Purification of Immunoglobulin G

SECTION III

PURIFICATION AND FRAGMENTATION

OF ANTIBODIES

For many applications, both monoclonal and polyclonal antibodies may be used in impure form: monoclonal antibodies may be used either as ascites fluid or as tissue culture supernatant, and polyclonal antibodies may be used as antiserum. Such unpurified antibodies are perfectly suitable for use in indirect flow cytometry assays (UNIT 5.3), for most ELISAs (UNIT 2.1), or for cytotoxicity studies (UNITS 3.3 & 3.4) if the concentration of antibody is not important. Purified antibodies must be used, however, when accurate concentrations are required, when chemical modifications such as labeling with fluores-cent and radioactive probes for binding studies are required, or when structural modifi-cations such as removal of the Fc portion to produce bivalent F(ab′)2 or monovalent Fab fragments are required. IgM can be modified and the single bivalent subunit (IgMs) of the pentamer produced as a low-molecular-weight alternative to intact IgM. Bivalent F(ab′)₂µ fragments of IgM are more difficult to produce but are sometimes required. Detailed methods for production of these fragments are provided in UNIT 2.8.

Choice of procedure for antibody purification depends on the intended use of the antibodies and on the available resources. UNIT 2.7 presents purification methods that can be tailored to the laboratory’s resources. If a highly purified product is required, an assay for antibody activity and a reliable means of assessing the degree of protein contamination are essential. The method of choice for determining purity is SDS-PAGE (UNIT 8.4). The antibody assay must be rapid and accurate to monitor activity throughout the purification. Some preferred assays are: (1) labeling of cell-surface antigens with the antibody, followed by incubation with a fluorescent anti-Ig of the correct specificity and micro-scopic or flow cytometry analysis of the sample (UNIT 5.3); (2) ELISA (UNIT 2.1); (3) complement-mediated lysis of the appropriate cells (UNIT 3.3); (4) immunodiffusion (UNIT 2.3); (5) radioimmunoassay (RIA; Cooper and Paterson, 1989); and (6) inhibition of purified fluorescent or radiolabeled antibody binding to its appropriate ligand. Assays will generally not provide a measure of contaminating proteins that remain in the preparation at different stages of the purification. These can be detected by SDS-PAGE. In the last five years, many companies have produced kits for the purification and fragmentation of antibodies derived from all common animal species. These kits are primarily designed for IgG and IgM classes of antibody, but there is also a kit for purification of egg yolk–derived IgY. The kits mentioned here are the most reliable in the opinion of the authors, but they may not be the least expensive option, so classical methods of purification and fragmentation are described in these units.

UNIT 2.7

Purification of Immunoglobulin G

IgG can be purified by ammonium sulfate precipitation followed by size-exclusion (SE) chromatography (see Basic Protocol 1). This is the least expensive option available for purification of antibodies. Protein A– and protein G–affinity chromatography (see Basic Protocol 2 and Alternate Protocol 1) are the fastest methods for purifying antibodies, but they are not effective for all subclasses of rat antibody. Affinity chromatography using anti-rat antibody can be used to purify rat antibodies (see Alternate Protocol 2). Ion-ex-change (IEX) chromatography (see Basic Protocol 3) can also be used to purify intact monoclonal and polyclonal antibodies and antibody fragments. All these methods give a product with a high degree of purity. Ammonium sulfate precipitation followed by IEX

chromatography can be applied to any type of antibody. However, affinity chromatogra- Induction of_Immune Responses

phy, either as described in Basic Protocol 2 and Alternate Protocol 1 or carried out using a commercially produced kit, is much more efficient and less tiresome to carry out. There are many commercial kits available for purifying IgG (see Table 2.7.1). The T-Gel purification kit (thiophilic gel; Pierce) produces results similar to those of ammonium sulfate precipitation because it has a broad specificity toward immunoglobulins derived from various animal species, irrespective of the type or subclass. The E-Z-SEP system (Pharmacia Biotech) consists of a group of kits with applications for all types of antibodies—e.g., ascites IgG, ascites IgM, bioreactor medium IgM, serum-free tissue culture IgG, serum polyclonal antibodies.

BASIC PROTOCOL 1

AMMONIUM SULFATE PRECIPITATION AND SIZE-EXCLUSION CHROMATOGRAPHY

Ammonium sulfate precipitation coupled with size-exclusion (SE) chromatography is a method for purifying proteins of all types and may be the procedure of choice for purifying certain antibodies. Ammonium sulfate precipitation can be used to purify all subclasses of mouse antibodies as well as antibodies of other species. This method can be used to purify IgM, IgG, and IgA of all species. Although more time-consuming than affinity chromatography (see Basic Protocol 2), it has a wider range of applications.

