ImmunoMethods最新文献

The immobilization of antibodies in two-dimensional (physical adsorption and immobilization via binding proteins) and that in three-dimensional (chemical binding to polypeptide strands) coverage of solid matrices have been compared. Methods of optimizing exposure of the immunoglobulin paratope after binding to surfaces to permit unimpaired antibody-antigen complex formation have been investigated. At comparable antibody densities, a larger number of paratopes are available for complex formation when antibodies are immobilized by means of binding protein (Protein A, Protein G, secondary antibodies, and F(ab)₂ fragments of secondary antibodies) compared to physical adsorption. Using a primary coat of binding protein, reproducible immobilization of antibodies at densities up to two orders of magnitude lower than densities produced by physical adsorption and chemical immobilization is achieved. Binding of antibodies by means of polypeptide strands can be used to obtain multi-layers of IgG molecules. This method of immobilization gives the IgG molecules some flexibility in lateral positioning which leads to formation of clusters and increased attachment to the solid matrix, impairing the paratopes′ ability to form complexes. However, flexible attachment can permit separation of the IgG molecules by treatment with detergents (e.g., Tween) or with charged molecules (e.g., fluorescein) or by introducing binding proteins as spacers. The implications for engineering organized multilayers of antibodies for optical biosensors are reviewed.

Total internal reflection fluorescence (TIRF) is used in concert with the multifrequency phase and modulation (MPM) technique to study in situ the interfacial distribution of surface-immobilized F(ab′) antifluorescein antibodies. Using this new technique we recover information on the surface-immobilized receptor dynamics and demonstrate that this particular immunosurface is composed of an unstable distribution of biodomains. The MPM-TIRF technique adds a new tool to our arsenal for studying the dynamics of biosensor interfaces.

This paper presents an overview of the principles and applications of immunofluorescence spectroscopy in the development of antibody-based fiber-optic sensors. Special focus is devoted to antibody-based fiber-optic fluoroimmunosensors developed in our laboratories to detect important pollutants and biochemicals. The fiber-optic sensor utilizes antibodies covalently bound or encapsulated on the probe tip. The usefulness of fluoroimmunosensors for environmental and biomedical monitoring is discussed.

Cytokines are powerful regulators of lymphocyte growth and development. Recent progress in isolating and cloning cytokine receptors has provided detailed knowledge about the structures of these proteins. However, the mechanisms by which these receptors transduce signals following ligand binding remain largely unknown. The conventional approach to defining a receptor-regulated signal transduction pathway has been to determine the membrane-proximal biochemical events and to use pharmacological mimics of these biochemical messengers. While this approach has provided detailed information about the early signaling events coupled to several receptor systems, evidence for a relationship between these early events to downstream nuclear and cellular responses is often circumstantial. Powerful genetic approaches now are amenable to dissecting receptor-initiated signal transduction pathways. Our primary focus here is to discuss these new experimental approaches in the context of cytokine receptors, although these approaches are applicable to any receptor system. These approaches rely on the identification of receptor-induced early response genes. These genes, once identified, can be used as tools for the identification of upstream components of the specific receptor-linked signal transduction pathway. After a brief introduction to receptor-mediated signaling, our discussion of experimental approaches begins in the nucleus, describing techniques for identifying cytokine-regulated genes. Following identification of regulated genes, we discuss techniques for isolating the proteins that activate these genes. Finally, approaches to determining how these proteins are regulated by second messenger systems are described.

The biological consequences of implanting biomaterials can include a series of progressively more complex inflammatory reactions. Cytokines play a pivotal role in these reactions. Cytokines are divided into either proinflammatory or growth factors and their activity can affect the cell of their origin, have a localized effect, or induce a systemic response. The expression of interleukin-8, interleukin-1, interleukin-2, interleukin-6, and tumor necrosis factor can be affected by the presence of implanted materials, causing a change in the resulting inflammatory process and thus affecting the clinical performance of biomaterials or medical devices. In vitro and in vivo studies that show numerous widely used biomedical polymers eliciting cytokine production are reviewed.

In this review we present and discuss the structures of three cytokines, interleukin-1β, interleukin-4, and interleukin-8, in the solution and crystal states. The availability of these high-resolution structures provides a framework for understanding their mechanism of action, for relating structure to function, and for the rational design of potential agonists and antagonists

Cytokine interactions are central to the development of immune and inflammatory responses. While in vitro assays provide evidence about specific activities of individual cytokines, they are less informative concerning cytokine interactions that constitute the cytokine network. In this article we review an in vivo model of experimental inflammation, streptococcal cell wall (SCW)-induced arthritis in rats, which, when combined with in vitro analysis, provides a powerful approach to deciphering the complexity of the cytokine network. Using this model, it is possible to identify cytokines present in acute and chronic inflammatory lesions and their cells of origin, to administer these cytokines exogenously and monitor their impact, and to antagonize specific cytokine actions to establish a causal relationship. In this context, we have reviewed our experiences with one particular cytokine, transforming growth factor-β (TGF-β), which plays a key role in the regulation of immune and inflammatory events. Whereas systemically administered TGF-β attenuated SCW-induced joint inflammation, consistent with its known immunosuppressive activities, local (intra-articular) injection surprisingly exacerbated SCW-induced arthritis. Moreover, antagonism of TGF-β within the synovium with neutralizing antibodies impaired the development of inflammation as evaluated noninvasively by radiology and by the measurement of swelling, deformity, and erythema of individual joints. Additional parameters of analysis include histology to visualize tissue destruction, immunohistochemistry to detect the presence of specific cytokines or infiltrating immune cells, and Northern hybridization to measure the expression of cytokine mRNA. Thus, in vivo studies coupled with in vitro characterization provide a comprehensive biological and molecular profile of a particular cytokine and its place in the cytokine network. Moreover, this approach provides insight into potential mechanisms for therapeutic modulation of aberrant immune and inflammatory responses.

Infection with the human immunodeficiency virus (HIV) causes the acquired immunodeficiency syndrome (AIDS) over a medium period of 10 years. During this time cells that actively express HIV and cells that harbor the virus in a latent form have been demonstrated in different tissues and organs of infected individuals. It is critical to delineate the cellular and/or viral factors that regulate HIV expression and latency in order to fully understand the pathogenic mechanisms of HIV disease. Among several in vitro models that have been developed since HIV has been recognized as a human pathogen, the chronically infected promonocytic cell line U1 has become one of the more broadly used surrogate models for studies of immunologic and molecular regulation of HIV expression. In particular, more than 10 different cytokines have been shown to up- or down-regulate virus production in U1 cells either alone or in synergistic combination. Autocrine/paracrine induction of HIV expression was first demonstrated in this cell line, anticipating results in more complex models of virus infection. In addition to cytokines, hormones, vitamins, and physical and chemical agents, including UV light, heat, and reactive oxygen intermediates, have been shown to reactivate quiescent integrated proviruses. Finally, U1 cells have been used as standard controls for polymerase chain reaction studies, for pharmacologic screening of potentially therapeutic agents, and as a convenient multicytokine bioassay system for studies of immunologic control of HIV expression.