PMID: 12738372May 10, 2003

Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis

Journal of Immunological Methods
Andrew HayhurstGeorge Georgiou

Abstract

Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage ant...Continue Reading

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Related Concepts

Metazoa
Antibodies, Bacterial
Antibody Specificity
Bacterial Warfare
Liquid Chromatography
Genes, Immunoglobulin
Immunoglobulin Constant Regions
Immunoglobulin Fv Fragments
Mice, Inbred BALB C
Hybrid Proteins, Recombinant

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