After removing cell debris from ascites fluid or a monoclonal antibody supernatant, ammonium sulfate is added to precipitate the proteins. The precipitate is recovered by centrifugation and dissolved in PBS or borate buffer, then dialyzed and concentrated. It is purified by SE chromatography and the pure IgG is analyzed.

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Kit Suppliera _Features

E-Z-SEPb _{Pharmacia Biotech Nine different kits to purify IgG or IgM from different} sources—polyclonal serum, tissue culture medium, bioreactor supernatant, ascites fluid; concentrates antibody by centri-fugation in a liquid linear polymer that crowds and precipitates antibody from the sample

Gamma Yolkb _{Pharmacia Biotech Concentrates IgY antibodies from egg yolk by centrifugation in} a liquid linear polymer

HiTrap protein A and protein G columnsb

Pharmacia Biotech, Sigma

Ready-packed columns in a variety of sizes for use with syringe loading or a peristatic pump

Immunopure (A) and Immunopure (G) IgGc

Pierce Protein A and protein G purification

Immunopure mouse IgG1c Pierce Mild elution buffer kit for purification of mouse IgG1 on

protein A Immunopure and

Immuno-pure Plus (A/G) IgG

Pierce Binds all IgGs that bind to both protein A and protein G; contains sufficient gel to purify 6 or 16 mg, respectively MAb Trap GIIb _{Pharmacia Biotech A complete protein G kit with buffers, column, and syringe} Protein A and Protein G

Superose columnsb

Pharmacia Biotech Ready-made, reusable columns for use with the Pharmacia FPLC system; good for purifying large amounts of antibodies T-Gelc _{Pierce One-step purification by adsorption to a thiophilic gel; the gel}

has broad specificity for immunoglobulins of any type or subclass derived from various animal species; used to purify antibodies from serum, ascites fluid, or culture supernatant

a_{See APPENDIX 5.}

b_{These kits may require specialized equipment or preparation of additional reagents.} c_{These kits come complete or reagents may be purchased separately.}

Purification of IgG

Materials

Ascites fluid or MAb supernatant (UNIT 2.6) PBS (APPENDIX 2)

Saturated ammonium sulfate (SAS; see recipe) Borate-buffered saline (optional; see recipe) Sephacryl S-200 Superfine (Pharmacia Biotech) PBS containing 0.02% sodium azide (optional) Glass wool (Polysciences)

Sorvall centrifuge and SS-34 rotor (or equivalent) 26 × 900–mm column (Pharmacia Biotech)

Additional reagents and equipment for protein dialysis (APPENDIX 3H), column chromatography (APPENDIX 3I), concentrating proteins (APPENDIX 3H), and reducing and nonreducing PAGE (UNIT 8.4)

1a. For ascites: Remove lipid from ascites fluid by placing enough glass wool into a funnel to cover the opening, pouring ascites through, rinsing glass wool with PBS, and squeezing glass wool gently with gloved fingers to obtain all the sample. Centrifuge filtered ascites 30 min at 20,000 × g (13,000 rpm in SS-34 rotor) either 4°C or room temperature. Decant and save the supernatant and discard membranous material and cell debris remaining in the pellet.

Wear gloves when handling glass wool.

1b. For MAb supernatant: Centrifuge MAb supernatant 30 min at 20,000 × g, either 4°C or room temperature. Decant and save the supernatant.

2. Add SAS slowly with stirring to the ascites or tissue culture supernatant to 45% (v/v). Leave 1 to 2 hr or overnight at 4°C to ensure precipitation of all the protein.

3. Centrifuge 1 hr at 20,000 × g, either 4°C or room temperature, and save precipitate to use in step 4. Save the supernatant to check for antibody activity.

4. Dissolve precipitate in a minimum volume of PBS or borate buffer (10 to 20 ml is usually suitable).

5. Place the dissolved precipitate in dialysis tubing. Dialyze against ≥20 vol PBS or borate buffer for 24 to 48 hr total at 4°C. Change the dialysis buffer four to six times during dialysis.

Generally, accurate molecular weight cutoffs are not required and it is not necessary to boil the tubing; merely soak for a few minutes in distilled water to soften.

The solution in the dialysis tubing will turn from a yellowish liquid to a cloudy or clear solution.

6. Concentrate the protein solution to ≤5 ml.

7. Prepare a 26 × 900–mm Sephacryl S-200 Superfine column and load the concentrated protein solution onto it. Elute protein with PBS, PBS containing 0.02% sodium azide, or borate buffer, and collect 100 fractions (1% of the column volume).

