Although the standard protocol for the purification of antibodies from serum using either recombinant Protein G or Protein A cross-linked to agarose beads is a proven technique, the method is arguably laborious and time-consuming. Furthermore, both Protein A and G have varying affinities for different IgG subclasses from different animal species. Although affinities for mouse, rabbit and human antibodies are generally comparable, a researcher needs to take special care to ensure that the correct affinity support is used to collect the desired antibody. The need to generate different supports simply adds more complexity to the process.
The Melon Gel Purification Kit (Pierce) allows for a simpler approach to monoclonal and polyclonal antibody purification from serum. The kit eliminates the need for repeated washings and elutions followed by neutralization and the need to couple the antibodies in the serum to Protein A/G. At a minimum, this will take a little over an hour for small samples (500 µl to 1 ml), not including the time to equilibrate reagents to room temperature. Since the antibodies are eluted in a mild buffer at physiological pH and free from amines, the melon gel kit further eliminates the need to dialyze the sample prior to subsequent experiments. Dialysis alone greatly extends the purification procedure. With the kit we were able to purify by gravity-flow column over 25 mls of polyclonal antibodies from rabbit serum in about 30 mins. Using the melon gel support in a microcentrifuge format (using Pierce’s Handee Spin Cups) for sample volumes less than 1 ml helps cut the purification time down to roughly 12 mins.
Additional time is definitely required to regenerate the support used in gravity columns for large gel volumes, but the amount of work as buffer passes through a column is negligible. During this time, the antibody that is collected can be immediately used for downstream applications (e.g., direct coupling to pre-activated agarose support for immunoprecipitation experiments or for fluorescent/biotin labeling). While some colleagues in our lab are critical of the substantial elution volume that results from large-scale purifications, this is easily remedied by concentrating the appropriate fractions using spin columns (Amicon). Pierce claims that a properly regenerated support can be used up to three times and, from experience, at least two purifications have been non-problematic resulting in the collection of comparable antibody concentrations. However, it should be noted that out of habit, we do not use these columns more than twice and therefore cannot validate the company’s claim. In addition, Pierce is transparent about potential problems from contaminating molecules (such as mouse transferrin) and it would be prudent to check the eluate by SDS-PAGE to ascertain the presence of contaminants. The biggest drawback is that the kit is by no means inexpensive and the melon support is not available for individual item purchase. The biggest advantage is the drastic reduction in labor and time and the convenient scalability. The relatively quick turn-around time is ideal especially for large scale purifications (greater than 2 mg of antibody).
Rhoel Dinglasan, Ph.D., M.P.H.
Johns Hopkins Bloomberg School of Public Health