Springer protocols feed by Immunology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.HLA Typing by Sequence-Specific Primers
HLA typing by sequence-specific primers (PCR-SSP) is a commonly used technique in HLA typing in which multiple pairs of cis-located allele-specific primers are used to determine the alleles present in a given DNA sample. Although the technique is around two decades old, it still offers a relatively straight forward way of typing and has the benefit of using commonly available laboratory equipment. Here we describe the background of the PCR-SSP, how to design and validate reactions, and common problems that arise. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
RNA Purification and Expression Analysis Using Microarrays and RNA Deep Sequencing
We present in this chapter a description of methods used to isolate mRNA from cells and tissues that has been optimized for preservation of RNA quality using clinical materials and implemented successfully in several large, multicenter studies by the authors. In addition, two methods, gene expression microarrays and RNAseq, are described for mRNA profiling of cells and tissues from clinical or laboratory sources. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Discovery and Customized Validation of Antibody Targets by Protein Arrays and Indirect ELISA
Because of our access to human genome data and ever improving genome sequencing and proteome analysis methods we are much better in terms of our understanding of biological processes. In addition to genomics, proteomics, and other “omics” methods, availability of more sophisticated molecular assaying methods have augmented our knowledge about immune processes towards autogeneic and allogeneic targets. High-density protein arrays are developed to analyze protein–small molecule interactions, enzyme–substrate profiling, protein–protein interaction, and immune monitoring by assessing antibodies in...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Tolerogenic Dendritic Cells and Induction of T Suppressor Cells in Transplant Recipients
Tolerogenic antigen presenting cells (APC), primarily dendritic cells (DC), are essential to the induction and maintenance of immunologic tolerance in clinical transplantation. They induce the differentiation of CD8+ T suppressor (Ts) and CD4+ T regulatory (Treg) or anergic cells, which prevent transplant rejection maintaining a state of quiescence. Tolerogenic APC express high levels of inhibitory receptors such as Immunoglobulin-like transcript (ILT)3 and 4 which inhibit the effector function of T cells that recognize HLA-peptide complexes on APC. Here, we describe the methods for detection of tolerogenic APC induced by ...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Detection of Intracellular Cytokines
The intracellular cytokine method allows for a multiparametric readout by flow cytometry with precise phenotyping of the responding T cells. The intracellular cytokine staining of cells that have been fixed and permeabilized following a short-term activation and staining with antibodies to cell surface markers allows for identification of the cellular origin of the cytokine accumulated product. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Cylex ImmuKnow Cell Function Assay
The use of immunosuppression in solid organ transplantation is associated with increased morbidity and mortality. Monitoring trough immunosuppression levels alone in transplant recipients is insufficient to gauge the suppression of immune responses. The ImmuKnow Immune Cell Function Assay (Cylex, Inc., Columbia, MD, USA) has been developed to measure the activity of CD4+ T cells as a marker of global immune-competence. The changes in T cell activation were correlated with the relative risk of rejection and infection. However, the most significant utility of the ImmuKnow test, in combination with other clinical parameters, ...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Detection of Antibodies to Self-Antigens (K-alpha 1 Tubulin, Collagen I, II, IV, and V, Myosin, and Vimentin) by Enzyme-Linked Immunosorbent Assay (ELISA)
The enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting antibodies (Abs) and is employed in clinical laboratories to identify Abs against various self-antigens–autoAb development and quantitation. This method relies on specific antigen–Ab interactions where one of the components is immobilized on a solid surface. Using this method, the concentrations of antigens or Ab present in the serum can be quantified with high specificity and accuracy. Here, we describe the detection of autoAbs to various self-antigens with different tissue restriction patterns which includes collagens, k-&a...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
The Detection of Antibodies to the Angiotensin II-Type 1 Receptor in Transplantation
