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Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells
Published on: August 5, 2016
Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis
Andrew Hayhurst1, Scott Happe, Robert Mabry
1Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712-1095, USA.
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 antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.
Insights
Researchers developed improved phage display antibodies for detecting Brucella melitensis, a dangerous pathogen. These novel antibodies offer enhanced specificity and signal amplification for diagnostic immunoassays.
Area of Science:
- Immunology
- Microbiology
- Biotechnology
Background:
- Brucella melitensis is a significant zoonotic pathogen and a potential biowarfare agent.
- Current diagnostic methods require improvement in sensitivity and specificity.
Purpose of the Study:
- To develop novel, highly specific diagnostic reagents for Brucella melitensis using phage display technology.
- To overcome challenges in expressing soluble and stable recombinant antibodies.
Main Methods:
- Construction of a phage display library using immunoglobulin genes from immunized mice.
- Screening of the library against gamma-irradiated Brucella melitensis.
- Engineering of single chain antibody variable region fragments (scFv) into single chain antibody fragments (scAb) with a human kappa light chain constant domain (Ck).
- Co-expression of the periplasmic chaperone Skp to improve antibody solubility and stability.
Main Results:
- Selected phage particles showed strong ELISA signals against Brucella melitensis.
- Fusion to Ck and co-expression of Skp enabled production of soluble, functional antibodies.
- Phage-displayed antibodies (scFv and scAb) demonstrated superior ELISA performance due to signal amplification.
- Isolated antibodies were highly specific to Brucella melitensis, distinguishing it from Yersinia pseudotuberculosis.
Conclusions:
- Engineered single chain antibody fragments (scAb) with a human kappa light chain constant domain (Ck) and periplasmic chaperone Skp improve antibody solubility and function.
- Phage display remains a superior platform for developing highly sensitive and specific diagnostic reagents for Brucella melitensis detection.
- The developed antibodies offer a promising tool for improved diagnostics of Brucella infections.
