research highlights

PROTEOMICS Cloning antibodies from serum

Proteomics analysis of polyclonal antibod- reference database using next-generation ies guides monoclonal production. sequencing. After inoculating rabbits with a When faced with a foreign invader, mam- human peptide associated with breast cancer, mals launch a diverse defense. One compo- the researchers selected the animal with the nent is an array of antibodies, each binding best response, collected RNA from its white the antigen in its own way, each generated blood cells and sequenced transcripts coding from a clonal population of white blood cells for antibody chains. called B cells. New work shows how to iden- The researchers also collected the highest- tify and manufacture the best of these anti- affinity antibodies from the rabbit’s serum. bodies in a reproducible way. These were divided into aliquots, each Researchers can generate antibodies digested with different enzymes, producing against antigens by inoculating an appropri- unique sets of digestion fragments that ate animal (usually a rabbit) and then col- could be analyzed by mass spectrometry and lecting the desired antibodies from its blood. compared to each other. ‘Polyclonal antibodies’ comprise the collec- To deduce the sequences in the best- tion of all antibodies against a given antigen binding antibodies reliably, the researchers produced in this fashion. Unfortunately, considered only data with particularly high polyclonal antibodies are not ideal for many coverage of the antibody regions and at research and medical applications because least a dozen unique peptides. This analysis their properties are hard to define and repro- could identify likely sequences for antibody duce consistently. For this reason, antibod- regions known as the heavy and light chains ies that are produced by just one clone of but not which sequences occurred together B cells, or ‘monoclonal antibodies’, are often in the same antibody. Thus, the researchers preferred. However, generating effective expressed and tested multiple possibilities. monoclonal antibodies can be difficult and This resulted in two monoclonal antibod- time consuming, and when using traditional ies, both with better specificity and sensi- means for this, such as hybridomas, much of tivity than the original polyclonal antibody the antibody repertoire is lost. mixture. Researchers at Cell Signaling Technology Researchers have used this technique of in Danvers, Massachusetts have now used inoculating animals and creating antigen-

© 2012 Nature America, Inc. All rights reserved. America, Inc. © 2012 Nature proteomics to identify the antigen-binding specific reference libraries to develop anti- sequences of promising antibodies pro- bodies from inoculations in both rabbits and duced by inoculated animals and then clone mice. The technique has now been used to promising sequences to generate monoclonal create antibodies from over a dozen antigens, npg antibodies. says company CSO Roberto Polakiewicz. Purifying high-affinity antibodies pro- Traditional methods of developing mono- duced in an inoculated mouse or rabbit is clonal antibodies require constructing and straightforward—these bind most tightly interrogating thousands of white blood cell to an antigen and can readily be captured. clones, a process that takes 7–10 weeks. In However, analyzing the sequences of these contrast, the proteomics-based approach proteins is more challenging. A typical pro- takes no more than 3 weeks. teomics analysis involves enzymatically The study also has implications for basic digesting collected proteins into peptide research, such as probing how the antibody fragments and then characterizing them response develops, says Polakiewicz. “This by mass spectrometry. Reference databases is a new technology to develop monoclonal based on gene collections can predict which antibodies, so it can be applied for making fragments are likely to be produced by a antibodies for any commercial application, particular protein digested by a particu- such as research tools, diagnostic assays and lar enzyme and so identify proteins in the therapeutics.” sample. Because immune systems shuffle Monya Baker portions of the genes to produce a dizzying assortment of antibodies, however, these RESEARCH PAPERS Cheung, W.C. et al. A proteomics approach for the genes are not accurately represented in the identification and cloning of monoclonal antibodies reference databases. To get around this prob- from serum. Nat. Biotechnol. advance online lem, the researchers created a specialized publication (25 March 2012).

440 | VOL.9 NO.5 | MAY 2012 | nature methods