Index: Volume 5 Number 1
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By Anthony Salerno, Ph.D. PureBiotech LLC

• Rapid, gentle & complete immobilization

• Easily automated

• No need for surfactants

• Will not clump or adhere to hydrophobic surfaces

• Economical purification of immunoglobulins

• Higher yields of concentrated target protein

PureBiotech LLC's Protein A particles are Magnetic Porous Glass (MPG®) particles to which protein A has been covalently attached. MPG® Protein A particles possess antibody binding properties similar to unconjugated protein A. The nominal binding capacity of 1 mg of particles is between 50-75 µg of immunoglobulin for strong species such as rabbit and human IgG¹.

The high surface area and high loading of MPG® Protein A promote quick capture of target antibody. Using an excess of MPG® Protein A particles rabbit IgG can be cleared (>94%) within 5-10 minutes at concentrations as low as 1 µg/ml, as shown in Figure 1. Excess antibody present at mg/ml concentrations also saturates MPG® Protein A particles within 5 minutes.

The high binding capacity of the particles combined with the ability to elute antibody in a minimal volume allows the recovery of highly concentrated target antibody/proteins. Figure 2 shows that more than 75% recovery of captured antibody can be achieved in as little as 10 µl volume while giving protein concentrations greater than 4 mg/ml. After neutralization, this allows for direct use of the supernatant in gel electrophoresis without the need for concentration or buffer exchange.

Figure 1. Binding Kinetics of Rabbit IgG to MPG® Protein A. MPG® Protein A was mixed with purified rabbit IgG at the ratios and concentrations indicated and incubated at room temperature in PBS (PBS containing 2 mg/ml lysozyme, to ensure binding specificity, for the 0.1 and 1 µg/ml curves). At the indicated times particles were magnetically separated and the supernatant was assayed for remaining IgG spectrophotometrically or by ELISA as required.

MPG® particles give higher yields of concentrated target protein than other available magnetic protein A particles. In addition to having higher binding capacity, magnetic porous glass particles do not clump. Polystyrene magnetic particles tend to clump and adhere to hydrophobic surfaces when surfactants or similar agents are eliminated from the buffers. This results in loss of particles to pipette tips, tube walls, etc. and increased elution volumes, which in turn lowers yield and dilutes recovered target protein (Table 1). MPG® Protein A performs well in high salt buffers without the presence of surfactants. Recovery can be essentially quantitative in as little as 50 µl of the appropriate buffer (Figure 2).

Figure 2. Recovery of Rabbit IgG as a Function of Elution Volume. MPG® Protein A was mixed with an excess of purified rabbit IgG. After washing and splitting into 1 mg replicates (n = 2-4), IgG was eluted with 0.1 M Glycine-HCl, pH 2.5 as indicated. The red bars represent the µg IgG (mean ± std. dev.) recovered, the gray bars are % total IgG binding capacity recovered, and the line represents the concentration of recovered IgG.

MPG® Protein A particles make mg scale magnetic batch purification of antibodies economically feasible. MPG® Protein A can be regenerated up to 12 times while still providing greater than 90% of initial binding capacity. The same high purity of antibody recovered from the particles during the first use is maintained in subsequent purifications. Only the heavy chain and diffuse light chain bands of polyclonal rabbit IgG are visable in the samples purified with MPG® Protein A (Figure 3). Regeneration is easy to do and takes only 5 minutes.

Figure 3: Purity of rabbit IgG recovered from MPG® Protein A particles. 3 mg MPG® Protein A was mixed with 0.5 ml 10% normal rabbit serum in 1 M glycine-HCl pH 8.6, 0.15 M NaCl (lane 2, 10 µg). After washing twice with an equal volume of the same buffer and twice with an equal volume of PBS, IgG was recovered from the particles with 0.1M glycine, pH 2.5 and neutralized with 0.1 volume of 1 M NaHPO4, pH 9.0. The regenerated particles were neutralized with 1 M glycine-HCl pH 8.6, 0.15 M NaCl and recycled with fresh serum for a total of 12 purifications. Samples (2.5 µg) from the initial (lane 3) and final (lane 4) purification were analyzed on a reducing SDS 12% polyacrylamide gel along with a commercial rabbit IgG standard (lane 1) and rabbit IgG purified from the same serum using PROSEP®-A High Capacity (lane 5). The gel was stained with SYPRO® Ruby.

¹ MPG® Protein A protocol (57.101)

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