In making recombinant antibodies, the resulting antibody is only as good as the combinatorial library and the screening assay. The trick is to find the molecule of highest affinity and specificity for the target among a library of millions of clones. Traditionally, recombinant antibody libraries have been phage-based and the screening relied on enzyme-linked immunosorbent assay (ELISA), not a high-throughput method. Changes in both phage display and screening methods are now moving recombinant antibody production into a high-throughput world.

Flow cytometry has become the assay of choice for rapid screening of clones from recombinant antibody libraries. “We looked at different ways of screening. Flow cytometry was the only one that seemed to meet our throughput requirements,” says Andrew Bradbury of Los Alamos National Laboratory in New Mexico. Bradbury's group has developed a flow-cytometry assay to screen its single-chain antibody-fragment phage libraries using a mixture of beads coated with specific and non-specific antigens. The method rapidly identifies antibodies that have good affinity for the protein of interest while discarding those that show low specificity.

Phage-display screening methods are also used to identify antibodies that target post-translational modifications (PTMs) such as phosphorylation or acetylation. As PTMs have a role in many processes — from gene regulation to apoptosis — they are of growing interest for the biological community. Companies have responded by developing antibodies targeting proteins in a specific state of modifications. “Antibodies to PTMs are gaining in importance with customers,” says Kumar Bala, director of antibody technologies for Millipore in the company's lab in Temecula, California.

Andrew Bradbury develops recombinant single-chain antibodies. Credit: LANL

But obtaining antibodies directed against PTMs is not a trivial task. Rockland Immunochemicals of Gilbertsville, Pennsylvania, has put in a lot of effort to develop antibodies for looking at phosphorylated and non-phosphorylated forms of various proteins in a sequence independent context, says Daniel O'Shannessy, the company's vice-president of corporate development.

Antibodies that recognize PTMs independently of the protein site on which the modification occurs are useful — particularly for enriching, for example, all phosphorylated proteins from a cell. But such antibodies are hard to make by animal immunization as the PTM itself is not immunogenic. So scientists are turning to recombinant molecules and in vitro screening such as phage display to isolate 'pan-PTM' affinity reagents.

Although making steps in the right direction, many more antibodies and further improvements in affinity reagent technology will be needed to understand and characterize the full range of PTMs found in nature. Still, Bala argues that the “best tools for purifying, identifying, differentiating and characterizing PTMs are antibodies”.

Nathan Blow