Affinity Purification of Antibodies Protocol

Introduction of Affinity Purified Antibodies

A variety of methods are available for the purification of antibodies, and the choice depends on the intended application, the species of origin, immunoglobulin (Ig) class and subclass, and the source of starting material. It is important to recognize that no single method can fulfill the requirements for all application needs, but those detailed in this chapter provide common methods suitable for most IgG purification. To maximize IgG purification, we take advantage of high-capacity IgG binding by specific bacterial proteins immobilized on a solid support such as agarose or Sepharose beads. The bacterial proteins used in the purification of antibodies are (1) protein- A derived from Staphylococcus aureus, (2) protein-G from Streptococcus sp., and (3) protein-L from Peptostreptococcus magnus. Each of these proteins provides unique Ig-binding properties, which can be used for purification of antibodies from a variety of matrices such as serum, culture supernatant, or ascites.

Polyclonal antibodies are frequently provided as an antiserum obtained from blood of immunized animals. Raw antiserum contains a large number of extraneous proteins that can interfere with immunoassays, and protein-A or protein-G is commonly used to eliminate the bulk of unwanted serum proteins. Antisera also contain a large number of unwanted antibodies (>95 % total IgG) against non-antigen targets, and these nonspecific Ig proteins are not removed by this purification method. Monoclonal antibodies are often provided as a hybridoma culture supernatant or as ascitic fluid harvested from the intraperitoneal cavity of mice after inoculation with a clonal hybridoma cell line. Similar to polyclonal antisera, monoclonal antibodies can be purified by protein-A or protein-G methods, but unlike antisera, the majority of immunoglobulins present in the crude fluid are of a singular class/subtype directed against the target antigen. Protein-L can often be substituted for protein-A or protein-G, and we take advantage of its inability to bind bovine IgG to purify mouse IgG from hybridoma culture supernatant, which is supplemented with fetal bovine serum (FBS). Neither protein-A nor protein-G bind IgY antibodies derived from chicken egg so purification is limited to protein- L. The typical concentration of antibody in antiserum is 1–3 mg/mL; hybridoma culture supernatant is 0.1–10 mg/mL and ascites 2–10 mg/mL. Prior to purification, crude antisera and ascites should be diluted in appropriate buffer to adjust binding pH and minimize bulk protein load effects that can block desired antibody binding to the resin.

Methods of Affinity Purified Antibodies Protocol

Antibody Purification Using Immobilized Protein-A

1. Protein-A purification is recommended for human IgG, and antibody is bound at slightly basic pH and eluted at an acidic pH (see Note 2).
2. Equilibrate the protein-A agarose and reagents to room temperature before use.
3. Clarify antibody fluids (antisera, ascites, or hybridoma supernatant) by centrifugation at 20,000 × g for 20 min at 4 °C or room temperature and collect supernatant (see Note 3).
1. Adjust pH of sample by dilution of antisera or ascitic fluid 1:10 with protein-A binding buffer (pH 8.0) or 1:2 for hybridoma cell-conditioned medium with 1 N NaOH used to adjust pH to 8.0 as necessary. Save a small volume fraction and label as input.
2. Prepare protein-A column by washing with 3–5×column volumes of binding buffer at 1 mL/min to remove preservative and exchange buffer and pH.
3. Pass clarified antibody sample in binding buffer over column 1–3× at a rate of 0.5–1 mL/min (see Note 4). Monitor protein absorbance at 280 nm using an in-line UV monitor (see Note 5). Save a small volume of the sample after it has been passed over the column and label as unbound (see Note 6).
4. Pass at least 5× column volume of protein-A binding buffer at 1 mL/min to wash beads of residual unbound protein and return to baseline 280 nm absorbance values. Save a small volume from each column pass of wash buffer and label them as wash 1–5 (see Note 7).
5. Apply protein-A elution buffer at a rate of 1 mL/min and begin collecting 1 mL fractions after approximately 1× column volume (see Note 8). Collect 1 mL fractions into tubes containing 0.1 mL of 1 M neutralization buffer (PBS; pH 8) to immediately neutralize acidic pH of the elution fraction. Monitor protein absorbance at 280 nm UV to identify desorption of the IgG from the resin as an A 280 protein peak and collect fractions until baseline protein measurement is achieved (see Note 9). Save a small volume from each elution fraction and label them as elution 1–5.
6. Determine protein concentration of each fraction by measuring absorbance at 280 nm or performing a protein assay such as Bradford or BCA. Plot the protein concentration of each fraction and evaluate the protein profile from all the fractions to identify those containing the antibody and ensure proper column performance. 
7. Pool and dialyze peak antibody fractions using a low molecular weight cutoff membrane against desired buffer such as PBS. Collect purified and desalted antibody for downstream application (see Note 11).
8. Regenerate the affinity resin by washing with 5× column volume of binding buffer followed by 5× column elution buffer (see Note 12).
9. To store the column, pass 5× column volumes of storage buffer that contains a bacteriostatic agent such as 0.05 % thimerosal or 30 % ethanol solution in water (see Note 13). Keep excess solution in the column to maintain resin hydration and store capped at 4 °C. 

