Phage display of antibody libraries has been widely used for over a decade to generate monoclonal antibodies. Yeast display has been developed more recently. Here the two approaches were directly compared using the same HIV-1 immune scFv cDNA library expressed in phage and yeast display vectors and using the same selecting antigen (HIV-1 gp120). Yeast display was shown to sample the immune antibody repertoire considerably more fully than phage display, selecting all the scFv identified by phage display and twice as many novel antibodies. Positive phage display selection appeared to largely reflect those antibodies that as phage-scFv gave the highest signal in phage ELISAs assessing antigen binding. This signal is thought to reflect the efficiency of expression of folded scFv at the phage surface. Increased access to immune repertoires may increase the rescue of novel antibodies of therapeutic or analytical value that often form a minor part of a typical antibody response.

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Antibodies are among the most highly selective tight-binding ligands for proteins. Because the human genome project has deciphered the proteome, there is an opportunity to use combinatorial antibody libraries to select high-affinity antibodies to every protein encoded by the genome. However, this is a large task because the selection formats used today for combinatorial antibody libraries are geared toward generating antibodies to one antigen at a time. Here, we describe a method that accelerates the identification of antibodies to a multitude of antigens simultaneously by matching combinatorial antibody libraries against eukaryotic antigen libraries so that replication-competent cognate antigen-antibody pairs can be directly selected. Phage and yeast display systems are used because they each link genotype to phenotype and can be replicated individually. When combined with cell sorting, the two libraries can be selected against each other for recovery of cognate antigen-antibody clones in a single experiment.antibody libraries ͉ human genome ͉ phage display ͉ yeast display T he generation of antibodies to scientifically and clinically important protein antigens has occupied researchers for the past 25 years and has led to the establishment of combinatorial antibody libraries (1-6). Essentially, these libraries constitute a synthetic immune system. Nowadays, such libraries are routinely prepared and contain antibody collections that exceed the diversity of natural repertoires by many orders of magnitude. These libraries are not restricted by tolerance, they avoid the use of live animals, and have yielded important therapeutic antibodies (6). The libraries are most often formatted in yeast (7,8) or phage (1, 4) so that single binding events can be replicated and high-affinity antibodies can be selected. However, we have yet to extract the full potential of these powerful library methods because we still select antibodies one antigen at a time (9).The bottleneck imposed when antibodies are selected to one antigen at a time is illuminated by the opportunities posed by the human and other genome projects. These projects have provided an explosion in the numbers of known proteins, and it would be desirable to generate a set of high-affinity monoclonal antibodies to each of them so that ultimately one has a set of antibodies to every protein in the genome. Because combinatorial antibody libraries are not restricted by immunological tolerance, any selfor nonself-protein can be bound by a member of the antibody library. This means that, with respect to a given antibody library, the human proteome can be considered to be a collection of antigens.The present article describes a solution to the problem of simultaneous selection of monoclonal antibodies to a large set of antigens rather than to one antigen at a time.Coselection of cognate antibody-antigen pairs from combinatorial libraries has been attempted by using selectively infective phage (10, 11) or protein fragment complementation (12-14) with only limited success. ...

Yeast display is a powerful technology for the isolation of monoclonal antibodies (mAbs) against a target antigen. Antibody libraries have been displayed on the surface of yeast as both single-chain variable fragment (scFv) and antigen binding fragment (Fab). Here, we combine these two formats to display well-characterized mAbs as single-chain Fabs (scFabs) on the surface of yeast and construct the first scFab yeast display antibody library. When expressed on the surface of yeast, two out of three anti-human immunodeficiency virus (HIV)-1 mAbs bound with higher affinity as scFabs than scFvs. Also, the soluble scFab preparations exhibited binding and neutralization profiles comparable to that of the corresponding Fab fragments. Display of an immune HIV-1 scFab library on the surface of yeast, followed by rounds of sorting against HIV-1 gp120, allowed for the selection of 13 antigen-specific clones. When the same cDNA was used to construct the library in an scFv format, a similar number but a lower affinity set of clones were selected. Based on these results, yeast-displayed scFab libraries can be constructed and selected with high efficiency, characterized without the need for a reformatting step, and used to isolate higher-affinity antibodies than scFv libraries.

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