Purification of antibodies

1.5 Purification of antibodies

An antibody reacts specifically with its own antigenic determinant to form an antigen–antibody complex. An animal immunized with an antigen will respond to produce antibodies all react- ing with the antigen to some degree. Serum from this animal will have the usual range of

1.5PURIFICATION OF ANTIBODIES 1.5PURIFICATION OF ANTIBODIES

An animal receiving a transplantable plasmacytoma or hybridoma will produce large amounts of the monoclonal immunoglobulin or antibody, but there will still be a significant background of normal serum proteins and immunoglobulins, even in ascitic fluid.

To study a particular antibody in detail it is of great advantage to be able to separate it from the surrounding non-specific antibody molecules using the antigen. Then to obtain reactive purified antibody we must separate the complex and remove the antigen.

The forces binding antibody to antigen are those involved in any protein–protein interaction: (a) coulombic; (b) dipole; (c) hydrogen bonding; (d) van der Waals’; (e) hydrophobic bonding.

All these forces depend upon the charge of the molecules taking part in the reaction. The net charge of the molecules in turn depends on the pH of the medium. If the pH of the medium is lowered sufficiently the protein molecules change conformation, gain H + ions and so repel each other. We are now faced with the problem of physically removing the antigen or the antibody, because when the pH is returned to neutrality the complexes will re-form.

If the antigen is insoluble it can be easily separated from soluble antibody. There are many methods available for rendering either the antigen or antibody insoluble, some of which are described in the following sections.

1.5.1 Preparation of a protein immunoadsorbent

In this experiment antibodies to mouse immunoglobulin are purified but the identical method can be used for other proteins.

MATERIALS AND EQUIPMENT Sepharose 4B Cyanogen bromide (this chemical is very toxic and must be handled in a fume cupboard )

2.0 M sodium hydroxide Phosphate-buffered saline (PBS) Borate–saline buffer, pH 8.3, ionic strength 0.1 Mouse immunoglobulin Sintered glass funnel UV spectrophotometer

METHOD

1 Pipette 14 ml of Sepharose (about 200 mg) into a 50-ml glass beaker and add 10 ml of distilled water. All procedures must now be carried out in a fume cupboard.

2 Weigh a stoppered tube, add some solid cyanogen bromide, replace the stopper and reweigh the tube.

Continued

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

3 Dissolve the cyanogen bromide in distilled water to a final concentration of 50 mg/ml.

4 Place the Sepharose beads on a magnetic stirrer and titrate the pH to 11.0–11.5 with 2.0 M sodium hydroxide.

5 Add 10 ml of the cyanogen bromide solution.

6 Maintain the pH at 11.0–11.5 by dropwise addition of sodium hydroxide for 5–10 min until the pH becomes stable.

7 Wash the activated beads on a sintered glass funnel with 100 ml of water, and then 100 ml of borate–saline buffer.

8 Wash the beads into a glass beaker, allow them to settle and remove the supernatant.

9 Add 100 mg of mouse immunoglobulin at 5–10 mg/ml (initial concentration).

10 Leave the beads stirring with the protein overnight at 4°C (most of the uptake occurs within

the first 4 h and so this stage can be abbreviated).

11 Wash the beads on a sintered glass funnel with 10 ml PBS and collect the washings. (Use negative pressure and collect washings in a tube standing in a side-arm flask.)

12 Wash the beads thoroughly with PBS to remove the rest of the unbound immunoglobulin.

13 A UV spectrophotometer reading of the washings will give the amount of unbound protein and so the approximate quantity of protein bound to the column can be calculated. The immunoadsorbent is now ready for use. Store in PBS containing azide (0.1 M ).

Note: Azide is a dangerous chemicalado not discard down the sink.

TECHNICAL NOTES • In step 6, wash the gel with borate–saline buffer as soon as the pH becomes stable. The rate

of inactivation by hydrolysis is highly pH dependent and increases sharply above pH 9.5. • For maximum uptake, the coupling pH should be above the pK a of the protein, but below pH 10.0. • Avoid buffers containing amines; they will compete with the amino function on the

protein for the activated groups on the gel. Borate and bicarbonate buffers are the most useful; however, Tris buffers may be used as the amino group on the Tris moiety is sterically hindered.

• After coupling, it is possible to add 1.0 M glycine, pH 8.0, for 6 h at 4°C if you wish to be completely sure that all the activated hydroxyls have been derivatized.

1.5.2 Use of immunoadsorbent for antibody purification

MATERIALS AND EQUIPMENT Rabbit anti-mouse immunoglobulin Immunoadsorbent mouse immunoglobulin on Sepharose 4B (20 ml)

0.1 M glycine–HCl buffer, pH 2.5 Trichloroacetic acid (TCA), 10% aqueous solution Tris-(hydroxymethyl) aminomethane (Tris) Phosphate-buffered saline (PBS) Chromatography column or 20-ml disposable syringe Glass wool

1.5PURIFICATION OF ANTIBODIES

Preparation of the column and antigen–antibody complex

METHOD

1 Pour the immunoadsorbent into the column and equilibrate with 20 ml PBS. Close the column.

2 Run 20 ml of antiserum through the column ado not use positive pressure; allow to run under gravity.

3 Wash the unbound protein from the column until the absorbance measured in a flow through a UV cell is < 0.1, otherwise wash with 200 ml PBS. Close the column.

