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疾病及处理

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Cloning of HIV Single-Strand DNA Binding Protein from Human Lymphocyte Lambda gt11 Expression Library with [32P]-Oligomers

Lambda gt11 cDNA libraries are routinely used for gene isolation with antibody and oligomer probes (1–3). Briefly, a recombinant DNA library from different types of cells is constructed in a λgt 11 expression vector. The foreign peptides (antigens) are expressed as a β-galactosidase fusion ...

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Telomeric Terminal Restriction Fragment (TRF)

Telomeres are protein-DNA structures at the ends of all eukaryotic chromosomes that are maintained by a unique ribonucleoprotein known as telomerase. This highly specialized RNA-dependent DNA polymerase provides a critical solution to the end-replication problem by adding TTA ...

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Quantitative Immunoblotting with Fusion Proteins

The development of vaccines against HIV-1 is currently hindered by incomplete understanding of correlates of protective immunity (1–3). Experiments are necessary to measure immune responses in sufficiently fine detail that specific protective responses can be discerned from t ...

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Serologic Analysis by Enzyme-Linked Immunosorbent Assay (ELISA)

One of the basic requirements for assessing the value of a vaccine candidate is measuring its immunogenicity. This can be done in a variety of ways, with focus on either the humoral or cellular arm of the response, or both. Assay of the humoral response, at minimum, requires the ability to measure changes in a ...

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Detection of HIV-1 Nucleic Acids by Southern Blotting

Replication of the human immunodeficiency virus type 1 (HIV-1) is associated with a high degree of viral sequence variation (1) that has been shown to correlate with disease state (2–8). The genetic diversity of the viral swarm within an HIV-1 infected individual is so extensive that this entity has ...

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Analysis of Antibody Interactions with HIV-1 Envelope Expressed on the Surface of Acutely Infected H9 Cells

Protective antibody responses against HIV-1 have yet to be identified or determined. HIV-1 envelope gpl20/gp41 is known to exist as a multimer (tetramers or trimers) on the surface of the virion (1–4). A number of immunoassays have been developed to evaluate HIV-1-specific binding antibody re ...

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Analysis of Antibody-Antigen Interactions Using Surface Plasmon Resonance

Accurate analyses of antibody specificities and affinities are critical to understanding their role in antibody-mediated neutralization of HIV-1 in vitro and their potential activity against HIV-1 in vivo. Multiple immunologic tools currently exist for measuring antibody- ...

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Measurement of HIV-1 Specific and Total Antibody Secreting Cells by ELISPOT

B-cells play an important role in protection against pathogens, and they secrete specific antibodies in serum and mucosal secretions upon antigenic stimulation contributing to immune exclusion and clearance of pathogens. The frequency of antibody forming cells (AFC) in specific o ...

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Linear Epitope Mapping by the PEPSCAN Method

The development of vaccine products for the prevention or treatment of HIV requires accurate assessments of the immune response, both humoral and cellular, so that specific responses can be correlated with efficacy. For assay of humoral immunity, a variety of techniques have been develop ...

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Neutralization of HIV-1 Infection of Human Peripheral Blood Mononuclear Cells (PBMC): Infectivity Reduction Method

The serum titration neutralization assay described in Chapter 33 utilizes a constant amount of infectious virus and indirectly estimates the antibody-mediated reduction in infectious virus by measuring p24 antigen expressed by human peripheral blood mononuclear cells (PBMC ...

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Neutralization of HIV-1 Infection of Human Peripheral Blood Mononuclear Cells (PBMC): Antibody Dilution Method

Antibodies elicited by passive or active immunization protect against numerous virus diseases. This in vivo protective immunity is often associated with in vitro detection of neutralizing antibodies (NAb) (e.g., polio, measles, influenza, respiratory syncytial virus, yellow f ...

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Collection and Processing of Mucosal Secretions from HIV-1 Infected Women

The major route of transmission of the human immunodeficiency virus type 1 (HIV-1) worldwide, like other agents of sexually transmitted disease, is via mucosal surfaces of the genital tract through sexual exposures (1–4). It has been hypothesized that immune responses at these sites may be im ...

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HIV-1-Specific Cytotoxic T-Cell Assays

Cytotoxic T-cells (CTL) provide the basis of protective immunity in many viral infections by appearing early in the immune response and becoming involved with the elimination of virus by lysis of infected cells. A considerable amount of evidence suggests that CTL may be an especially import ...

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Lymphocyte Proliferation Assay

The lymphocyte proliferation assay is used as an in vitro surrogate, similar to the in vivo delayed-type hypersensitivity assay, to assess the overall quality and character of the cellular arm of the immune response. The lymphocyte proliferation assay is used to assess congenital immune de ...

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HIV-1-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC)

Antibodies elicited during the course of HIV-1 infection can act as a bridge between cytolytic cells and HIV-1-infected cells or other cells that have passively absorbed appropriate HIV-1-antigens. These cytolytic cells cause lysis of the HIV-1-infected cells thereby decreasing vir ...

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Evaluation of Natural Killer Cell Activity

The immune response to a virus infection involves both nonspecific and specific immune mechanisms. Natural killer (NK) cells are naturally-occurring cytolytic cells capable of lysing various tumor cells and virus-infected cells without previous sensitization or with a require ...

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Generation and Expansion of HIV-1 CD4+ Antigen-Specific T Cell Lines

T-helper (Th) lymphocytes play a pivotal role in the immune response to viral pathogens. Understanding of the mechanisms governing the Th response to human immunodeficiency virus type 1 (HIV-1) is key to unveiling the pathogenesis of HIV-1 disease given the primary role the CD4+ T-cell plays as bo ...

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Detection of Apoptosis in HIV-Infected Ceil Populations using TUNEL

Cells within an organism undergo two common forms of cell death. Sudden injury resulting from physical or chemical insult leads to a form of cell death called necrosis. A more subtle programmed form of cell death is termed apoptosis. Apoptosis describes a genetically encoded pathway that plays ...

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Immunophenotyping and Assessment of Cell Function by Three-Color Flow Cytometry of Peripheral Blood Lymphocytes

Infection by the human immunodeficiency virus type 1 (HIV-1) is known to cause a number of changes in the immunophenotypic profile of patients even in the early asymptomatic stages of disease. Such “surrogate markers” are known to correlate with the stage of HIV disease and often are predictive of o ...

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The In Situ Detection of PCR-Amplified Hepatitis C RNA

The discovery of PCR in 1985 has had an enormous impact on the field of molecular diagnostics. However, an important drawback of solution-phase PCR is that one cannot localize the target of interest to a specific cell type owing to the obligatory tissue destruction required for DNA extraction. This ...

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