Monitor the protein fractions with a UV spectrophotometer at 280 nm. Alternatively, a precalibrated column can be used.

8. Check the purity of the fractions with A280 values >0.5 on nonreducing and reducing 10% polyacrylamide gels.

In nonreducing PAGE, a band at ∼150 kDa indicates IgG; in reducing PAGE, two bands at ∼55 kDa (heavy chain) and ∼25 kDa (light chain) indicate IgG. IgG_2b has asymmetric

Induction of Immune Responses

glycosylation of the heavy chains and will therefore appear as a doublet on the gel (Table 2.7.2). If the fractions are not sufficiently concentrated for detection by SDS-PAGE, concentrate as described in APPENDIX 3H.

9. Assess IgG concentration spectrophotometrically at A280 (Table 2.7.2). Pool the eluates containing pure IgG.

Antibodies purified by ammonium sulfate precipitation followed by SE chromatography are of sufficient purity for any manipulation. They may be used for fragmentation after dialysis to the desired buffer (APPENDIX 3H), for ELISA (UNIT 2.1), or for labeling with fluorescein isothiocyanate (FITC) or biotinylation (UNIT 5.3).

10. Adjust IgG concentration to 0.1 to 30 mg/ml in borate buffer or PBS with 0.02% (w/v) sodium azide and store at 4°C up to several years. For back-up frozen stocks, freeze IgG at −70°C.

Thawing and freezing once from −70°C is usually fine, although as a general rule this should be avoided. Do not store antibodies at −20°C for >1 month. Do not repeatedly freeze/thaw from −20°C.

BASIC PROTOCOL 2

AFFINITY CHROMATOGRAPHY USING PROTEIN A–SEPHAROSE

This protocol describes the purification of antibody using protein A–Sepharose affinity chromatography. Protein A can be used to isolate monoclonal and polyclonal IgG from ascites, serum, and tissue culture and bioreactor supernatants. Protein A purification is recommended for human (except IgG3; mouse IgG1 may bind only weakly), rabbit, guinea pig, and pig antibodies. The protocol requires addition of the antibody to a protein A–Sepharose column at pH 8.0, followed by elution at a lower pH. The antibody is then dialyzed back against PBS.

Materials

Ascites fluid or MAb supernatant (UNIT 2.6) PBS, pH 8.0 and pH 7.3 (APPENDIX 2) 1 M NaOH

Protein A–Sepharose CL-4B, hydrated (Pharmacia Biotech or Sigma) 0.1 M citric acid at pH appropriate for subclass of antibody (see step 5) Borate-buffered saline (optional; see recipe)

3 M potassium thiocyanate, filtered

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Molecule A280 (1 mg/ml

solution)

Molecular weight (kDa) Nonreduced Reduceda IgG 1.43 150 50, 25 IgM 1.18 900 78, 25 IgM subset 1.18 180 78, 25 Fab 1.53 50 25b F(ab′)2 1.48 100-110 25b F(ab′)2µ 1.38 135 44, 25 F(ab′)µ 1.38 65 44, 25

a_{Numbers to the left of the comma represent molecular weights of the heavy chain}

of immunoglobulins, and numbers to the right are weights of the light chain of immunoglobulins.

b_{May appear as a doublet on SDS-PAGE.}

Purification of IgG

Sorvall centrifuge and SS-34 rotor (or equivalent) 0.45-µm filter

1.5 × 10–cm column

HiTrap protein A column (Pharmacia Biotech or Sigma; optional) Additional reagents and equipment for dialysis (APPENDIX 3H) and column

chromatography (APPENDIX 3)

For ascites fluid:

1a. Clarify ascites fluid and remove lipids (see Basic Protocol 1, step 1a). 2a. Dilute ascites fluid 10-fold with PBS, pH 8.0.

For MAb supernatant:

1b. Centrifuge MAb supernatant at 20,000 × g (13,000 rpm in SS-34 rotor), 4°C, and filter through a 0.45-µm filter.

2b. Adjust MAb supernatant to pH 8.0 by dialysis against PBS, pH 8.0, or by adding 1 M NaOH.

It is important to have the protein sample at pH 8.0 if IgG1 is to be purified. Other subclasses should bind to protein A at pH 7.4.

3. Prepare protein A–Sepharose column and attach to fraction collector. Equilibrate column with PBS, pH 8.0, at either 4°C or room temperature. Layer antibody solution onto resin bed.

Volumes of 1 ml to several liters can be loaded onto a protein A–Sepharose column. Use a peristaltic pump or gravity to assist in loading large volumes.

