Cleavage of polyclonal IgG by proteolytic enzymes

1.7 Cleavage of polyclonal IgG by proteolytic enzymes

In the pre-antibiotic era a major use of immunoglobulins was in immunotherapy. Immuno- globulins from animals immunized with antigens such as tetanus and diphtheria toxin were used to passively immunize patients suffering from diseases caused by these toxins. Unfortunately, these foreign immunoglobulins were themselves immunogenic and could often provoke a type

III hypersensitivity serum sickness. To try to reduce the immunogenicity of these foreign immunoglobulins, they were treated with a range of proteolytic enzymes including pepsin and papain. These enzymes were known to cleave the antibodies into active and inactive fragments. In 1958 Porter exploited these observations in his Nobel prize-winning studies on antibody structure.

Many enzymes have now been introduced for the production of immunoglobulin fragments, but papain and pepsin are still the two most commonly used in the immunology laboratory. Most of the accumulated wisdom on enzymic fragmentation applies to human and rabbit immunoglobulin. Digestion conditions have been determined using biochemically heterogene- ous polyclonal antibodies and so relate only to the average properties of the mixture.

With the advent of the hybridoma technique for producing monoclonal immunoglobulins it has become necessary to produce similar fragments from mouse immunoglobulins. Two problems thus present themselves in that: (i) far less is known about the fragmentation of mouse immunoglobulins in general; and (ii) each monoclonal antibody is unique and requires its own digestion conditions.

1.7.1 Papain digestion

Papain is an enzyme that contains active site cysteine. It splits the IgG molecule to the N-terminal side of the disulphide bonds linking the two heavy chains, thus giving rise to two Fab fragments and one Fc fragment.

Soluble papain is perfectly adequate for most uses but does result in some contamination with residual papain. Insoluble papain can be prepared by attaching the enzyme to a support such as agarose using cyanogen bromide. Alternatively it may be purchased attached to agarose or CM–cellulose (car- boxymethyl cellulose) from Sigma. Insoluble enzymes may use the same conditions as soluble enzymes for protein degradation experiments and often result in more discrete fragments. The advantage of the insoluble enzyme preparations is that the reaction may be easily stopped by centrifugation at slow speed to pellet the enzyme.

Rabbit IgG

MATERIALS AND EQUIPMENT IgG (rabbit)

Papain Cysteine Ethylene diamine tetra-acetic acid (EDTA), disodium salt Carboxymethyl cellulose (CM–cellulose) (30 × 2.5.cm) Sodium acetate buffers, 0.01 M and 0.9 M , pH 5.5

34 C H A P T E R 1: Isolation and structure of immunoglobulins

Phosphate-buffered saline (PBS) Chromatography column or 40-ml disposable syringe Fraction collector

METHOD

1 Adjust the concentration of the IgG to 20 mg/ml in PBS.

2 Add cysteine to a final concentration of 0.02 M .

3 Add EDTA to a final concentration of 0.002 M .

4 Add 1 mg papain for every 100 mg IgG used.

5 Incubate at 37°C for 4 h.

6 Dialyse the digest against 0.01 M sodium acetate buffer, pH 5.5. When the cysteine and EDTA are removed by dialysis, the enzyme is inactivated. More controlled termination of the reaction can be obtained by adding iodoacetamide to a final concentration of 0.1 mg/ml.

7 Apply the dialysate to a CM–cellulose column. This column should be prepared as for DEAE–cellulose (see Section 1.3) but should be pre-equilibrated with 0.01 M sodium acetate buffer, pH 5.5.

8 Allow 200 ml 0.01 M sodium acetate buffer, pH 5.5, to run through the column.

9 Apply a linear gradient of increasing ionic strength (starting buffer 0.01 M sodium acetate, pH 5.5; limit buffer 0.9 M sodium acetate, pH 5.5).

10 Collect and concentrate each peak. The first two peaks contain Fab, while the third smaller peak contains the Fc.

11 Dialyse against PBS. Crystals of the Fc fragment may form at this stage.

Note: It is possible to obtain pure fragments by a two-stage ion-exchange separation; the fraction- ation of the products of digestion is more complex. The first is on CM–cellulose followed by refractionation on DEAE–cellulose (see Section 1.3 and Fig. 1.9).

Human IgG

Human IgG subclasses differ in their sensitivity to papain, in the order: IgG 3 > IgG 1 > IgG 4 > IgG 2 . Any one set of conditions will have a tendency to overdigest some subclasses while underdigesting

Fig. 1.9 Elution profile of papain digest of mouse IgG from

DEAE–cellulose. After digestion with papain the sample was equilibrated in 0.005 m phosphate buffer, pH 8.0, and

Absorbance at 280 nm

applied to a DEAE–cellulose column

equilibrated in the same buffer. The column was eluted with a linear

0 10 20 30 40 50 60 70 gradient, limit buffer 0.2 m phosphate,

Tube number

pH 8.0 (see also Fig. 1.10.) 1.7CLEAVAGE OF POLYCLONAL IGG BY PROTEOLYTIC ENZYMES

IgG Fc regions. Protein A has no affinity for the Fab region. As IgG 3 does not bind to protein A, the IgG preparation should be selected for protein A-binding species before digestion. Some of the IgG invariably remains undegraded after digestion so it is necessary to remove this on a gel filtration column before use of the protein A column.

