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July, 2002
RECENTLY PUBLISHED:
UNIT 6.8 Phage Display Selection and Analysis of B-cell Epitopes (David Enshell-Seijffers & Jonathan M. Gershoni Tel Aviv University, Isreal). Combinatorial phage display libraries of random peptides can be used to discover the epitopes of antibodies through a procedure termed "biopanning". The affinity isolation of phage-displayed epitope peptidomimetics allows molecular definition of the epitopes of monoclonal antibodies. Epitope-profiles recognized by polyclonal serum samples can also be generated. Detailed step-by-step protocols and discussion of applications are provided.
UNIT 6.24 Detection of Intracellular Cytokines by Flow Cytometry (Barbara Foster and Calman Prussin, NIAID, NIH, Bethesda, MD). This updated unit describes the techniques required to perform intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cells, and cell-surface staining are included. Because of the greater level of nonspecific staining inherent in fixed, permeabilized cells, greater care must be taken in designing controls.
UNIT 17.2 Monitoring Antigen-Specific T Cells Using MHC-Ig Dimers (Jonathan P. Schneck, Jill E. Slansky, Sean M. O’Herrin, and Tim F. Greten, Johns Hopkins University, School of Medicine, Baltimore, Md.). The lack of high-affinity reagents has made it difficult to distinguish T cells on the basis of antigen specificity. This unit provides protocols that utilize innovations in molecular design allowing one to construct soluble multivalent MHC complexes (MHC-Ig dimers) with high avidity for cognate T cell receptors. MHC-Ig dimers display stable binding properties when they interact with antigen-specific T cells, thus allowing their use in the staining of antigen-specific T cells by flow cytometry. Methods for constructing and detecting these MHC-Ig dimers are included, along with protocols for applying them in the quantitation of antigen-specific T cells.
UNIT 19.11 Influenza Virus (Robert Cottey, Cheryl A. Rowe, and Bradley S. Bender, University of Florida College of Medicine, Gainsville, Fla.). This new addition to the Animal Models for Infectious Diseases chapter contains several methods for infecting mice with influenza virus. The unit also includes protocols needed to propagate influenza virus in hen eggs, quantitate virus titers (in tissue culture medium and in influenza-infected mouse serum), and adopt human isolates of influenza for growth in mice. Methods for measuring the 50% mouse lethal dose are also included. Finally, protocols for generating anti-influenza cytotoxic T lymphocytes (CTL) from splenocyte precursors and harvesting pulmonary CTL following respiratory tract challenge of mice with influenza virus are provided.
Two new chapters: Current Protocols in Immunology has added two new chapters to the series. An Innate Immunity chapter (Chapter 14) focuses on a broad range of topics and will include protocols for investigating and modulating key receptors such as KIR and TOLL. The chapter also contains commentary units including one on receptor-specific antibodies and their cross-reactivity profiles. A Microscopy chapter (Chapter 21) includes three units that cover such topics as: proper alignment and adjustment of the light microscope (Unit 21.1); fluorescence microscopy (Unit 21.2); and, immunofluorescence staining (Unit 21.3).
UNIT 10.19B Phage Display of Single-Display Antibody Constructs (Itai Benhar and Yoram Reiter, Tel-Aviv University Ramat Aviv, Tel-Aviv and Technion, Haifa, Israel). In recent years, the use of surface-display vectors for displaying polypeptides on the surface of bacteriophage, combined with powerful selection and screening strategies, has transformed the way in which ligands, such as peptides, enzymes, and antibodies, are generated and manipulated. This unit addresses recent progress in the construction of, and selection from antibody phage display libraries, together with useful approaches for the analysis of phage antibodies. The unit focuses on the construction of immune libraries where the sources for antibody genes are spleens of immunized mice and the isolation and initial characterization of single-chain antibodies (scFvs) from such libraries. Methods for the isolation of antibody variable-domain coding DNA from spleen or hybridoma RNA by RT-PCR, the assembly of full-length scFv DNA cassettes and their cloning into a phagemid vector and standard affinity selection using plastic-immobilized antigen to capture phages that display scFvs that are antigen-specific are included.
FORTHCOMING:
UNIT 11.9D TRAF-Mediated TNFR Family Signaling (Joseph R. Arron, Matthew C. Walsh, and Yongwon Choi, University of Pennsylvania School of Medicine, Philadelphia). Thanks again for agreeing to review this unit for chapter 3 on the subjects of TNFR family signaling. This commentary unit will provide a comprehensive summary of the TNF family proteins, their activating receptors, and the six known TNF receptor-associated factors (TRAF). The unit focuses on structural properties of TRAF proteins and signaling pathways.
UNIT 11.10 Isolation and Use of Rafts (Deborah A. Brown, State University of New York at Stony Brook, Stony Brook, NY). This updated unit will describe methods for isolating and analyzing rafts by detergent insolubility. To distinguish these rafts from raft-like membranes isolated by other methods they are referred to here as detergent-resistant membranes (DRMs). Two DRM isolation methods will be included: (1) flotation on sucrose density gradients, and (2) isolation by pelleting after detergent extraction. Protocols for analysing DRM proteins using SDS-PAGE, immunoblotting, or using radiolabeled DRM proteins will be included together with methods for quantitation by high-performance thin layer chromatography. New protocols for raft disruption by cholesterol removal and measuring the kinetics of such removal will be provided along with a method for reversing cholesterol removal.
UNIT 6.13
Measurement of Mouse and Human Interleukin-9 (Jen-Christophe Renauld and Jaques
Van Snick, Ludwig Institute for Cancer Research and Catholic University of
Louvain, Brussels, Belgium). This updated unit will describe several proliferation
assays that can be used to detect or quantitate human and murine interleukins-9
(IL-9). Detailed guidelines for making each approach IL-9-specific will be
provided together with support protocols for maintaining and cryopreserving
each of the IL-9-responsive cell lines