免疫学技术

Standardized sequence and structure analysis of antibody using IMGT�, the international ImMunoGeneTics information system� (http://www.imgt.org), allows to bridge, for the first time, the gap between antibody sequences and three-dimensional (3D) structures. This is achieved ...

The VBASE2 database integrates sequences present in the various DNA sequence databases and selects germline-encoded variable gene segments from these databases. The sequences are grouped into three classes: the first class consisting of the variable gene segments that are proven g ...

Flow cytometry allows the multiparametric physical and biochemical analysis of single cells within large cell suspensions which is most frequently for cell counting and for the measurement of antigen expression with fluorescence conjugated antibodies. Moreover, flow cytome ...

Monoclonal antibodies (mAbs) can exert their biological functions by a large variety of mechanisms and have therefore become a major part in the treatment of diseases like viral infections and cancer. For the clinical efficacy of many mAbs Fc-mediated effector functions like antibody-d ...

The quantification of human immunoglobulin G (IgG) and recombinant derivatives is required for many applications in research and diagnostics. Particularly in research, robust and simple protocols are necessary to determine the concentration of recombinant human IgGs after pr ...

Functional characterization of antibodies that inhibit soluble cytokines or chemokines requires robust, sensitive in vitro and in vivo bioassays. Testing an antibody in vitro requires consideration of antigen source, integrity, and concentration, as well as the magnitude of the b ...

Neutralization tests are performed in vitro, at the cellular level - including on cells in the form of organs - and at the sub-cellular level. They assess the efficacy of antibody fragments, or antibodies, to inhibit critical stages of activities. These activities are generally deleterious to H ...

The use of radioiodinated proteins is one of the most sensitive methods to demonstrate and quantify specific protein/protein interactions. The direct introduction of Iodine-125 into proteins is easily and quickly done, provided the mandatory safety equipment is available. Most an ...

To determine the affinity, the KD,-value, between interaction molecules a number of techniques using labels are available, such as chromatography, isothermal titration calorimetry, radioimmuno- assay and the widely used enzyme-linked immunosorbent assay (ELISA). However, ne ...

The hexahistidine tag is one of most commonly used fusion tags in affinity purification of recombinantly expressed proteins. Real-time binding analysis using Biacore technology allows in-depth characterization of respective association and dissociation patterns of pote ...

Antibodies and their fragments are widely used tools for research, diagnostics and therapy. The strength of the interaction between an antibody molecule and the recognized antigen is conveniently characterized by the affinity constant. Among the available approaches to determi ...

Glycosylation is one of the main IgG post-translational modifications and has essential roles in antibody effectors functions, immunogenicity and plasmatic clearance. In this chapter we discuss and provide detailed protocols for IgG homogeneity and determination of level of gl ...

Among all analytical methods used to characterize monoclonal antibodies (mAbs), mass spectrometry plays an increasingly important role for both global and fine structural characterization of therapeutic candidates. In this chapter we discuss and provide detailed protocols ...

Size exclusion chromatography is a standard chromatographic technique that allows the separation of molecules or molecule complexes by their hydrodynamic volume (grossly equivalent to the molecular mass). In antibody engineering, it is used to analyze and to separate antibodies ...

Many approaches for antibody epitope mapping utilize peptides or peptide fragments and are limited to antibodies binding linear epitopes or epitopes not requiring fully folded conformationally correct antigens. The protocols in this chapter show how yeast displayed antigens a ...

Affordable high-density peptide arrays are needed to routinely define the exact binding sites of antibodies. In terms of prize and density peptide arrays currently lag far behind oligonucleotide arrays that are available in densities exceeding 50.000 oligonucleotides per cm2. This ...

Antibodies recognize and bind their target protein antigens via surface accessible interaction sites, the epitopes (Fig. 35.1), which can involve amino acid side chain and backbone contacts along a linear segment of the antigen amino acid chain (linear epitopes) or which can involve amino a ...

The following chapter describes the purification of whole IgG by Protein A affinity chromatography from crude protein mixtures such as serum, ascites fluids or cell culture supernatant. This reliable method is based on the ability of Protein A to bind with high affinity to the Fc region of immuno ...

Production of IgG antibodies is usually done in mamalian cells. Predominantly, chinese hamster ovary (CHO) cells are used for generating stably transfected cell lines and for manufacturing. In addition to large scale manufacturing, stably transfected CHO cells are also well suited for ...

Antibodies have come to the forefront of protein therapeutics in the past decade and while they are now widely accepted and administered to patients, the cost of using antibody therapy remains extremely high. Here we describe a protocol for the expression of human antibodies using the chlorop ...