There is a limit to the binding capacity for immunoglobulin (∼5 mg mouse IgG, and 8 mg human IgG, per milliliter resin). The expected concentrations can be assessed from the yields (see Anticipated Results). The antibody activity of the unbound fraction can be tested to check for overloading. Use a flow cytometry assay (UNITS 5.3 & 5.4) or ELISA (UNIT 2.1)

for assessing activity of the eluate.

The HiTrap protein A column is ready-to-use. Alternatively, a column can be prepared in a 10-ml syringe. Add glass wool to the syringe before adding hydrated protein A–Sepharose CL-4B.

4. Wash column with several volumes PBS, pH 8.0.

Eluate should have an A₂₈₀ at baseline before proceeding to the next step.

5. Elute with 0.1 M citric acid at suitable pH (bring to appropriate pH with 1 M NaOH): for mouse IgG₁ use pH 6.5, for IgG_2a use pH 4.5, and for IgG_2b and IgG₃ use pH 3.0.

It is thought that standing in low pH may damage the antibody; therefore, 50 µl of 2 M Tris base buffer (Boehringer-Mannheim)/ml of eluate may be placed in the fraction collector tubes prior to elution. Reverse elution is undertaken by reversing the leads of the column so that the pump is pushing buffer up the column in the opposite direction to that in which the column was loaded. Samples that do not occupy the entire capacity of the column can be eluted at a higher concentration by this method. Reverse elution can concentrate the antibody if the protein A–Sepharose column is underloaded.

6. Pool protein-containing fractions, place eluates in dialysis tubing, and dialyze eluates against 1 liter PBS, pH 7.3, with or without 0.02% (w/v) sodium azide, at 4°C. Change the dialysis buffer twice. Store samples in PBS or borate-buffered saline at 4°C.

If desired, check purity by SDS-PAGE.

Induction of Immune Responses

7. Clean column with 1 column volume of filtered 3 M potassium thiocyanate before reequilibrating it in PBS, pH 7.3. Store at 4°C.

Wash the column with several volumes of PBS, pH 7.3, to remove residual potassium thiocyanate, which absorbs at 280 nm and could interfere in later purifications.

Antibodies purified by protein A–Sepharose affinity chromatography will be of similar purity to those obtained by ammonium sulfate precipitation and can be used for the same purposes (see Basic Protocol 1).

ALTERNATE PROTOCOL 1

AFFINITY CHROMATOGRAPHY USING PROTEIN G–SEPHAROSE

Affinity chromatography using protein G-Sepharose is useful for purifying antibody from serum, ascites fluid, tissue culture supernatant, and bioreactor supernatant. Protein G (Akerstrom and Bjorck, 1986) has a binding profile opposite to that of protein A with respect to pH: antibodies bind better at a low pH and badly at high pH. However, some antibodies (mouse IgG1, and rabbit and human antibodies) do remain bound to protein G at high pH (8 to 10), so it is best to bind the antibody at pH 5 and elute at pH 2.8. This method is useful for mouse IgG1, rat (most subclasses bind weakly although IgG2b may not), monkey, rabbit, cow, goat, horse, and sheep antibodies. As with protein A purifica-tion, there is the possibility of some loss of antibody binding ability due to low-pH elution. Additional Materials (also see Basic Protocol 2)

0.1 M sodium acetate, pH 5.0 0.1 M glycine⋅HCl, pH 2.8

HiTrap protein G column (Pharmacia Biotech or Sigma)

1. Prepare ascites fliud or MAb supernatant (see Basic Protocol 2, step 1a or 1b). 2. Dilute with 0.1 M sodium acetate, pH 5.0.

Dilute >2-fold for tissue culture supernatant or 10-fold for ascites fluid and bioreactor supernatant.

3. Equilibrate HiTrap protein G column in 0.1 M sodium acetate, pH 5.0. Layer antibody solution onto resin bed.

Protein G has a higher capacity for IgG than protein A: ∼10 mg protein/ml of gel with some species variation.

4. Wash column with several column volumes of 0.1 M sodium acetate, pH 5.0. 5. Elute bound antibody with 0.1 M glycine⋅HCl, pH 2.8.

To minimize exposure to low pH, add 50 µl Tris-based buffer (Boehringer-Mannheim) per milliliter of eluate to each tube of the fraction collector..

6. Pool protein-containing fractions and dialyze.

7. Recycle the column by washing it with 0.1 M glycine⋅HCl, pH 2.8, then reequilibrat-ing it to pH 5.0 with 0.1 M sodium acetate, pH 5.0.