The detection of auto-antibodies detection is a major aspect of patient’s immunomonitoring. Antibodies directed against the heart and kidney Angiotensin II type 1 receptor (AT1R) play a major role in antibody mediated rejection after heart and kidney transplantation and in obliterative vasculopathy of autoimmune diseases. Here, a sandwich enzyme-linked immunosorbent assay (ELISA) is the reliable tool for the detection of auto-antibodies targeting AT1R. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
A Flow Cytometric Crossmatch Test Using Endothelial Precursor Cells Isolated from Peripheral Blood
Flow cytometric crossmatch tests provide a donor-specific, cell based method for the detection of alloreactive antibodies in the sera of transplant patients. Conventional crossmatch tests used in solid organ transplantation utilize lymphocytes as target cells to detect the presence of alloreactive HLA antibodies. Isolation of endothelial precursor cells (EPCs) from peripheral blood now allows testing for antibodies reactive with non-HLA endothelial cell antigens. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Tetramer Staining for the Detection of HLA-Specific B cells
HLA-specific B cells can be identified, quantified, and isolated after staining with HLA tetramers. Quantification of these B cells can in turn identify individuals who are sensitized to HLA antigens and the isolation of these cells facilitates a variety of experimental investigations. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
C1q Assay for the Detection of Complement Fixing Antibody to HLA Antigens
Solid phase Luminex® and flow cytometric single antigen bead assays offer exquisite sensitivity and specificity for HLA antibody detection. Unlike the historical complement-dependent cytotoxicity (CDC) method, these assays do not distinguish complement fixing from non-complement fixing antibody, the former of which are considered the most clinically relevant in the peri-transplant period. This chapter describes a novel solid phase C1q binding assay to distinguish HLA antibodies that can bind the first component of complement (C1q). These antibodies have the capacity to initiate the complement cascade irrespective of wh...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Solid Phase Assay Measuring C4d Deposition to Determine Complement Fixation by HLA-Specific Antibodies
The definition of HLA-specific antibodies in solid organ transplant patients is a necessary tool for recipient selection prior to transplantation and monitoring for rejection post transplant. Solid phase assays can detect both complement fixing and non-complement fixing HLA-specific antibodies. Here we describe a method for determining the presence of complement fixing HLA-specific antibodies using a sensitive solid phase assay. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Lymphocyte Crossmatching by Flow Cytometry
Flow cytometry is an invaluable tool for studying lymphocyte biology. In transplantation, flow cytometry represents the most sensitive method for demonstrating the binding of HLA antibodies to cellular targets. Referred to as the flow cytometric crossmatch (FCXM), the implementation of this method has revolutionized HLA antibody assessments and facilitated increased allograft survival among highly sensitized recipients. Here we describe the methods for performing the FCXM and discuss its critical aspects, as well as the presentation of data and interpretation of results. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Complement-Dependent Cytotoxicity Crossmatch
The complement-dependent cytotoxic crossmatch is an informative test that detects alloantibodies in pre- and post-transplant patients, which may dictate clinical management of transplant patients. While challenging to perform, the cytotoxic crossmatch represents the only assay that provides direct evidence for the presence of potentially pathologic (i.e., cytotoxic) alloantibodies. The cytotoxic crossmatch combines patient (recipient) serum and donor cells. If donor-reactive alloantibodies are present in patient serum, these antibodies can bind donor cells. Antibody-antigen complexes, in turn, can activate the complement c...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Gene-Specific PCR Typing of Killer Cell Immunoglobulin-Like Receptors
By interacting with specific HLA class I molecules, the killer cell immunoglobulin-like receptors (KIR) regulate the effector function of natural killer (NK) cells and subsets of CD8 T cells. The KIR receptors and HLA class I ligands are encoded by unlinked polymorphic gene families located on different human chromosomes, 19 and 6, respectively. The number and type of KIR genes are substantially variable between individuals, which may contribute to human diversity in responding to infection, malignancy and allogeneic transplants. PCR typing using sequence-specific primers (PCR-SSP) is the most commonly used method to deter...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
