Phage display mediated immuno-PCR

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10 000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications.

[1]  G. P. Smith,et al.  Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. , 1985, Science.

[2]  R. Joerger,et al.  High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction. , 1995, Nucleic acids research.

[3]  A. Osterhaus,et al.  Evaluation of two commercially available immunoassays for the detection of hantavirus antibodies in serum samples. , 2000, Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology.

[4]  I. Pastan,et al.  Improving antibody affinity by mimicking somatic hypermutation in vitro , 1999, Nature Biotechnology.

[5]  H. Hoogenboom,et al.  Selecting and screening recombinant antibody libraries , 2005, Nature Biotechnology.

[6]  A. Schots,et al.  Fluobodies: green fluorescent single-chain Fv fusion proteins. , 1999, Journal of immunological methods.

[7]  P. Hudson,et al.  Engineered antibody fragments and the rise of single domains , 2005, Nature Biotechnology.

[8]  In-situ immuno-PCR to detect antigens , 2000, The Lancet.

[9]  A. Kaufmann,et al.  Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay. , 1997, Analytical biochemistry.

[10]  T. Wisniewski,et al.  An Aggregation-Specific Enzyme-Linked Immunosorbent Assay: Detection of Conformational Differences between Recombinant PrP Protein Dimers and PrPSc Aggregates , 2005, Journal of Virology.

[11]  Christof M Niemeyer,et al.  A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins. , 2003, Biochemical and biophysical research communications.

[12]  E. Barklis,et al.  Hantavirus Nucleocapsid Protein Oligomerization , 2001, Journal of Virology.

[13]  C. Milstein,et al.  Continuous cultures of fused cells secreting antibody of predefined specificity , 1975, Nature.

[14]  A. Plückthun,et al.  New protein engineering approaches to multivalent and bispecific antibody fragments. , 1997, Immunotechnology : an international journal of immunological engineering.

[15]  Hassan M E Azzazy,et al.  Phage display technology: clinical applications and recent innovations. , 2002, Clinical biochemistry.

[16]  A. Pini,et al.  Phage display of antibody fragments. , 2000, Current protein & peptide science.

[17]  Wadih Arap,et al.  Hybridoma-free generation of monoclonal antibodies , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Anna M Wu,et al.  Arming antibodies: prospects and challenges for immunoconjugates , 2005, Nature Biotechnology.

[19]  Thomas D. Schmittgen,et al.  Real-Time Quantitative PCR , 2002 .

[20]  C R Cantor,et al.  Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. , 1992, Science.

[21]  Christof M Niemeyer,et al.  Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. , 2005, Trends in biotechnology.

[22]  K. Pfizenmaier,et al.  Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli , 1998, Applied and Environmental Microbiology.

[23]  Russell Higuchi,et al.  Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions , 1993, Bio/Technology.

[24]  A. Plückthun,et al.  Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. , 1988, Science.

[25]  S. Prusiner,et al.  Prion diseases and the BSE crisis. , 1997, Science.

[26]  K. Jooss,et al.  Stable antibody expression at therapeutic levels using the 2A peptide , 2005, Nature Biotechnology.

[27]  M. Mir,et al.  Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle , 2004, Journal of Virology.

[28]  C. Niemeyer,et al.  Combination of DNA-directed immobilization and immuno-PCR: very sensitive antigen detection by means of self-assembled DNA-protein conjugates. , 2003, Nucleic acids research.

[29]  W. März,et al.  Immuno-PCR with a commercially available avidin system. , 1993, Science.