Purification of IgG

ALTERNATE PROTOCOL 2

AFFINITY CHROMATOGRAPHY USING ANTI–RAT κ CHAIN MONOCLONAL ANTIBODY COUPLED TO SEPHAROSE

Occasionally, a monoclonal antibody (particularly one derived from the rat) cannot be purified by either protein A– or protein G–Sepharose chromatography, either because the antibody fails to bind the protein A or protein G, or because the elution conditions are too harsh for the retention of activity. In such a case, a column consisting of an anti-rat-Ig light-chain monoclonal antibody coupled to Sepharose can be used to bind the rat monoclonal antibody from a tissue culture supernatant or from ascites fluid. A series of buffers of decreasing pH can be used to assess the mildest conditions for elution of the rat antibody from the anti-Ig column. This protocol describes the production of such a column and the conditions for binding and elution from it.

Additional Materials (also see Basic Protocol 2)

Mouse anti-rat κ MAb: MAR 18.5 (ATCC TIB 216) purified using protein A–Sepharose (see Basic Protocol 2)

CNBr–Sepharose CL-4B (UNIT 8.3; Pharmacia Biotech)

Binding buffer: 0.05 M Tris⋅Cl/0.15 M NaCl/0.02% (w/v) NaN₃, pH 8.6 Crude rat antibody solution to be purified (MAb supernatant or ascites

fluid; UNIT 2.6)

pH 7.0 elution buffer: 0.05 M sodium phosphate/0.15 M NaCl/0.02% (w/v) NaN₃, pH 7.0

pH 5.5 elution buffer: 0.05 M sodium citrate/0.15 M NaCl/0.02% (w/v) NaN3, pH 5.5

pH 4.3 elution buffer: 0.5 M sodium acetate/0.15 M NaCl/0.02% (w/v) NaN₃, pH 4.3

pH 2.3 elution buffer: 0.5 M glycine/0.15 M NaCl/0.02% (w/v) NaN3, pH 2.3 Additional reagents and equipment for preparation of antibody-Sepharose (UNIT 8.3)

Prepare column

1. Covalently couple ≥10 mg purified MAR 18.5 antibody to the CNBr–Sepharose 4B.

Coupling ratio should be ∼10 mg MAR 18.5/ml wet gel. Such a column will have a capacity to bind ∼1 mg of protein.

2. Prepare the column and wash extensively with the binding buffer at 4°C or room temperature.

Purify antibody

3. Clarify ascites fluid or MAb supernatant (see Basic Protocol 2).

4. Load the column with ∼10 ml ascites or ∼100 ml MAb supernatant of crude rat antibody solution.

5. Wash the column extensively with 10 to 15 column volumes of binding buffer. Monitor the A280 to be certain the absorbance returns to baseline. Set up a fraction collector to collect all fractions from steps 6 to 9.

6. Elute with 5 column volumes of pH 7.0 elution buffer, watching the UV monitor.

For most antibodies, this does not elute the bound antibody and serves as a preliminary wash step. Be sure A₂₈₀ has returned to baseline before beginning the next step.

7. Elute with 5 column volumes of pH 5.5 elution buffer, watching the UV monitor.

Some antibodies will elute under these mild conditions. Be sure A₂₈₀ has returned to

baseline before beginning the next step. Induction ofImmune

8. Elute with 5 column volumes of pH 4.3 elution buffer, watching the UV monitor.

Most antibodies will elute under these conditions. Be sure A₂₈₀ has returned to baseline before beginning the next step.

9. Elute with 5 column volumes of pH 2.3 elution buffer, watching the UV monitor.

All antibodies will elute at pH 2.3. This also serves as a final wash step. Be sure A₂₈₀ has returned to baseline before beginning the next step.

10. Equilibrate column with binding buffer by washing with ≥10 column volumes. Store column wrapped in Parafilm at 4°C.

11. Identify eluted protein peaks (including the unbound initial fractions), pool, assay for antibody activity, and concentrate if necessary (see Basic Protocol 1, step 9).

BASIC PROTOCOL 3

DE52 ION-EXCHANGE CHROMATOGRAPHY WITH TRIS⋅CL

DE52 ion-exchange (IEX) chromatography can be used to purify antibodies from a tissue culture supernatant, ascites fluid, and serum or ammonium sulfate precipitates derived from any of these antibody-containing fluids. The protocol may also be used as a second step following purification by size exclusion (SE) chromatography (see Basic Protocol 1 and Alternate Protocols 1 and 2). The major contaminant protein in all these preparations is albumin, which binds DE52 tightly under conditions of low-to-moderate ionic strength. Antibody either fails to bind to DE52, in which case it elutes in the void volume as the column is loaded, or it binds loosely, and can be eluted with a gentle salt or pH gradient. Fractions eluted are assayed first by A₂₈₀ and then by either ELISA (UNIT 2.1) or SDS-PAGE (UNIT 8.4).

