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Bioinformatics Identification of Antigenic Peptide: Predicting the Specificity of Major MHC Class I and II Pathway Players

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 ...

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Recombinant Poxviruses: Versatile Tools for Immunological Assays

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, ...

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Purification, Preparation, and Use of ChaperonePeptide Complexes for Tumor Immunotherapy

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 ...

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Identifying Source Proteins for MHC Class I-Presented Peptides

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 ...

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Biochemical Analysis of Naturally Processed Antigenic Peptides Presented by MHC Class I Molecules

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 ...

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Quantitating MHC Class I Ligand Production and Presentation Using TCR-Like Antibodies

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 ...

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Establishing MHC Class I Peptide Motifs

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 ...

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Biochemical Large-Scale Identification of MHC Class I Ligands

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 ...

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Determining the Activity of the Transporter Associated with Antigen Processing in the Compartments of the Secretory Pathway

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 ...

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Evaluation of Viral Interference with MHC Class I-Restricted Antigen Processing and Presentation Using a Flow Cytometry-Based Approach

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 ...

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Bacterial Two Hybrid: A Versatile One-Step Intracellular Selection Method

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 ...

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Pichia Surface Display: A Tool for Screening Single Domain Antibodies

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 ...

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Semiautomated Panning of Naive Camelidae Libraries and Selection of Single-Domain Antibodies Against Peptide Antigens

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 ...

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Selection by Phage Display of Single Domain Antibodies Specific to Antigens in Their Native Conformation

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 ...

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Selection of VHH Antibody Fragments That Recognize Different A Depositions Using Complex Immune Libraries

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 ...

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Selection of VHHs Under Application Conditions

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 ...

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Cell-Free Selection of Domain Antibodies by In Vitro Compartmentalization

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 ...

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Selection of Functional Single Domain Antibody Fragments for Interfering with ProteinProtein Interactions Inside Cells: A One Plasmid Mammalian Two-Hy

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 ...

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Intracellular Antibody Capture (IAC) Methods for Single Domain Antibodies

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 ...

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Preparation of a Nave Library of Camelid Single Domain Antibodies

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 ...

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