Bioinformatics methods for immunology have become increasingly used over the last decade and now form an integrated part of most epitope discovery projects. This wide usage has led to the confusion of defining which of the many methods to use for what problems. In this chapter, an overview is given f ...
The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, ...
The molecular chaperone heat-shock protein 70 (Hsp70) possesses immune stimulatory properties that have been employed in the preparation of anticancer vaccines. Hsp70 binds antigenic peptides in the cytoplasm of cancer cells. Hsp70 thus serves as a convenient, non-discriminati ...
Identification of antigenic peptides recognized by cytolytic T lymphocytes (CTL) is a prerequisite for the development of targeted cancer immunotherapy approaches. This chapter provides a global approach for the identification of peptides recognized by CTL. It implies the iden ...
Immune surveillance of infected or tumor cells by CD8+ T cells requires that MHC class I molecules present a diverse repertoire of peptides on the cell surface. Even a few copies of individual peptides among this mixture are sufficient for recognition by the antigen receptors of appropriate CD8+ T ...
Accurately determining the number of peptide–MHC class I complexes on the cell surface is necessary when evaluating cellular processes or pharmaceuticals that alter the antigen presentation machinery. Here I describe a quantitative flow cytometry application for determining ...
Major histocompatibility complex (MHC) class I peptide motifs are used on a regular basis to identify and predict MHC class I ligands and CD8+ T-cell epitopes. This approach is above all an invaluable tool for the identification of disease-associated epitopes. As a matter of fact, the vast majori ...
The large-scale identification of MHC class I presented peptides is indispensable for gaining insight into the fundamental rules of immune recognition as well as it is an invaluable tool in identifying potential targets for the immunotherapy of disease. In this chapter we briefly review t ...
Peptide-receptive MHC class I molecules and the TAP (transporter associated with antigen processing) peptide transporter are known to leave the ER and cycle through the cis side of the Golgi apparatus. The amount, and the extent of the activity, of TAP in post-ER compartments is likely to vary betw ...
The peptide content of MHC class I molecules present at the cell surface is monitored by surveilling CD8+ cytotoxic T cells. In case of a viral infection, a proportion of the MHC class I molecules will carry peptides derived from viral proteins. This allows the CD8+ T cells to recognize and eliminate virus ...
Many antibody fragments, selected ex vivo by phage display, fail to form functional antigen-binding entities when expressed and used intracellularly (i.e., as intrabodies) because the interior of the cell poses significant challenges on the folding of antibodies. Such dropout can be av ...
Yeast surface display is being employed as an efficient tool for the isolation and engineering of traditional antibody fragments, both scFv and Fab, as well as single domain antibodies. Here we describe the protocols for a yeast surface display system developed in the methylothrophic yeast ...
With the identification of vast numbers of novel proteins through genomic and proteomic initiatives, the need for efficient processes to characterize and target them has increased. Antibodies are naturally designed molecules that can fulfill this need, and in vitro methodologies f ...
Phage display of antibody fragments and other binding molecules is a well-established technique to identify ligands interacting with any molecule of interest. Selection of in vivo matured single domain antibody fragments from phage display libraries is very powerful as in these libr ...
Phage display technology is frequently used to obtain antigen specific binders with predetermined characteristics. Phage display libraries are often constructed from animals immunized with the antigen of interest. An important point of consideration when making immune libr ...
The successful application of antibody fragments such as VHHs in diagnostic assays, affinity purification, imaging, or therapy is not determined by the specificity and affinity of the antibody fragment alone. The ability to bind the target protein in the environment in which the antibody f ...
Efficient identification of antibodies, or any fragments thereof, displaying desired specificity and affinity is critical for the development of novel immunotherapeutics. Here we describe the adaptation of in vitro compartmentalization for the cell-free selection of Vκ and VH d ...
As a complement to the intracellular antibody capture method to isolate intracellular single domain antibody fragments (iDabs) from high diverse libraries, we describe here a simple mammalian two-hybrid (M2H) protocol using a “bait-prey hybrid single plasmid” to assess those inter ...
Intracellular single domain antibodies are recombinant proteins, comprising one variable region domain fragment, that bind specifically to intracellular molecules and can interfere with their particular functions within various cellular compartments. They are valua ...
The preparation of antibody libraries starting from lymphocytes recovered from immunized members of the Camelidae enables to collect binders that underwent somatic maturation. However, the time and costs necessary to prepare a library for each new antigen may urge to look for alternat ...