Construction of Single Chain Variable Fragment (ScFv) and BiscFv-Alkaline Phosphatase Fusion Protein for Detection ofBacillusAnthracis

The use of biotherapeutics for the treatment of diseases of the central nervous system (CNS) is typically impeded by insufficient transport across the blood–brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (ZSYM73) via binding to the transferrin receptor (TfR). ZSYM73 binds monomeric amyloid beta, a peptide involved in Alzheimer’s disease pathogenesis, with subnanomolar affinity. We generated a tri-specific fusion protein by genetically linking a single-chain variable fragment of the TfR-binding antibody 8D3 and an albumin-binding domain to the affibody molecule ZSYM73. Simultaneous tri-specific target engagement was confirmed in a biosensor experiment and the affinity for murine TfR was determined to 5 nM. Blockable binding to TfR on endothelial cells was demonstrated using flow cytometry and in a preclinical study we observed increased uptake of the tri-specific fusion protein into the cerebrospinal fluid 24 h after injection.

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Screening Antibody Libraries with Colony Assay Using scFv-Alkaline Phosphatase Fusion Proteins

Molecules ◽

10.3390/molecules25122905 ◽

2020 ◽

Vol 25 (12) ◽

pp. 2905

Author(s):

Yoshiro Hanyu ◽

Mieko Kato

Keyword(s):

Alkaline Phosphatase ◽

Fusion Proteins ◽

Direct Detection ◽

Screening Methods ◽

Single Chain Variable Fragment ◽

Antibody Library ◽

Single Chain ◽

Library Size ◽

Colony Assay ◽

Antibody Libraries

Screening antibody libraries is an important step in establishing recombinant monoclonal antibodies. The colony assay can identify positive clones without almost any false-positives; however, its antibody library is smaller than those used in other recombinant screening methods such as phage display. Thus, to improve the efficiency of colony assays, it is necessary to increase library size per screening. Here, we report developing a colony assay with single-chain variable fragment (scFv) fused to the N-terminus of bacterial alkaline phosphatase (scFv-PhoA). The scFv-PhoA library was constructed in an expression vector specifically designed for this study. Use of this library allowed the successful and direct detection of positive clones exhibiting PhoA activity, without the need for a secondary antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers a higher success rate than previous methods based on scFv libraries, and—most importantly—it enables high-throughput procedures.

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Selection of Single-Chain Variable Fragment Antibodies to Black Currant Reversion Associated Virus from a Synthetic Phage Display Library

Phytopathology ◽

10.1094/phyto.1998.88.3.230 ◽

1998 ◽

Vol 88 (3) ◽

pp. 230-233 ◽

Cited By ~ 12

Author(s):

Petri Susi ◽

Angelika Ziegler ◽

Lesley Torrance

Keyword(s):

Alkaline Phosphatase ◽

Phage Display ◽

Plant Extracts ◽

Fusion Proteins ◽

Phage Display Library ◽

Antibody Fragments ◽

Healthy Plant ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Black Currant

Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.

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