner funnel and wash the Sepharose with ice-cold PBS (or the appropriate buffer for the subsequent ligand-coupling step). Note that the filtrate and washings will contain cyanogen bromide and must be disposed of in an appropriate manner (see Note 23).

7. Proceed to step 5 of the next section.

3.3.32. Coupling of Protein to Cyanogen Bromide-Activated Agarose

If purchased, cyanogen bromide-activated agarose will be in a dried form in the presence of stabilizers and will require swelling and washing before use. The activated groups are liable to hydrolysis at alkaline pH and thus the swelling and washing is carried out at acid pH.

1. If necessary swell the activated-agarose for 30 min at room temperature using 200 mL of 1 mM HCl per gram of agarose. One gram of dry weight will result in approx 3.5 mL of swollen gel.

2. Remove the acid using a sintered glass filter and a Buchner flask and wash with a further 200 mL of 1 mM HCl.

3. Dissolve the protein at a concentration of 2-5 mg/mL (using 5 mg per mL of agarose) in coupling buffer ( 0.1 M bicarbonate buffer, 0.5 M NaCl, pH 8.3; see Note 21).

4. Wash the activated agarose with 500 mL of coupling buffer. Remove the buffer by filtration, taking care not to allow the agarose to dry out.

5. Quickly add the ligand solution and mix gently for 2 h at room temperature, or overnight at 4°C (see Note 24).

6. Any unreacted groups on the agarose can be blocked by the addition of an excess of amino groups (1 mL of 1 M ethanolamine or 0.5 M glycine per milliliter of gel) and continuing to mix gently for another 2 h at room temperature, or overnight at 4°C.

7. Wash successively with 500 mL of coupling buffer, 0.1 M acetate buffer, pH 4.0, and coupling buffer to remove the unbound protein and store the agarose at 4°C until required (see Note 25).

3.3.3.3. Purification of Immunoglobulin Using Protein A, G, or l-Agarose

1. Equilibrate column with five-column volumes of 0.05 M phosphate, 0.1 M NaCl, pH 7.4, and apply the sample (see Note 26).

2. Wash the column with 10-column volumes of 0.05 M phosphate, 0.1 M NaCl, pH 7.4, or until the absorbance of the eluate at 280 nm is less than 0.05.

3. Elute the bound immunoglobulin by washing the column with 0.1 M glycine/HCL pH 2.5. Collect the eluate into 1 M Tris-HCl (0.1 mL per milliliter of eluate) in order to neutralize the solution and minimize denaturation of the protein.

4. Dialyze the immunoglobulin fractions against a suitable buffer and store at 4°C (see Note 11).

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