Protein Protocols Protein Protocols

Affinity Purification of Monoclonal Antibodies 1125
3.2. General Purification of Mouse IgG1, Rat IgG1,
and Rat IgG2b Using Protein A (see Note 2)
For cell culture supernatants that contain fetal calf serum and the separation of contaminating
bovine IgG from the monoclonal antibody, please see Subheading 3.3.
1. Bring all materials to room temperature.
2. Dilute supernatant 11 with 1000 mM sodium citrate solution. Filter the resulting solution
through a 0.2-µm filter (important).
3. Equilibrate a 10-mL protein A gel with 5 column volumes of buffer A.
4. Load supernatant onto the protein A–gel column at 5 mL/min.
5. Load up to 12 mg of antibody/mL of gel.
6. Wash with buffer A until the UV baseline is reached.
7. Elute antibody with 10 column volumes of buffer B.
8. Reequilibrate column with 2 column volumes of buffer A.
9. Wash with 5 column volumes of buffer C followed by 10 column volumes of buffer A.
The column is ready for the next chromatography run.
3.3. General Purification of IgG with Removal of Bovine IgG
Using Protein A (see Note 3)
This method is designed for the purification of human, humanized, and mouse IgG2a
and IgG2b monoclonal antibodies from hybridoma cell culture supernatant containing
fetal calf serum. It does not work well with weakly binding antibodies such as mouse IgG1.
1. Bring all materials to room temperature.
2. Equilibrate a 10-mL protein A gel with 5 column volumes of buffer A.
3. Load supernatant onto the protein A–gel column at 5 mL/min.
4. Load up to 20 mg of antibody/mL of gel.
5. Wash with 5 column volumes of buffer A.
6. Wash with 15 column volumes of buffer B.
7. Elute antibody with 10 column volumes of buffer C.
8. Wash with 5 column volumes of buffer E followed by 10 column volumes of buffer A.
The column is ready for the next chromatography run.
3.4. Thiophilic Purification of IgG (see Note 4)
This protocol is designed for the purification of all IgG from hybridoma cell culture
supernatant and ascitic fluids. For cell culture supernatants that contain more than 5%
fetal calf serum, the monoclonal antibody will be contaminated with bovine IgG.
1. Bring all materials to room temperature.
2. Dilute supernatant 11 with 1000 mM sodium sulfate solution.
3. Filter the resulting solution through a 0.2-µm filter (important).
4. Equilibrate thiophilic gel with 5 column volumes of buffer A.
5. Load supernatant onto the thiophilic gel column at 5 mL/min.
6. Load up to 10 mg of antibody/mL of gel.
7. Wash with buffer A until the UV baseline is reached.
8. Elute antibody with 10 column volumes of buffer B.
9. Wash with 5 column volumes of buffer C followed by 10 column volumes of buffer A.
The column is ready for the next chromatography run.