Methods for Measuring Antibody-Dependent Cell-Mediated Cytotoxicity In Vitro
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a relevant characteristic to measure for a number of therapeutic monoclonal antibodies (mAbs) under development. ADCC is a mechanism by which antibody-opsonized, infected, or cancerous cells are destroyed by FcγRIII (CD16)-expressing effector cells. Here we describe three methods that can be used to quantify the ADCC activity of mAbs by measuring distinct aspects of the ADCC mechanism. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Tests for Circulating Immune Complexes
Antigen–antibody complexes in tissues play a central role in the pathogenesis of lupus. Some of the immune complexes are formed in situ, i.e., in the tissues. Others are present in the blood stream, and these circulating immune complexes may deposit in tissues and incite inflammatory mechanisms in those tissues. A variety of techniques are available to measure circulating immune complexes. The assays that have been most studied in SLE include approaches that rely on the interaction of immune complexes with complement proteins, and therefore bind to C1q or contain bound C3. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Detection of Antinuclear Antibodies in SLE
The antinuclear antibodies (ANA) also known as antinuclear factors (ANF) are unwanted molecules which bind and destroy certain structures within the nucleus. In systemic lupus erythematosus (SLE), they are produced in excess; hence their detection in the blood of patients is important for diagnosis and monitoring of the disease. Several methods are available which can be used to detect ANA; nevertheless, indirect immunofluorescence antinuclear antibody test (IF-ANA) is considered a “reference method” for their detection. Though IF-ANA is relatively easier to perform, its interpretation requires considerable ski...
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Recombinant Antibody Microarray for Profiling the Serum Proteome of SLE
Systemic lupus erythematosus (SLE) is a severe autoimmune connective tissue disease. Our current knowledge about the serum proteome, or serum biomarker panels, reflecting disease and disease status is still very limited. Affinity proteomics, represented by recombinant antibody arrays, is a novel, multiplex technology for high-throughput protein expression profiling of crude serum proteomes in a highly specific, sensitive, and miniaturized manner. The antibodies are deposited one by one in an ordered pattern, an array, onto a solid support. Next, the sample is added, and any specifically bound proteins are detected and quan...
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

C1 Inhibitor: Quantification and Purification
C1 inhibitor is a multipotent serpin capable of inhibiting the classical and the lectin pathways of complement, the fibrinolytic system, and contact/kinin system of coagulation. Deficiency of C1 inhibitor manifest as hereditary angioedema (HAE), an autosomal dominant hereditary disease. Measuring the C1 inhibitor level is of vital importance for the diagnosis of HAE and also for monitoring patients receiving C1 inhibitor for therapy. Determination of the antigenic C1 inhibitor level by the radial immunodiffusion (RID) technique is described in detail in this chapter. The presented purification method of plasma C1 inhibitor...
Source: Springer protocols feed by Immunology - November 13, 2013 Category: Allergy & Immunology Source Type: news

Purification and Functional Characterization of Factor I
Factor I (FI) is a soluble, 88 kDa glycoprotein present in plasma at a concentration of approximately 35 mg/L. FI inhibits all complement pathways as it degrades activated C4b and C3b when these are bound to a cofactor such as C4b-binding protein or factor H. Here, we describe a method for purification of FI from human plasma, which is based on affinity chromatography followed by anion exchange chromatography. We also describe a functional assay, in which activity of FI can be assessed. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - November 13, 2013 Category: Allergy & Immunology Source Type: news

Purification and Functional Characterization of C4b-Binding Protein (C4BP)
C4b-binding protein (C4BP) is a soluble, 570 kDa large glycoprotein, present in plasma at a concentration of approximately 200 mg/L. C4BP is the main inhibitor of the classical and lectin pathways of complement, where it controls C4b-mediated reactions. Here, we describe a method for purification of C4BP from human plasma, which is based on barium chloride precipitation, anion exchange chromatography, and gel filtration. We also describe a functional assay, in which C4BP’s cofactor activity to factor I, in the degradation of C4b, can be assessed. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - November 13, 2013 Category: Allergy & Immunology Source Type: news

Purification and Functional Analysis of Human Properdin
Properdin is a member of the alternative pathway of complement. It is unique in that it is the only known positive regulator of the complement system. Properdin can stabilize and promote complement activation, but in addition is also capable of initiating the alternative pathway, making it a unique protein. This chapter focuses on the methods required for purifying human properdin and testing its functionality. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - November 13, 2013 Category: Allergy & Immunology Source Type: news

