Rapid Antibody Concentration with Amicon® Ultra Centrifugal Filters

Introduction

Purification protocols for immunoglobulins generally include affinity binding to Protein A or G chromatography media, washing unbound proteins, and eluting with a low pH buffer. The resulting purified antibodies are often too dilute for downstream studies or long-term storage. Dialysis is often necessary to concentrate the antibody and/or exchange the buffer into one that preserves protein activity. Ultrafiltration achieves the same result faster, with up to 99% immunoglobulin recovery and one-step salt removal (see Concentration, Desalting, and Buffer Exchange protocol).

Method and Results

Purification and concentration of Rabbit IgG with Prosep® A and Prosep®Montage® Antibody Purification Kits and Amicon® Ultra-15 mL centrifugal filters.

A. Purification of IgG using Montage® Kits

  1. Equilibrate the Prosep® A or Prosep® G resins with 10 mL of binding buffer and spin for 5 min at 500 xg
  2. Pre-clarify the serum sample by passing through a 0.22 µm Steriflip® GP filter.
  3. Dilute the sample 1:1 with binding buffer A and centrifuge the device at 100-150 x g for 20 min.
  4. Wash the spin column with 20 mL of binding buffer A to remove unbound contaminants by centrifuging the column for 5 min at 500 x g.
  5. Elute the samples by adding 10 mL of elution buffer B2 into a fresh centrifuge tube containing 1.3 mL of neutralization buffer.
  6. Measure the total protein concentration of the sample using the Direct Detect® Spectrometer or other appropriate concentration method, such as UV absorbance at 280 nm.

B. Concentration of IgG using Amicon® Ultra-15 30kDa MWCO devices.

  1. Add the neutralized eluted fraction into Amicon® Ultra 15 mL 30 kDa MWCO device.
  2. Centrifuge for 10 to 20 min at 4000 x g in a swinging bucket rotor or 5000 x g in a fixed angle rotor until the sample has reached the desired concentration.

Figure 1 shows a decrease in the retentate volume proportional to the increase in antibody concentration. A 20-minute centrifugation step resulted in >95% recovery of immunoglobulins. Figure 2 shows an SDS-PAGE gel of purified rabbit IgGs before and after ultrafiltration. The data demonstrate the suitability of Amicon® Ultra devices for concentrating purified IgG.