Caution should be exercised in that different monoclonal antibodies as well as antibody fractions from different immunized animals’ sera elute from DE52 under different conditions. In this protocol, antibody in 0.01 M Tris⋅Cl at pH 8.6 is passed over the DE52 column and bound antibody is eluted with a gradient in the same buffer approaching 0.5 M NaCl.

Materials

DE52 powder (Whatman)

0.01 M Tris⋅Cl, pH 8.6 (APPENDIX 2)

0.5 M NaCl/0.01 M Tris⋅Cl, pH 8.6 (see recipe)

Antibody sample (ascites fluid, tissue culture supernatant, immune serum, or ammonium sulfate precipitate)

1.5 × 50–cm column

Additional reagents and equipment for column chromatography (APPENDIX 3I), dialysis (APPENDIX 3H), and ELISA (UNIT 2.1) or SDS-PAGE (UNIT 8.4) 1. Swell DE52 powder in 0.01 M Tris⋅Cl, pH 8.6, and remove fine particles.

It is very important to adjust the pH prior to pouring the DE52 gel into the column.

2. Pour a 1.5 × 50–cm DE52 column and equilibrate with 0.01 M Tris⋅Cl, pH 8.6.

The size of the column is dependent upon the total amount of protein in the crude antibody sample. The capacity of DE52 is ≥100 mg total protein/ml hydrated gel, so a column of 1 to 5 ml is commonly sufficient.

3. Place antibody sample in dialysis tubing. Dialyze twice against ≥20 times the sample volume of 0.01 M Tris⋅Cl, pH 8.6, overnight at 4°C.

Purification of IgG

4. Load the dialyzed antibody sample onto the column.

5. Elute column with 0.01 M Tris⋅Cl, pH 8.6, until all of the protein that does not bind to DE52 at this pH comes through the column (detected by monitoring A280 with a UV monitor). Collect the first ten fractions (∼12 ml each).

Some antibodies elute at this stage (i.e., they do not bind DE52 in 0.01 M Tris⋅Cl, pH 8.6). They will thus be free of the major contaminant, albumin, which remains on the column. However, the great majority of antibodies will adsorb to the column under these conditions.

6. Elute the remaining material using a 200- to 250-ml linear gradient of 0.01 M Tris⋅Cl, pH 8.6, to an equal volume of 0.5 M NaCl/0.01 M Tris⋅Cl, pH 8.6. Collect 2-ml fractions.

7. Monitor column fractionation as follows: (1) A₂₈₀, to measure protein concentration; (2) conductivity, as an indication of the progress of the gradient as the concentration changes; (3) activity of the antibody by ELISA (UNIT 2.1); and (4) protein structure by SDS-PAGE (UNIT 8.4).

A typical example of the elution profile of a mouse MAb is shown in Figure 2.7.1. The first peak that comes off as the ionic strength is increased is the IgG. This is confirmed by SDS-PAGE or ELISA.

IEX chromatography is also useful for separating Fab fragments. Following size-exclusion chromatography of the digestion mixture to remove intact IgG (APPENDIX 3I), the 50-kDa

0 10 20 30 40 50 Fraction number start gradient purified lgG 1 2 3 4 A 280 0 )LJXUH ,(;FKURPDWRJUDSK\HOXWLRQSURILOHIURPD'(FROXPQ×FP7KH,J* IUDFWLRQVIURPDQ$&$FROXPQZHUHGLDO\]HGDJDLQVW07ULV⋅&OS+DQGDSSOLHGWRWKH FROXPQ7HQIUDFWLRQVPOZHUHFROOHFWHGXVLQJWKHVWDUWLQJEXIIHUWKHQDOLQHDUJUDGLHQWRIWR 01D&OLQ7ULV⋅&OS+ZDVVWDUWHG)UDFWLRQVWRFRQWDLQHGSXUH,J*DVGHWHUPLQHGE\ 6'63$*(81,7 Induction of Immune Responses

fraction is dialyzed (APPENDIX 3H) exhaustively against 0.01 M Tris⋅Cl, pH 8.6, and separated as described in this protocol using DE52. Elution profiles can vary greatly between antibodies as shown in Figure 2.7.2. For one of the antibodies (open circles), the Fab elutes at 0.01 M Tris⋅Cl, pH 8.6. In contrast, the Fab from the other antibody (closed circles) elutes 40 ml after the salt gradient is started. These examples reinforce the need to monitor the location of the protein during the purification process.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2; for suppliers, see APPENDIX 5.

