Advertisement
Articles
Advertisement

Antibody Purification With Affinity Spin Columns

Wed, 07/16/2003 - 10:05am
Kits include all the devices and premixed buffers necessary for prefiltration, purification concentration and desalting of mono and polyclonal antibodies from a variety of species.
by Mark Kavonian



Monoclonal and polyclonal antibodies serve as important reagents for capturing antigens of interest in a wide range of immunochemical procedures. Researchers commonly isolate antibodies from crude sources such as ascites, cell culture supernatant or serum using Protein A or G affinity chromatography. The Montage® Antibody Purification Kit (Millipore Corporation) incorporates centrifugal spin columns with Protein A or Protein G affinity media, replacing traditional chromatographic mehods and providing speed and convenience in the procedure as well as product purity, good yields and reproducibility.

Centrifugation-enhanced affinity purification
The Montage Kit includes all devices and premixed buffers necessary for prefiltration, purification, concentration, and desalting of mono- and polyclonal antibodies from a variety of species. Central to the kit are Montage spin columns, which incorporate plugs pre-packed with PROSEP® media (Millipore Corporation). PROSEP media is a porous glass matrix with either Protein A or Protein G covalently immobilized onto it.

Researchers can add up to 10 ml of sample and purify up to 20 mg of antibody using the Montage spin columns with PROSEP-A media in less than an hour. In addition, multiple spin columns can be centrifuged simultaneously to process several samples simultaneously.

Montage spin columns do not require setup or monitoring, nor do they require pumps, tubing or syringes. It is typical to use a centrifuge that is already in the lab with a standard swinging bucket rotor.

Montage spin columns minimize hold-up volume, ensuring high solute recovery with minimal loss from non-specific absorption. Each column may be used up to 10 times without loss of binding capacity and specificity.

Total purification solution
The Montage kit includes prepacked media plugs, optimized buffers, and everything needed for purification, from initial fractionation to final desalting and concentration. The complete package saves preparation time and promotes reproducibility.

For prefiltering immediately before loading samples, the kit includes the Steriflip® GP filter device (Millipore). Its low protein binding microporous filter helps prevent aggregates and precipitating protein complexes from slowing the flow and potentially shortening column life. The kit also includes new Amicon® Ultra-15 centrifugal filters (Millipore) for desalting and concentration of the purified antibody. The kit provides enough premixed buffers for 10 purifications per column.


Figure 3. The Montage Antibody Purification Kit incudes spin columns as well as prefiltration and concentration devices.
Binding kinetics and flow rate
Flow rate in affinity chromatography is crucial to binding. If the mobile phase flows too quickly it will have insufficient time to diffuse fully into the column support. Too slow a flow may result in too much diffusion, causing nonspecific binding.

PROSEP media chemistries have sufficiently rapid association kinetics between the target antibody and the immobilized ligand to allow the mobile phase to diffuse through the internal bead structure. The large internal surface area of the PROSEP media bed is designed for centrifuge speeds of 1,000 3 g. (No data is available for performing wash steps at greater speeds.)

PROSEP chromatography media
The PROSEP media matrix is porous glass permeated by interconnecting pores of uniform size. It is incompressible and highly durable, resisting both shrinkage and swelling, and is highly stable and insoluble when exposed to low pH, detergents, buffers and various solvents.

PROSEP-A media has proven successful in large-scale bioprocessing applications, including purification of monoclonal antibodies. Because the Montage Kit makes it available for purification in research, PROSEP media can be used for applications that may require scale-up to process-scale columns.

Choosing between Protein A and Protein G
Protein A and Protein G, bacterial cell wall proteins, both have a high affinity for the Fc region of immunoglobulin, which makes them the ligands of choice for antibody purification. Although they form strong bonds with the antibody, their affinity is pH-dependent, which allows for easy elution using a low pH acidic buffer.

Traditionally, Protein G media has been shown to bind to more immunoglobulin classes than Protein A media. In addition, antibodies have formed stronger bonds to Protein G than Protein A. Therefore, Protein G has been used to purify some antibodies that traditional Protein A media may be unable to bind, such as Human IgG3. However, the high pH, high salt binding buffer included in the Montage Kit with PROSEP-A expands the range of antibody species that bind to the Protein A media (See Table 1 for a comparison of binding affinities).

Reproducibility and effectiveness
When Steriflip devices (included in the kit) are used to remove debris immediately before loading the sample, the Montage spin columns are proven successful for up to 103 without loss of binding capacity or specificity. Figure 1 shows the reproducibility of the Montage spin column with Protein-G media in purifying human IgG over 10 cycles.

The Montage kit is effective in purifying mono- and polyclonal antibodies from crude samples from a variety of species. Figure 2 shows SDS-PAGE validation of the effectiveness of the Montage spin column with PROSEP-A media for purifying monoclonal mouse IgG in ascites.

Conclusion
Montage Antibody Purification Kits with PROSEP-A or PROSEP-G media provide a rapid, convenient purification method that removes most contaminants while achieving purities approaching homogeneity. The kits save time and allow less experienced technicians to easily purify antibodies. The kits use standard laboratory centrifuges, avoiding the need for specialized equipment. Flow properties and low hold-up volumes of the spin columns contribute to high yields and repeatable performance over multiple uses.

Antibodies purified with Montage spin columns can be used in a wide range of laboratory procedures, including immunoprecipitation, immunofluorescence, Western blotting, and enzyme-linked immunosorbent assays (ELISAs). The purified antibodies can be used for radiolabelling, conjugations (such as with fluorescein) or preparation of immuno-affinity columns.

About the author
Mark Kavonian is a Group Product Manager with Millipore Corporation.Millipore Corp., Danvers, MA. 800-426-4266; millipore.com
Advertisement

Share this Story

X
You may login with either your assigned username or your e-mail address.
The password field is case sensitive.
Loading