Isolation of monoclonal immunoglobulin

2.11 Isolation of monoclonal immunoglobulin

If purified antibody cannot be isolated directly by antigen binding, prepare an immunoglobulin fraction of the ascitic fluid using either affinity or ion-exchange chromatography (see Sections 1.3 and 1.4).

2.11.1 Problems and solutions

Purification of monoclonal antibodies follows the same general procedures outlined for poly- clonal antibodies. As each monoclonal antibody is unique, an individual determination will be required to establish the particular conditions for the isolation of each antibody.

Antibodies in tissue culture supernatants must first be concentrated either in a membrane concentrator or by ammonium sulphate precipitation before they can be isolated. Ascitic fluids can be treated in a similar manner to serum immunoglobulins. However, they frequently contain appreciable quantities of lipid that should be removed at the beginning of the isolation procedure by treatment with Aerosil.

Hybridoma antibodies grown in serum-free medium contain little immunoglobulin other than that of hybridoma origin, but contain a lot of albumin. Thus, problems of antibody purification are essentially the same for serum-supplemented or serum-free media. Concentration and partial purification can be obtained by precipitation at a 50% saturation with ammonium sulphate, using solid ammonium sulphate to avoid excessive increase in volume. Excess albumin may be removed by affinity chromatography on Cibacron blue dye affinity gel before attempting ion- exchange chromatography.

Note: A combination of Cibacron blue with diethylaminoethyl (DEAE) is available from Biorad that allows albumin removal and ion-exchange in one step. DEAE and quaternary aminoethyl (QAE) ion-exchangers are suitable for purification of IgG monoclonal antibodies. As monoclonal antibodies have a unique isoelectric point and charge density, use gradient elution from the ion exchanger.

Monoclonal IgA is more difficult to prepare but can be isolated by ammonium sulphate pre- cipitation, followed by ion-exchange chromatography. Using gradient elution, the IgA should elute after IgG but before the albumin fraction.

IgM antibodies are most readily prepared by gel filtration using a high-resolution gel such as Sephacryl S-300 HR.

2.11.2 Anti-immunoglobulin affinity columns

The IgG or antibody fraction of goat or rabbit anti-mouse immunoglobulin may be linked to Sepharose and the affinity column so formed used to isolate hybridoma immunoglobulin.

MATERIALS AND EQUIPMENT As for Section 1.5.2 (Use of immunoadsorbent for antibody purification) but in addition:

62 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation

Anti-mouse immunoglobulin, goat or rabbit Mouse immunoglobulin Hybridoma-derived antibody

METHOD

1 Isolate the antibody or IgG fraction from the anti-mouse immunoglobulin serum.

2 Link anti-immunoglobulin to Sepharose and pack into a column.

3 Add 100 mg normal mouse immunoglobulin prepared by ammonium sulphate precipitation and allow it to filter slowly through the column.

4 Elute with glycine–HCl buffer and re-equilibrate the column with PBS until the absorbency of the effluent is less than 0.01 at 280 nm.

5 Add solution containing hybridoma-derived antibody, wash and elute as above.

TECHNICAL NOTES • These columns are precycled with normal mouse IgG and eluted with acid buffer to saturate

the high-affinity anti-immunoglobulin antibodies that would otherwise bind the precious monoclonal antibody virtually irreversibly. In any case, these columns should always be pre-eluted with acid buffer and re-equilibrated before use to remove any loosely bound material.

• To minimize denaturation, elution should be accomplished under the most gentle conditions compatible with the release of antibody. • Mouse immunoglobulin may also be isolated by ion-exchange chromatography (see Section

1.3) or by affinity chromatography on staphylococcal protein A–Sepharose (subclass IgG 2a , IgG 2b and IgG 3 asee Section 1.4). (Rat immunoglobulins, with the exception of the minor sub- class IgG 2c , do not bind to protein A, but may be prepared by streptococcal protein G.)