免疫学技术

Phage display is a powerful tool to select antibodies for conformation-specific epitopes from antibody libraries. Based on the M13 pIII phage display technology we describe a cell suspension-based strategy, which allows panning against complex, multimeric, fully functional cell ...

In vitro selections performed by phage display are often performed against a recombinant antigen adsorbed on a solid surface. Passive adsorption of the antigen involves its partial denaturation, which can lead to binders able to strongly recognize the adsorbed antigen but displaying a p ...

A major ‘post-genome’ objective is to fully characterise the human proteome using a comprehensive collection of specific antibodies directed against all human proteins and their variants. To achieve this, substantial reductions in cost and effort per antibody have to be achieved. A con ...

Rabbits provide an interesting alternative to antibodies derived from the murine system. Furthermore, rabbits show an exceptional feature with respect to the mechanisms that are used for the generation of antibody diversity, i.e. a single VH gene that is preferentially used, and the diver ...

Libraries constructed from immunised non human primates (NHP; macaques here) are an appealing alternative to human libraries when immunization of Humans is difficult, as is so often the case. These phage-displayed libraries allow to obtain antibody fragments of very high affinities ( ...

Antibody fragments have gained strong attention in the last years and presumably represent one of the most promising molecules to construct new types of therapeutic proteins. Contrary to complete IgG, antibody fragments like scFv are not very stable and are frequently expressed at a very low ...

The quality of antibodies selected from antibody gene libraries, in particular their affinity and the number of different clones per antigen, is a direct function of library diversity. Here, the optimization of every single step during library construction is of utmost importance for the q ...

Antibody phage display libraries have frequently been used for the isolation of antibody fragments against potential target structures on the cell surface of different types of cancer. Independent from the source of V-regions and the antibody format, two general strategies have been f ...

Although nowadays many different libraries for the selection of antibodies are available, hybridoma cells and immunized animals are still an important source for the production and selection of specific binders. Therefore, this protocol describes the isolation of the coding mRNA, P ...

Hybridoma cDNA can be difficult to clone using V region PCR. This is due to mutations in primer regions as well as the presence of other expressed V genes in the hybridoma. In this chapter, the use of RACE to clone V genes is described. This avoids some of the problems associated with the use of V gene PCR.

Some hybridoma cell lines are very low producers, difficult to cultivate, or antibody production is lost upon prolonged culture. In these cases, a recombinant "hybridoma immortalization" can rescue a valuable antibody for further unlimited propagation in E. coli or other recombinant p ...

Activation, proliferation and differentiation of na�ve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC–peptide com ...

RNA interference is a phenomenon in which specific, endogenous genes are silenced by mRNA degradation. This technology is highly regarded as a potential therapeutic due to its high efficacy and low toxicity. However, the difficulty of delivering RNAi to target cells has impeded the develop ...

Flow cytometry is combined with highly specific fluorophore-conjugated antibodies that will only bind to the activated forms of molecules. The advances in flow cytometry enable to perform quantitative multiplexed analysis of single cells within heterogeneous populations st ...

Peripheral blood T lymphocytes are a pool of cells with extremely different characteristics and, therefore, it may be difficult to obtain clear-cut results and to attribute a certain function to a defined T cell population in several experimental settings. The availability of a population ...

Since its development, flow cytometry gave a relevant contribution to the field of Immunology. Its unique potential to analyse multiple parameters at the single cell level allowed the identification of unknown cell subsets with specific roles in immunoregulation as well as in the pathog ...

The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA–peptide restrictions that have been previously ...

Limiting dilution analysis (LDA) has been extensively employed as a quantitative method to estimate the precursor frequency of various T lymphocyte subsets according to their functional properties in vitro. We describe here an example of LDA experiment assessing antigen-specif ...

Subsets of T cells can be distinguished on basis of their cytokine production and secretion profile. With the critical role of cytokines in the regulation of immune and inflammatory responses, cytokines hold the promise to become the ideal biomarkers to monitor development and progressi ...

RNA interference (RNAi) holds considerable promise as a novel therapeutic strategy to silence disease-causing genes not amenable to conventional therapeutics. Since it relies on small interfering RNAs (siRNAs), which are the mediators of RNAi-induced specific mRNA degradatio ...