Borate-buffered saline 0.015 M sodium borate 0.15 M NaCl

Adjust to pH 8.5 with 1 M NaOH and filter sterilize Store indefinitely at room temperature

Saturated ammonium sulfate (SAS) 76 g ammonium sulfate

100 ml H₂O

Heat with stirring to just below boiling point Leave overnight at room temperature Store indefinitely at room temperature

° 20 30 10 0 0 Fraction number 40 Fc start gradient Fc Fab MAb 315F6 MAb B723 Fab 1 2 A280 )LJXUH ,(;FKURPDWRJUDSK\HOXWLRQSURILOHRI)DEIURPWZRGLIIHUHQWDQWLERGLHVXVLQJD'( FROXPQ×FP(OXWLRQFRQGLWLRQVDQGJUDGLHQWZHUHWKHVDPHDVIRUWKHH[DPSOHLQ)LJXUH )RU0$E)RSHQFLUFOHVWKH)DEIUDJPHQWHOXWHGLQWKHVWDUWLQJEXIIHUDQGWKH)FSRUWLRQ PXFKODWHULQWKHJUDGLHQW)DEIUDJPHQWVIURP0$E%FORVHGFLUFOHVHOXWHGDWWKHEHJLQQLQJ RIWKHJUDGLHQW Purification of IgG

0.5 M NaCl/0.01 M Tris⋅Cl, pH 8.6 1.21 g Tris base

29 g NaCl 800 ml H2O

Adjust pH to 8.6 with HCl and add H2O to 1 liter

COMMENTARY

Background Information

Before purifying antibody from any prepa-ration, consideration should be given to the potential use of the final product. If the antibody is to be used in an assay with internal controls or to saturate cell-surface antigen, then impure ascites fluid or a dialysed ammonium sulfate precipitate will usually suffice. At the other extreme, biochemical modifications such as conjugation to drugs or certain fluorochromes (e.g., phicobiliproteins, UNIT 5.3) may require an extremely pure product. In this case ion-ex-change (IEX) chromatography (see Basic Pro-tocol 3) should be used in addition to ammo-nium sulfate precipitation and affinity chroma-tography. For uses such as conjugation to fluorescein isothiocyanate (FITC), biotin (UNIT 5.3), or radioisotopes (UNIT 8.11), antibody that has been purified by protein A or G is perfectly adequate.

Protein A is a cell wall component produced by several strains of Staphylococcus aureus; it is a single polypetide chain with a molecular weight of 42,000 Da. Protein A binds specifi-cally to the Fc region of immunoglobulin mole-cules, especially IgG for which it has four high-affinity binding sites. It is heat stable and retains its native conformation even after expo-sure to denaturing reagents such as 3 M thio-cyanate, or in acidic conditions. Thus antibody can be stripped from protein A and the reagent used again for purification. Not all IgG mole-cules bind to protein A; rat IgGs bind particu-larly weakly, and many mouse IgG1 subclass

antibodies will not bind. Some of these anti-bodies can be affinity purified with the similar reagent, protein G.

Protein G is a bacterial cell wall protein isolated from group G streptococci. Protein G also binds IgG molecules through their Fc por-tions. There are two binding sites for IgG in native protein G; it also has binding sites for Fab regions of antibody, albumin, and cell membranes. Sigma sells a recombinant form of protein G which has been truncated so the sites for Fc binding remain, but the Fab-, albumin-, and membrane-binding sites have been

re-moved. Protein G is particularly useful for purification of human IgG3 and rat IgG2b;

al-though protein G can be used for other rat IgG molecules, the binding is frequently weak, es-pecially in the case of IgG1. Protein G is usually

better than protein A for purification of mouse IgG1.

Rat antibody can be problematic to purify, and an anti-rat affinity column (Alternate Pro-tocol 2) may be required. The authors have had little success with protein G purification for rat monoclonal antibodies.

Elution profiles for IEX chromatography are different for each antibody or antibody fragment. The activity of eluted antibodies should be monitored using an ELISA (UNIT 2.1) in addition to the absorbance (A280) profile.

Table 2.7.1 describes kits available for anti-body purification. On first inspection, these may not seem to be the least expensive option, but it is important to remember that the reliabil-ity of kits and their excellent perfomance in terms of yield usually make them quite eco-nomic in the long term. It is a false economy to spend many hours of staff time obtaining a decreased percentage yield for the sake of sav-ing a few dollars at the outset.

