抗体抗原实验

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 ...

RNA interference (RNAi) is a process through which double-stranded RNA induces the activation of endogenous cellular pathways of RNA degradation, resulting in selective and potent silencing of genes that have homology to the double strand. Much of the excitement surrounding small int ...

This chapter describes a method by which activating receptor-mediated calcium signaling can be measured in individual murine NK cells using a flow cytometer fitted with a UV laser. One major advantage of this method is that the calcium response of the minority NK cell population and even smaller ...

Cytokines stimulate biological responses by activating intracellular signaling pathways. We have been adapting flow cytometric techniques to measure the levels of expression and activation of signaling molecules within mixed populations containing NK cells and to charac ...