丁香实验_LOGO
登录
提问
我要登录
|免费注册
点赞
收藏
wx-share
分享

Producing Bispecific and Bifunctional Antibodies

互联网

1019
Bispecific antibodies are artificially engineered monoclonal antibodies (MAbs) that consist of two distinct binding sites and are capable of binding two different antigens noncovalently. They can be produced by chemical cross-linkage, genetic engineering, or somatic hybridization. This chapter describes a rapid method using somatic fusion to generate hybrid hybridomas (quadromas). Two fluorescence-labeled hybridoma cell lines were fused with polyethylene gly col (PEG) to generate the quadroma. Generation of a quadroma secreting bsMAb against biotin and HRPO is described, along with a benzhydroxamic acid-agarose affinity chromatography procedure to purify the bsMAb-HRPO complex. This bsMAb can be used for ultrasensitive ELISA detection of biotinylated antigens. Essentially a similar method can be used for fusing any two hybridomas for therapeutic applications. Bifunctional antibodies are colinear molecules with one or more paratopes linked with diagnostic or therapeutic molecules. There are some limitations of therapeutic monoclonal antibodies in the clinic that can be overcome by engineering smaller and more effective antibody fragments. Here we describe a stepwise procedure for developing a bifunctional ScFv (bfScFv). We constructed a bfScFv from a hybridoma cell line using PCR strategies. The VL and VH gene segments are linked with a 45-bp linker and fused with a biotin mimic sequence at the 3′ end. This engineered bifunctional antibody fragment gene could be expressed and the protein purified on a large scale in Escherichia coli as inclusion bodies. Such bifunctional antibody molecules could have useful applications in the area of immunodiagnostics and immunotherapy. Similar strategies can be used to incorporate a second single-chain antibody or any nonantibody entity such as a cytokine for therapeutic applications.
ad image
提问
扫一扫
丁香实验小程序二维码
实验小助手
丁香实验公众号二维码
扫码领资料
反馈
TOP
打开小程序