Critical Parameters

Because activity of the purified product is of prime importance, this should be evaluated for the impure antibody before purification is started and throughout the steps of purification. Some investigators report that the acid condi-tions required to elute from protein A can dam-age the antibody. Care should be taken to avoid excessively long periods of incubation at low pH. Elution from protein A– or protein G– Sepharose can also be accomplished using 3 M potassium thiocyanate, high ionic strength, or high pH. For particular antibodies these condi-tions may be more gentle, but in the authors’ experience the best results are obtained with the methods described here.

Ammonium sulfate precipitation is a more gentle procedure than protein A purification, but it is more time-consuming. It can

some-Induction of Immune Responses

times be difficult to remove samples from pro-tein G. Use lower pH and increased salt con-centration if problems are encountered.

Protein A columns can be used at flow rates of ∼1 ml/min, while SE columns should be limited to ≤0.5 ml/min. Optimal results for SE are obtained using samples containing 5 to 100 mg of antibody. Protein A should be loaded within the limits given.

The antibody-binding capacity of protein A is variable between species. For mouse IgG, 1 ml of hydrated protein A will bind ∼5 mg of antibody; for human IgG, 1 ml of hydrated protein A will bind ∼8 mg of antibody.

For IEX (DE52) chromatography, the pH is very important and buffers should be checked on a pH meter immediately before starting. All column fractions should be retained until SDS-PAGE and antibody activity assays have been performed. In some cases, antibody may elute from the IEX column in the same fractions as serum albumin, in which case SE chromatog-raphy should be used.

DE52 has an enormous capacity for binding protein, as estimated by the manufacturer at 130 mg/ml bed volume. Thus, dilute antibody preparations can be concentrated effectively by this method.

Anticipated Results

Different batches of ascites fluid and mono-clonal antibody supernatant can vary widely in the amount of antibody they contain. Generally, 1 ml of ascites should yield 1 to 4 mg of purified antibody and 1 ml of MAb supernatant should yield 0.5 to 50 µg of purified product. If cells are grown in automated culture systems that recycle the medium (bioreactors), yields equivalent to those of ascites fluid can be ob-tained. The lowest yields are usually from am-monium sulfate precipitation and the highest yields are usually from IEX or affinity methods. Yields significantly lower than these should be a warning that the antibody-producing hy-bridomas are being overgrown by nonpro-ducers. This can be remedied by returning to an earlier freeze of cells, or by recloning (and rescreening) the hybridoma.

Time Considerations

Procedures such as dialysis and running col-umns can be lengthy, but large amounts of antibody can be purified in 2 to 3 days. FPLC

(fast peptide, protein, and polynucleotide liquid chromatography) systems are expensive, but can reduce the time taken to obtain purified antibody.

Quickest results can be obtained when cul-ture supernatant or ascites is purified by one of the affinity methods described here (Basic Pro-tocol 2): pure antibody can be obtained in <1 day. The ammonium sulfate, size-exclusion, or ion-exchange methods will take longer. Literature Cited

Akerstrom, B. and Bjorck, L. 1986. A physiochemi-cal study of protein G molecule with unique immunoglobulin G–binding properties. J. Biol. Chem. 261:10240-10247.

Cooper, H.M. and Paterson, Y. 1989. Determination of the specific antibody titer. In Current Proto-cols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seid-man, J.A. Smith, and K. Struhl, eds.) pp. 11.16.1-11.16.18. John Wiley & Sons, New York.

Key References

Hardy, R.R. 1986. Purification and characterization of monoclonal antibodies. In Handbook of Ex-perimental Immunology, Vol. 1: Immunochem-istry (D.M. Weir, ed.) pp. 13.1-13.13. Blackwell Scientific, Oxford.

An excellent and detailed review of methods for purifying antibodies that includes an extensive list of current literature.

Lane, D. (ed.) 1988. Antibodies: A Laboratory Man-ual. Cold Spring Harbor Press, Cold Spring Har-bor, N.Y.

The complete text on antibodies; everything you want to know and more.

A Technical Guide to Antibody/Protein Purification. 1995. Pierce, Rockford, Ill.

A highly informative supplement to the Pierce anti-body-purification kits.

Monoclonal Antibody Purification Handbook. 1994. Pharmacia Biotech, Piscataway, N.J. This handbook contains the computer program MAb Assistant.

Contributed by Sarah M. Andrew Lancaster University

Lancaster, United Kingdom Julie A. Titus

National Cancer Institute Bethesda, Maryland

Purification of IgG