MATERIALS AND EQUIPMENT Supadex-75 Chromatography column and fraction collection apparatus Polyethylene glycol 8000 (PEG-8000) Protein A–Sepharose CL-4B Phosphate-buffered saline (PBS)

1.0 M sodium hydroxide

0.1 M glycine–HCl buffer, pH 2.8 Dialysed digest (human IgG papain digest prepared as for rabbit IgG, steps 1–6, previous page)

METHOD

1 Prepare a Supadex-75 column and equilibrate with PBS (see Appendix B.1).

2 Pass the dialysed digest through the column and collect fractions.

3 Any undigested IgG will come through in the breakthrough volume. The Fab and Fc will come later in one peak.

4 Concentrate the Fab/Fc peak to the pregel filtration volume, e.g. by placing in a dialysis bag and covering with PEG-8000.

5 Prepare a protein A–Sepharose CL-4B column (see Section 1.4.2).

6 Apply the Fab/Fc peak to this column and wash through with PBS. The capacity of the column for binding Fc is 8 mg/ml of swollen gel.

7 Collect the Fab which comes straight through the column.

8 Elute the bound Fc with glycine–HCl buffer, pH 2.8.

9 Titrate the pH of the purified Fc to near neutrality with NaOH and then dialyse against PBS.

10 Regenerate the column by running through 2 column volumes of PBS. Store the column

at 4°C.

Examination of fragments The fragments can be analysed by either:

(a) SDS-PAGE (both under reducing and non-reducing conditions) (see Appendix B.2.1) to determine the molecular weights of the fragments. Then blot the fragments onto nitrocellulose and con- firm their immunological identities with specific antisera (see Section 4.11); or alternatively:

(b) immunoelectrophoresis which is a simple technique for examining fragments (see Section 3.7.2; see Fig. 1.10 illustrating mouse fragments), although this will have less sensitivity for detecting contaminants.

36 C H A P T E R 1: Isolation and structure of immunoglobulins

Fig. 1.10 Immunoelectrophoresis of fractions from DEAE –cellulose chromatography of papain digest

of mouse IgG (from Fig. 1.9). Sample a: peak 1, DEAE–cellulose, Fab fragments. Sample b: original IgG. Sample c: peak 2, DEAE–cellulose, Fc fragments.The proteins were visualized by precipitation with rabbit anti-mouse whole IgG. (Photograph of unstained preparation.)

1.7.2 Pepsin digestion

Digestion of IgG yields a fragment with two-thirds the molecular weight of the original mole- cule but with intact, divalent antigen-binding activity. This is the F(ab′) 2 fragment. The other one-third of the IgG molecule is digested into a smaller pFc′ fragment corresponding to the

C H 3 domains held together non-covalently, while the C H 2 domains are digested away to small peptides.

MATERIALS AND EQUIPMENT IgG (rabbit or human) Pepsin

0.1 M sodium acetate Phosphate-buffered saline (PBS) Acetic acid, glacial Sephacryl S-200 column equilibrated with PBS

METHOD

1 Adjust the IgG solution to 20 mg/ml and dialyse 10 ml against 0.1 M sodium acetate for 3 h.

2 Adjust the dialysate to pH 4.5 with acetic acid.

3 Add 2 mg pepsin for each 100 mg of IgG used.

4 Incubate at 37°C overnight.

5 Adjust the supernatant to pH 7.4. This inactivates the enzyme.

6 Centrifuge at 1000 g for 15 min and discard any precipitate that may form.

Analysis and isolation of fragments Apply the digest to a gel filtration column, e.g. Sephacryl S-200, and equilibrate the column

with PBS, monitor the eluate and collect fractions. Generally this will give fragments of sufficient purity but recycling on a column will get rid of any remaining contamination (see Fig. 1.11). Peak 1 contains some undigested IgG; the amount will vary from preparation to preparation and

may only show as a slight bulge on the leading edge of the second peak.

Peak 2 contains the F(ab′) 2 .

Peak 4 contains pFc′, the small C H 3 fragment, while the remaining material is composed of small peptides.

1.7CLEAVAGE OF POLYCLONAL IGG BY PROTEOLYTIC ENZYMES

1 3 Fig. 1.11 Elution profile of a pepsin

Absorbance at 280 nm digest of IgG on gel filtration. A 2 ml 4 sample of digest (20 mg/ml) was

applied to gel filtration column (bed Elution volume (ml)

volume 90 × 2.5 cm) and equilibrated with phosphate-buffered saline.