Purification, Measurement of Concentration, and Functional Complement Assay of Human Ficolins
Ficolins constitute a group of lectins involved in innate immunity. L-Ficolin, H-ficolin, and M-ficolin are present in human serum. The human ficolins differ in carbohydrate-binding specificity, but they have in common the ability to recognize the acetyl group. L-Ficolin and H-ficolin are associated with serine proteases termed MASPs (MBL-associated serine proteases) and their truncated proteins, and the complexes (L/H-ficolin–MASP) activate the lectin pathway of complement upon binding to their ligands. Recombinant M-ficolin is also able to form a complex with MASP, resulting in complement activation. L-Ficolin and ...
Source: Springer protocols feed by Immunology - November 13, 2013 Category: Allergy & Immunology Source Type: news

Assay for Estimation of the Functional Activity of the Mannan-Binding Lectin Pathway of the Complement System
Mannan-binding lectin (MBL) is a soluble pattern recognition molecule of the innate immune system. It is found in plasma in complex with MBL-associated serine proteases (MASPs). When MBL recognizes foreign, e.g., the surface of some microorganisms, or altered host surfaces the MASPs are activated and this will in turn lead to the initiation of the complement system, i.e., activation of complement by the MBL pathway. This will end up in increased phagocytosis of the microorganism and killing by insertion of pore structures in the membrane of the microorganisms. Lack of MBL seems significant in specific situations, e.g., in ...
Source: Springer protocols feed by Immunology - November 13, 2013 Category: Allergy & Immunology Source Type: news

Genotyping of FCN and MBL2 Polymorphisms Using Pyrosequencing
Pyrosequencing represents one of the most thorough methods used to analyze polymorphisms. One advantage of using pyrosequencing for genotyping is the ability to identify not only single-nucleotide polymorphisms (SNPs) but also tri-allelic variations, insertions and deletions (InDels). In contrast to most other genotyping assays the sequence surrounding the polymorphism provides an internal control making this method highly reliable. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - November 13, 2013 Category: Allergy & Immunology Source Type: news

Recombinant Genetic Libraries and Human Monoclonal Antibodies
In order to comprehensively manipulate the human proteome we require a vast repertoire of pharmacological reagents. To address these needs we have developed repertoires of synthetic antibodies by phage display, where diversified oligonucleotides are used to modify the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. As diversity is produced outside the confines of the mammalian immune system, synthetic antibody libraries allow us to bypass several limitations of hybridoma technology while improving the experimental parameters under which pharmacological reagents are produced. H...
Source: Springer protocols feed by Immunology - September 20, 2013 Category: Allergy & Immunology Source Type: news

Chimeric Antibodies
Here we describe a detailed protocol for the one-step preparation of antigen-specific human chimeric immunoglobulin G (IgG) monoclonal antibodies (mAbs) using an in vitro antibody design method referred to as the ADLib (Autonomously Diversifying Library) system. This method employs a chicken B cell line DT40-based library in which the variable regions of the Ig gene loci have been highly diversified by treatment with the histone deacetylase inhibitors. DT40 cells express both membrane-bound and secreted forms of chicken IgM. This property allows a rapid screening and selection of antibody-producing B cells from the library...
Source: Springer protocols feed by Immunology - September 20, 2013 Category: Allergy & Immunology Source Type: news

Humanization and Simultaneous Optimization of Monoclonal Antibody
Antibody humanization is an essential technology for reducing the potential risk of immunogenicity associated with animal-derived antibodies and has been applied to a majority of the therapeutic antibodies on the market. For developing an antibody molecule as a pharmaceutical at the current biotechnology level, however, other properties also have to be considered in parallel with humanization in antibody generation and optimization. This section describes the critical properties of therapeutic antibodies that should be sufficiently qualified, including immunogenicity, binding affinity, physiochemical stability, expression ...
Source: Springer protocols feed by Immunology - September 20, 2013 Category: Allergy & Immunology Source Type: news

Production of Human Monoclonal Antibodies by the Epstein–Barr Virus Method
Epstein–Barr virus (EBV) is a herpes virus which in vitro efficiently immortalizes nearly all human B lymphocytes. The lymphoblastoid diploid cell lines (LCL’s) thus generated preserve the characteristics of the cells initially infected by the virus: the cells produce and secrete immunoglobulins and also express these molecules on their surface. A selection of specific antibody-producing cells (i.e., antigen-committed cells) before EBV-infection or when LCL’s have already been established, enables isolation of monoclonal cell lines that secrete specific antibodies. If selection of antigen-committed cells ...
Source: Springer protocols feed by Immunology - September 20, 2013 Category: Allergy & Immunology Source Type: news