Quantification of Multiple Cytokines and Chemokines Using Cytometric Bead Arrays
Quantitative suspension array technology allows the simultaneous measurement of different cytokines and chemokines in small sample volumes. The possibility of measuring multiple variables is important for discovery of biomarkers of pathogenesis or protection in complex diseases as well as measurement of antigen-specific cellular responses. Measurements can be made in biological specimens, such as plasma or serum, cell culture supernatants, and others. This technology is based on a capture-detection sandwich-type assay using fluorescent microspheres analyzable by Luminex instruments or flow cytometers. The complexity and co...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news

A Novel Three-Dimensional Biosensor Based on Aluminum Oxide: Application for Early-Stage Detection of Human Interleukin-1
Immunosensors based on electrolyte-oxide-semiconductors (EOS) have been extensively researched over the last few decades. By electrochemical impedance spectroscopy (EIS) the specific molecular biorecognition of the antibody–antigen (Ab–Ag) can be detected providing an alternative quantitative system to immunoassay techniques. The electrochemical variations from a fabricated immunosensor can provide quantitative values for the analyte of interest at reduced costs and analysis time. In this context, a novel EOS substrate based on aluminum oxide (Al2O3) grown by atomic layer deposition on silicon was applied. The ...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news

Enhanced ELISA Based on Carboxymethylated Dextran Coatings
In a “sandwich” enzyme-linked immunosorbent assay (ELISA) designed to detect an antigen in a complex protein mixture, the antigen is usually captured via an antibody adsorbed to the wells of a microplate. Plate preparation for standard assay involves a passive adsorption of capture antibodies followed by the incubation of blocking agents. Here, we describe a new strategy that replaces these two time-consuming adsorption steps (up to 15 h) by a unique step corresponding to the covalent grafting of the capture antibody on a carboxymethylated dextran (CMD) layer, a single step completed in 15 min. Taking advantage...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news

ELISA-Based Assay for IP-1 Detection from Filter Paper Samples
IP-10 is a small pro-inflammatory chemokine secreted primarily from monocytes and fibroblasts. Alterations in IP-10 levels have been associated with inflammatory conditions including viral and bacterial infections, immune dysfunction, and tumor development. IP-10 is increasingly recognized as a biomarker that predicts severity of various diseases and can be used in the immunodiagnostics of Mycobacterium tuberculosis and cytomegalovirus infection. Here, we describe an ELISA-based method to detect IP-10 from dried blood and plasma spot samples. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news

Analysis of the Released Nuclear Cytokine HMGB1 in Human Serum
A ubiquitous nuclear protein, the high-mobility group box 1 (HMGB1), is secreted by activated macrophages/monocytes and leaked passively from injured cells. HMGB1 functions as a mediator of infection- and injury-elicited inflammatory diseases. Here, we describe a semiquantitative immuno-blotting method to measure the released HMGB1 in human serum, in comparison with a commercially available HMGB1 ELISA technique. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news

The Challenge of Measuring Elusive Immune Markers by Enzyme-Linked Immuno-Spot (ELISPOT) Technique
The enzyme-linked immuno-spot (ELISPOT) technique is a sensitive method used for measurement of elusive immune markers in limited-volume samples. By virtue of the exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses), which was not possible before, is now relatively easy. However, development of a method sensitive enough to pinpoint elusive immune markers at the single-cell level is a challenge since there are a number of demands that have to be fulfilled and traps to avoid, achieving a valuable outcome. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news

DNA Vaccination as a Treatment for Chronic Kidney Disease
Chronic kidney disease is one of the major health problems worldwide. DNA vaccination delivers plasmid DNA encoding the target gene to induce both humoral and cellular immune responses. Here, we describe the methods of CD40 DNA vaccines enhanced by dendritic cell (DC) targeting on the development of Heymann nephritis (HN), a rat model of human membranous nephropathy. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - April 10, 2014 Category: Allergy & Immunology Source Type: news

