Applied Biochemistry and Biotechnology

, Volume 160, Issue 6, pp 1808–1821

Single-Chain Fv Antibody Fragments Retain Binding Properties of the Monoclonal Antibody Raised Against Peptide P1 of the Human Prion Protein

  • Nives Škrlj
  • Vladka Čurin Šerbec
  • Marko Dolinar
Article

Abstract

Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrPSc-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics.

Keywords

Prion protein Recombinant antibody scFv Escherichia coli 

Abbreviations

ABTS

2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)

BSA

Bovine serum albumin

CNS

Central nervous system

DEPC

Diethyl pyrocarbonate

ELISA

Enzyme-linked immunosorbent assay

Fv

Antibody variable domains (Vl+Vh)

HLL

scFv in Vh-linker-Vl chain arrangement

HRP

Horseradish peroxidase

IMAC

Immobilized metal ion affinity chromatography

IPTG

Isopropyl β-d-1-thiogalactopyranoside

LLH

scFv in Vl-linker-Vh chain arrangement

mAb

Monoclonal antibody

PCR

Polymerase chain reaction

PrP

Prion protein

PrPC

Cellular form of the PrP

PrPSc

Pathogenic form of the PrP

scFv

Single-chain antibody variable domains

TBS

Tris-buffered saline

TMB

3,3′,5,5′-tetramethylbenzidine

Vh

Variable domain of the heavy chain of an antibody

Vl

Variable domain of the light chain of an antibody

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Copyright information

© Humana Press 2009

Authors and Affiliations

  • Nives Škrlj
    • 1
  • Vladka Čurin Šerbec
    • 1
    • 2
  • Marko Dolinar
    • 1
  1. 1.Chair of Biochemistry, Faculty of Chemistry and Chemical TechnologyUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Department for Production of Diagnostic Reagents and ResearchBlood Transfusion Centre of SloveniaLjubljanaSlovenia

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