Whether an antibody comes from a hybridoma or from recombinant expression in mammalian cells or bacteria, it has to be separated from everything else in the sample: cell lysate, medium, ascites fluid, serum. The right strategy depends on the antibody's class, species and status (tagged or untagged), its intrinsic chemistry, and how pure it needs to be. A few pointers follow.
Affinity resins
For monoclonal antibodies, affinity purification usually means Protein A, Protein G or Protein L chromatography. Binding capacity depends on the species and class of the antibody. Protein A and Protein G beads are the most common choice for the capture step with human IgG. They are not efficient for human IgM, IgE, IgD or IgA, so for those classes you need a different approach.
When the standard resins fall short
Protein L, G and A resins are easy to use and relatively cheap, and for most antibodies they are the right answer. Some species, classes and antibody fragments are hard to capture with them, though. Ion exchange can work in those cases, but it takes several purification steps to reach the same purity, so it is not efficient.
Two alternatives are worth knowing about. NHS-activated beads use N-hydroxysuccinimide chemistry to couple a ligand to a pre-activated medium through its primary amine group, so the antibody is captured purely on its affinity for the target. Multimodal beads are the option for the harder cases: when other strategies have failed, when you need to cut the number of purification steps, or when protein stability is a problem.
Pel-Freez Biologicals manufactures biological products for research in the United States under an ISO 9001:2015 quality system and holds an Animal Welfare Certification. Our range includes animal serum, plasma, complement, human and goat gamma globulin, IgG/IgM/antibody-depleted complement and adult rabbit complement. Contact us to learn more, or call (800) 643-3426 or (479) 636-4361.