Tetravalent DNA vaccines Product as a vaccines Candidate Against Dengue
Dengue is the most important arbovirus worldwide and is the virus that causes dengue fever and the more severe dengue hemorrhagic fever. There are four serotypes of dengue with each possessing the ability to cause disease. Developing a preventive vaccines is the most efficient and effective way to prevent these diseases, and because immunity to one serotype does not protect against the other serotypes, a vaccines must provide tetravalent protection. We used DNA immunization as a platform to develop a tetravalent vaccines. In this chapter, we describe the laboratory, regulatory, and clinical methodology for evaluating a can...
Source: Springer protocols feed by Immunology - April 10, 2014 Category: Allergy & Immunology Source Type: news

Immunotherapy for Alzheimer’s Disease: DNA- and Protein-Based Epitope vacciness
Active immunotherapy for Alzheimer’s disease (AD) is aimed to induce antibodies specific to amyloid-beta (Aβ) that are capable to reduce the level of Aβ in the CNS of Alzheimer’s disease patients. First clinical trial AN-1792 that was based on vaccination with full-length Aβ42 showed that safe and effective AD vaccines should induce high titers of anti-Aβ antibodies without activation of harmful autoreactive T cells. Replacement of self-T cell epitope with foreign epitope, keeping self-B cell epitope intact, may allow to induce high titers of anti-Aβ antibodies while avoiding the activa...
Source: Springer protocols feed by Immunology - April 10, 2014 Category: Allergy & Immunology Source Type: news

Protective and Therapeutic DNA Vaccination Against Allergic Diseases
DNA vacciness represent a novel approach for protective and therapeutic intervention against type I allergies. In contrast to classical subcutaneous immunotherapy, which relies on the injection of alum-adsorbed protein extracts, DNA vacciness do not suffer from side effects such as anaphylaxis or therapy-induced IgE antibodies. In animal models, DNA vacciness have been demonstrated to prevent TH2 sensitization or balance an existing TH2-mediated allergic immune response by induction of TH1 or regulatory T cells, rendering them promising candidates for prophylactic vaccination as well as therapy. In this chapter, we discuss...
Source: Springer protocols feed by Immunology - April 10, 2014 Category: Allergy & Immunology Source Type: news

Pharmaceutical Grade Large-Scale Plasmid DNA Manufacturing Process
For pharmaceutical applications of plasmid DNA, either direct or indirect, certain quality standards are required. Whereas for direct gene transfer into human “Good Manufacturing Practice” (GMP) grade is mandatory, for GMP production of, e.g., viral vectors (AAV, etc.) the plasmid DNA used needs not necessarily be produced under GMP. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - April 10, 2014 Category: Allergy & Immunology Source Type: news

Plasmid Fermentation Process for DNA Immunization Applications
Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - April 10, 2014 Category: Allergy & Immunology Source Type: news

Intradermal Vaccination by DNA Tattooing
DNA vaccination is an attractive vaccination method. First, the production of plasmid DNA as a vaccines is considerably more cheap and simple than the production of recombinant protein. Second, the expression cassette of DNA vacciness can readily be modified, making DNA vacciness highly flexible. Finally, in animal models, DNA vaccination is able to induce potent cellular immune responses. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - April 10, 2014 Category: Allergy & Immunology Source Type: news

DNA Vaccination in Skin Enhanced by Electroporation
DNA vacciness are a next generation branch of vacciness which offer major benefits over their conventional counterparts. However, to be effective in large mammals and humans, an enhancing delivery technology is required. Electroporation is a physical technique which results in improved delivery of large molecules through the cell membrane. In the case of plasmid DNA, electroporation enhances both the uptake and expression of the delivered DNA. The skin is an attractive tissue for DNA vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring, and most importantly the immunocompetent na...
Source: Springer protocols feed by Immunology - April 10, 2014 Category: Allergy & Immunology Source Type: news

Electroporation-Based DNA Delivery Technology: Methods for Gene Electrotransfer to Skin
DNA delivery to for example skin and muscle can easily be performed with electroporation. The method is efficient, feasible, and inexpensive and the future possibilities are numerous. Here we present our protocol for gene transfection to mouse skin using naked plasmid DNA and electric pulses. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - April 10, 2014 Category: Allergy & Immunology Source Type: news