免疫学技术

One of the major bottlenecks in antibody discovery for research and therapeutic applications is the need for large quantities of protein in a short amount of time. Here we describe an alternative method using the Drosophila melanogaster S2 expression system to produce high levels of antibo ...

In the past two decades, the production levels for monoclonal antibodies in mammalian expression systems have improved dramatically. Single cell productivity for monoclonal antibodies has increased 20–50 fold due to the improvements in expression hosts, expression vectors, ce ...

New antibody derivatives are continuously being generated to interact with a range of therapeutic targets. The cost-effective and efficient production of these and other antibody derivatives is crucial for their further success. Here, we describe the construction of the expression ...

Escherichia coli is a host widely used in the industrial production of recombinant proteins. However, the expression of heterologous proteins in E. coli often encounters the formation of inclusion bodies, which are insoluble and nonfunctional protein aggregates. For the successful ...

Display technology has been developed and widely used in antibody screening and selecting. While phage can only display antibody fragments, mammalian cells can display not only fragments but full-length antibodies. Here we described the display of full length antibody on the surface of 2 ...

Ribosome display is a cell-free technology which enables in vitro selection and evolution of antibodies from very large diversified DNA libraries. It operates through the following key steps: (1) generation of PCR library; (2) formation of stable antibody-ribosome-mRNA (ARM) comple ...

The critical need for renewable, high-quality affinity reagents in biological research, as well as for diagnostic and therapeutic applications, has required the development of new platforms of discovery. Yeast display is one of the main methods of in vitro display technology with phage d ...

Guided selection provides a powerful tool for humanization of the preexisting nonhuman antibodies as exemplified by HUMIRA, the world’s first human antibody approved. This chapter describes the sequential guided selection procedure in which mouse VL and VH domains are replaced seq ...

Recombinant bispecific antibodies have many different applications; especially promising is their therapeutic potential due to their ability to retarget an effector molecule or a cell to a disease-related target structure. In the last years, many formats have been developed: two co ...

Humanized antibodies are constructed by CDR grafting, while retaining those murine framework residues that influence the antigen-binding activity. To reduce the immunogenicity of CDR-grafted humanized antibodies, the murine content in the CDR-grafted humanized antibodi ...

Traditional methods of phage display panning bind purified antigen to plates or other solid phases to which libraries are then applied. These methods are not directly applicable to antigens in their native environment on cell surfaces or in settings where the target antigen is unknown. We des ...

The drive to exploit novel targets and biological pathways has lead to the expansion of classical antibody research into innovative fragment adaptations and novel scaffolds. The hope being that alternative or cryptic epitopes may be targeted, tissue inaccessibility may be overcome, ...

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding an ...

ScFv fragments are popular recombinant antibody formats but often suffer from limited stability. Phage display is a powerful tool in antibody engineering and applicable also for stability selection. ScFv variants with improved stability can be selected from large randomly mutated ...

Libraries of antibody fragments displayed on filamentous phages have proved their value to generate human antibodies against virtually any target. We describe here a simple protocol to make large and diverse libraries based on a single or few frameworks. Diversity is introduced in the thi ...

Human antibodies are valuable tools for proteome research and diagnostics. Furthermore, antibodies are a rapidly growing class of therapeutic agents, mainly for inflammation and cancer therapy. The first therapeutic antibodies are of murine origin and were chimerized or humaniz ...

The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Although useful for the production of recombinant antibodies, this method is not totally adapted to the study of the antibody repertoire formed in vivo dur ...

Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-d ...

Human antibody single domains are a promising new class of antibody fragments. Here we describe methods for the cloning of human VH and VL genes into phage and phagemid vectors. Furthermore, we provide detailed protocols for the generation of single domain antibody libraries by Kunkel mutag ...

Antibody modeling is an interesting option to gain structure–function insights and to allow rational engineering of these molecules in the absence of experimental data. Among a diversity of algorithms, software packages, and specialized Web servers, the protocol described here pr ...