Antibody Purification Using Immobilized Protein-G

1. Protein-G purification is recommended for mouse IgG, and antibody is bound at slightly acidic pH and eluted at a more acidic pH.
2. Equilibrate the protein-G agarose and reagents to room temperature before use.
3. Clarify antibody fluids (antisera, ascites, or hybridoma supernatant) by centrifugation at 20,000 × g for 20 min at 4 °C or room temperature and collect supernatant (see Note 3).
4. Adjust pH of sample by dilution of antisera or ascitic fluid 1:10 with protein-G binding buffer (0.1 M sodium acetate; pH 5.0) or 1:2 for hybridoma-conditioned medium. Save small volume of sample and label input.
5. Prepare protein-G column by washing with 3–5× column volumes of protein-G binding buffer at a rate of 1 mL/min to remove preservative and exchange buffer and pH.
6. Pass clarified antibody sample in binding buffer over column 1–3× at a rate of 0.5–1 mL/min (see Note 4). Monitor protein absorbance at 280 nm using an in-line UV monitor (see Note 5). Save a small volume of the sample after it has been passed over the column and label as unbound (see Note 6).
7. Pass at least 5× column volume of protein-G binding buffer at 1 mL/min to wash beads of residual unbound protein and return to baseline 280 nm absorbance values. Save a small volume from each column pass of wash buffer and label them as wash 1–5 (see Note 7).
8. Apply protein-G elution buffer (0.1 M glycine-HCL, pH 2–3) at a rate of 1 mL/min and begin collecting 1 mL fractions into tube containing 0.1 mL of neutralization buffer (1 M Tris buffer, pH 8.0). Monitor protein absorbance at 280 nm UV to identify desorption of the IgG from the resin as an A 280 protein peak and collect fractions until baseline protein measurement is achieved (see Note 9). Save a small volume from each elution fraction and label them as elution 1–5.
9. Determine protein concentration of each fraction by measuring absorbance at 280 nm or performing a protein assay such as Bradford or BCA. Plot the protein concentration of each fraction and evaluate the protein profile from all the fractions to identify those containing the antibody and ensure proper column performance. (see Note 10).
10. Pool and dialyze peak antibody fractions using a low molecular weight cutoff membrane against desired buffer such as PBS. Collect purified and desalted antibody for downstream application (see Note 11).
11. Regenerate the affinity resin by washing with 5× column volume of binding buffer followed by 5× column elution buffer (see Note 12).
12. To store the column, pass 5× column volumes of storage buffer that contains a bacteriostatic agent such as 0.05 % thimerosal or 30 % ethanol solution in water (see Note 13). Keep excess solution in the column to maintain resin hydration and store capped at 4 °C.

Immobilized Protein-L

1. Protein-L purification is recommended for hybridoma cellconditioned medium containing fetal bovine serum, and antibody is bound at neutral pH and eluted at an acidic pH (see Note 14).
2. Equilibrate the protein-L agarose and reagents to room temperature before use.
3. Centrifuge hybridoma supernatant to remove cells and debris at 10,000 × g for 5 min at room temperature then pass supernatant through a 0.2 μm filter.
4. Dilute sample at least 1:1 with protein-L binding buffer (PB, pH 7.2) before applying onto the protein-L column to maintain optimal ionic strength and pH for binding. Save a small volume of fraction and label as input.
5. Prepare protein-L column by washing with 3–5× column volumes of protein-L binding buffer at a rate of 1 mL/min to remove preservative and exchange buffer and pH.
6. Pass clarified antibody sample in binding buffer over column 1–3× at a rate of 0.5–1 mL/min (see Note 4). Monitor protein absorbance at 280 nm using an in-line UV monitor (see Note 5). Save a small volume of the sample after it has been passed over the column and label as unbound (see Note 6 ).
7. Pass at least 5× column volume of protein-L binding buffer at 1 mL/min to wash beads of residual unbound protein and return to baseline 280 nm absorbance values. Save a small volume from each column pass of wash buffer and label them as wash 1–5 (see Note 7).
8. Apply protein-L elution buffer (0.1 M glycine-HCL, pH 2–3) at a rate of 1 mL/min and begin collecting 1 mL fractions into tube containing 0.1 mL of neutralization buffer (1 M Tris buffer, pH 8.0). Monitor protein absorbance at 280 nm UV to identify desorption of the IgG from the resin as an A 280 protein peak and collect fractions until baseline protein measurement is achieved (see Note 9). Save a small volume from each elution fraction and label them as elution 1–5.
9. Determine protein concentration of each fraction by measuring absorbance at 280 nm or performing a protein assay such as Bradford or BCA. Plot the protein concentration of each fraction and evaluate the protein profile from all the fractions to identify those containing the antibody and ensure proper column performance.
10. Pool and dialyze peak antibody fractions using a low molecular weight cutoff membrane against desired buffer such as PBS. Collect purified and desalted antibody for downstream application (see Note 11).
11. Regenerate the affinity resin by washing with 5× column volume of binding buffer followed by 5× column elution buffer (see Note 12).
12. To store the column, pass 5× column volumes of storage buffer that contains a bacteriostatic agent such as 0.05 % thimerosal or 30 % ethanol solution in water (see Note 13). Keep excess solution in the column to maintain resin hydration and store capped at 4 °C.

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