PURIFICATION AND FRAGMENTATION
OF ANTIBODIES
SECTION III
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 fluorescent and radioactive probes for binding studies are required, or when structural modifications 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 microscopic 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.
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-exchange (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 chromatograContributed by Sarah M. Andrew and Julie A. Titus
Current Protocols in Immunology (1997) 2.7.1-2.7.12
Copyright © 1997 by John Wiley & Sons, Inc.
UNIT 2.7
Induction of
Immune
Responses
2.7.1
Supplement 21
7DEOH &RPPHUFLDO.LWVIRU3XULI\LQJ$QWLERG\
Kit
Suppliera
Features
E-Z-SEPb
Pharmacia Biotech
Gamma Yolkb
Pharmacia Biotech
HiTrap protein A and
protein G columnsb
Immunopure (A) and
Immunopure (G) IgGc
Immunopure mouse IgG1c
Pharmacia Biotech,
Sigma
Pierce
Nine different kits to purify IgG or IgM from different
sources—polyclonal serum, tissue culture medium, bioreactor
supernatant, ascites fluid; concentrates antibody by centrifugation in a liquid linear polymer that crowds and precipitates
antibody from the sample
Concentrates IgY antibodies from egg yolk by centrifugation in
a liquid linear polymer
Ready-packed columns in a variety of sizes for use with
syringe loading or a peristatic pump
Protein A and protein G purification
Immunopure and Immunopure Plus (A/G) IgG
MAb Trap GIIb
Protein A and Protein G
Superose columnsb
T-Gelc
Pierce
Pierce
Pharmacia Biotech
Pharmacia Biotech
Pierce
Mild elution buffer kit for purification of mouse IgG1 on
protein A
Binds all IgGs that bind to both protein A and protein G;
contains sufficient gel to purify 6 or 16 mg, respectively
A complete protein G kit with buffers, column, and syringe
Ready-made, reusable columns for use with the Pharmacia
FPLC system; good for purifying large amounts of antibodies
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
aSee APPENDIX 5.
bThese
kits may require specialized equipment or preparation of additional reagents.
cThese
kits come complete or reagents may be purchased separately.
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
Purification
of IgG
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.
2.7.2
Supplement 21
Current Protocols in Immunology
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. IgG2b has asymmetric
Induction of
Immune
Responses
2.7.3
Current Protocols in Immunology
Supplement 21
7DEOH ([WLQFWLRQ&RHIILFLHQWVDQG0ROHFXODU6L]HRI
,PPXQRJOREXOLQVDQG7KHLU)UDJPHQWV
Molecule
IgG
IgM
IgM subset
Fab
F(ab′)2
F(ab′)2µ
F(ab′)µ
Molecular weight (kDa)
A280 (1 mg/ml
solution)
Nonreduced
Reduceda
1.43
1.18
1.18
1.53
1.48
1.38
1.38
150
900
180
50
100-110
135
65
50, 25
78, 25
78, 25
25b
25b
44, 25
44, 25
aNumbers 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.
bMay
appear as a doublet on SDS-PAGE.
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
Purification
of IgG
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
2.7.4
Supplement 21
Current Protocols in Immunology
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 IgG2a use pH 4.5, and for IgG2b 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
2.7.5
Current Protocols in Immunology
Supplement 21
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 purification, 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 reequilibrating it to pH 5.0 with 0.1 M sodium acetate, pH 5.0.
Purification
of IgG
2.7.6
Supplement 21
Current Protocols in Immunology
AFFINITY CHROMATOGRAPHY USING ANTI–RAT κ CHAIN
MONOCLONAL ANTIBODY COUPLED TO SEPHAROSE
ALTERNATE
PROTOCOL 2
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 of
Immune
Responses
2.7.7
Current Protocols in Immunology
Supplement 21
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.
Purification
of IgG
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.
2.7.8
Supplement 21
Current Protocols in Immunology
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
4
A
280
3
2
1
start gradient
purified
lgG
0
0
10
20
30
Fraction number
40
50
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Induction of
Immune
Responses
2.7.9
Current Protocols in Immunology
Supplement 21
2
A280
Fab
1
Fab
start gradient
Fc
MAb 315F6
MAb B723
Fc
0
0
10
20
30
40
Fraction number
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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
Purification
of IgG
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
The solubility of ammonium sulfate at 100°C is 76 g per 100 ml.
2.7.10
Supplement 21
Current Protocols in Immunology
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 preparation, 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-exchange (IEX) chromatography (see Basic Protocol 3) should be used in addition to ammonium sulfate precipitation and affinity chromatography. 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 specifically to the Fc region of immunoglobulin molecules, especially IgG for which it has four
high-affinity binding sites. It is heat stable and
retains its native conformation even after exposure to denaturing reagents such as 3 M thiocyanate, or in acidic conditions. Thus antibody
can be stripped from protein A and the reagent
used again for purification. Not all IgG molecules bind to protein A; rat IgGs bind particularly weakly, and many mouse IgG1 subclass
antibodies will not bind. Some of these antibodies 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 portions. 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; although protein G can be used for other rat IgG
molecules, the binding is frequently weak, especially 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 Protocol 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 antibody purification. On first inspection, these
may not seem to be the least expensive option,
but it is important to remember that the reliability of kits and their excellent perfomance in
terms of yield usually make them quite economic 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 saving 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 conditions required to elute from protein A can damage 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 conditions 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
2.7.11
Current Protocols in Immunology
Supplement 21
times be difficult to remove samples from protein G. Use lower pH and increased salt concentration 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 SDSPAGE 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 chromatography 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
(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 culture supernatant or ascites is purified by one of
the affinity methods described here (Basic Protocol 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 physiochemical 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 Protocols in Molecular Biology (F.M. Ausubel, R.
Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 11.16.111.16.18. John Wiley & Sons, New York.
Key References
Hardy, R.R. 1986. Purification and characterization
of monoclonal antibodies. In Handbook of Experimental Immunology, Vol. 1: Immunochemistry (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.
Different batches of ascites fluid and monoclonal 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 obtained. The lowest yields are usually from ammonium 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 hybridomas are being overgrown by nonproducers. This can be remedied by returning to
an earlier freeze of cells, or by recloning (and
rescreening) the hybridoma.
Lane, D. (ed.) 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
Time Considerations
Julie A. Titus
National Cancer Institute
Bethesda, Maryland
Procedures such as dialysis and running columns can be lengthy, but large amounts of
antibody can be purified in 2 to 3 days. FPLC
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 antibody-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
Purification
of IgG
2.7.12
Supplement 21
Current Protocols in Immunology