Two complementary strategies can be used in the fabrication of molecular biomaterials. In the 'top-down' approach, biomaterials are generated by stripping down a complex entity into its component parts (for example, paring a virus particle down to its capsid to form a viral cage). This contrasts with the 'bottom-up' approach, in which materials are assembled molecule by molecule (and in some cases even atom by atom) to produce novel supramolecular architectures. The latter approach is likely to become an integral part of nanomaterials manufacture and requires a deep understanding of individual molecular building blocks and their structures, assembly properties and dynamic behaviors. Two key elements in molecular fabrication are chemical complementarity and structural compatibility, both of which confer the weak and noncovalent interactions that bind building blocks together during self-assembly. Using natural processes as a guide, substantial advances have been achieved at the interface of nanomaterials and biology, including the fabrication of nanofiber materials for three-dimensional cell culture and tissue engineering, the assembly of peptide or protein nanotubes and helical ribbons, the creation of living microlenses, the synthesis of metal nanowires on DNA templates, the fabrication of peptide, protein and lipid scaffolds, the assembly of electronic materials by bacterial phage selection, and the use of radiofrequency to regulate molecular behaviors.

http://users.isr.ist.utl.pt/~sm3/biomedica/tissue-engeneering/Tiss_eng_3.pdf

Fabrication of novel biomaterials through molecular self-assembly.

During the past decade several display methods and other library screening techniques have been developed for isolating monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are now able to identify mAb leads with virtually any specificity. Innovative strategies enable directed evolution of binding sites with ultra-high affinity, high stability and increased potency, sometimes to a level that cannot be achieved by immunization. Automation of the technology is making it possible to identify hundreds of different antibody leads to a single therapeutic target. With the first antibody of this new generation, adalimumab (Humira, a human IgG1 specific for human tumor necrosis factor (TNF)), already approved for therapy and with many more in clinical trials, these recombinant antibody technologies will provide a solid basis for the discovery of antibody-based biopharmaceuticals, diagnostics and research reagents for decades to come.

Selecting and screening recombinant antibody libraries

This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest

/pdf/isolating-and-engineering-human-antibodies-using-yeast-3v51pwv9wx.pdf

Isolating and engineering human antibodies using yeast surface display

In the year 2003 there was a 17% increase in the number of publications citing work performed using optical biosensor technology compared with the previous year. We collated the 962 total papers for 2003, identified the geographical regions where the work was performed, highlighted the instrument types on which it was carried out, and segregated the papers by biological system. In this overview, we spotlight 13 papers that should be on everyone's 'must read' list for 2003 and provide examples of how to identify and interpret high-quality biosensor data. Although we still find that the literature is replete with poorly performed experiments, over-interpreted results and a general lack of understanding of data analysis, we are optimistic that these shortcomings will be addressed as biosensor technology continues to mature.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jmr.753

Survey of the year 2003 commercial optical biosensor literature

In vitro display technologies, best exemplified by phage and yeast display, were first described for the selection of antibodies some 20 years ago. Since then, many antibodies have been selected and improved upon using these methods. Although it is not widely recognized, many of the antibodies derived using in vitro display methods have properties that would be extremely difficult, if not impossible, to obtain by immunizing animals. The first antibodies derived using in vitro display methods are now in the clinic, with many more waiting in the wings. Unlike immunization, in vitro display permits the use of defined selection conditions and provides immediate availability of the sequence encoding the antibody. The amenability of in vitro display to high-throughput applications broadens the prospects for their wider use in basic and applied research.

Beyond natural antibodies: the power of in vitro display technologies

A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.

Jane M. Weaver Feldhaus

Papers

Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library