We now have the antigen–antibody complex. Dissociation of complex

METHOD

1 Pipette out 20 × 0.5 ml aliquots of TCA into small glass tubes. (Use this to sample the effluent for protein elution if a flow-through UV cell is not available.)

2 Add glycine–HCl buffer to the top of the column and collect the effluent when protein is first detected.

3 Stop collecting the effluent when protein is no longer detectable. The first stage of the elution is now complete and part of the antibody has been recovered. The acid elution buffer will, however, eventually denature the antibody so we must raise the pH.

4 Titrate the protein to pH 8.5 with solid Tris. Mix thoroughly and monitor with a pH meter or indicator papers. The elution conditions are altered to recover a second batch of antibody.

5 Add glycine–HCl plus 10% dioxane to the column. Monitor the effluent and collect the second batch of antibody.

6 Adjust the pH to 8.5 with solid Tris.

7 Read the absorbance of each protein solution at 280 nm and calculate the recovered protein. (Remember to use the buffer plus dioxane as reference for the spectrophotometer.)

8 Concentrate the samples in dialysis tubing with either sucrose or polyethylene glycol

40 000 or by negative-pressure dialysis (see also Appendix B.1.4).

9 When the sample volume has been reduced to 3.0 –5.0 ml, dialyse against 5 × 1 litre PBS.

10 Spin off the precipitate and determine the protein content of each sample.

This method of antibody purification is highly reproducible and so it is not necessary to calculate the antibody content of the sample routinely. However, a specimen calculation is given below.

TECHNICAL NOTES • Under the conditions described, the Sepharose should bind 90–100 mg of mouse immuno-

globulin. Approximately the same uptake can be expected with other common protein antigens, with the notable exception of bovine serum albumin where only 20–30 mg are bound.

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

Key:

0.1 ml original serum 1.0 300 µg purified

antibody

Weight of precipitate (mg) 0.2

Fig. 1.8 Precipitin curves of anti- immunoglobulin serum and antibody.

Concentration of antigen (µg)

• Although the proportion of antibody in the final sample is fairly constant, the actual yield of

antibody relative to the serum concentration varies with serum pool and species. The greatest loss of antibody occurs due to denaturation and precipitation after elution, concentration and dialysis.

• In the experiment, the immunoadsorbent has been used below its maximal capacity; in general it should be able to deplete 1 ml of antiserum for each mg of antigen on the column. • Pre-activated Sepharose is available commercially; this avoids the use of cyanogen bromide. For large-scale preparations, however, it is relatively expensive.

1.5.3 Calculation of recovery from immunoadsorbent

Total weight of immunoglobulin on column = 92.0 mg on 200 mg of Sepharose 4B. Volume of antiserum for antibody purification = 10 ml. Antibody content of serum calculated from Fig. 1.8, at equivalence condition. Antibody content of serum = 5.2 mg/ml. % yield of antibody protein from serum: immediately 81.5%; after concentration and dialysis 47.8%.

Eluates from immunoadsorbent Total protein concentration in eluate:

After concentration Eluant

Immediately

and dialysis

Glycine–HCl

⎪ Glycine–HCl + 10% dioxane

1.5PURIFICATION OF ANTIBODIES

Calculation of antibody content of eluate: From Fig. 1.8: weight of antibody in around 300 µg of eluted protein = 490 − 160

= 330 µg.* * Within limits of experimental error

Hence, all the recovered protein has retained antibody activity. (In general, at least 90% of the recovered protein should be antibody.)

1.5.4 Elution conditions

Antibodies with high-affinity antigen-binding sites are the essential constituents of a ‘strong’ high-titred antiserum. When these antibodies are linked to Sepharose and used as solid-phase immunoadsorbents they give virtually irreversible binding to antigen. It is rarely possible to isolate antigen or antibody by true affinity methods, simply because a sufficiently high concentration of free competitor cannot be obtained to compete effectively with the solid-phase reagents.

Most techniques for the release of material from antibody affinity columns rely on deforming agents to alter the shape of the reacting molecules and so lower their net binding affinity. Acid or alkaline buffers are usually sufficient to release an acceptable proportion of bound material, most of which will regain full activity when a near-neutral pH is restored.

The addition of dioxane to an acid buffer increases yield from an affinity column (by reducing hydrophobic interactions) but with additional loss of recovered material due to irreversible denaturation. Other eluting buffers are more effective because they deform (and denature) to a greater extent but may produce an unacceptably high proportion of denatured material.

In order of increasing harshness they are: (a) 3.5 m potassium thiocyanate in 0.1 m phosphate buffer, pH 6.6; (b) 8.0 m urea; and (c) 7.0 m guanidine hydrochloride.

When using anti-immunoglobulin affinity columns to isolate a particularly valuable antibody,

e.g. a monoclonal antibody produced by a hybridoma cell line, it is good practice to saturate high-affinity anti-immunoglobulin sites by a cycle of pretreatment with normal mouse immuno- globulin and acid elution.

1.5.5 Practical applications of immunoadsorbents

Immunoadsorbents are widely used to render antisera specific by depletion of cross-reacting antibodies, and for quantitative adsorption. Although the method above used an antigen immuno- adsorbent to isolate antibody, it is possible to prepare antigen or immunoglobulin by the same procedure using an antibody immunoadsorbent column or a cellular immunoadsorbent column.