Antibody-specific aptamer-based PCR analysis for sensitive protein detection

Nucleic acid amplification techniques were applied to the enzyme linked immunosorbent assay (ELISA) with an antibody-specific aptamer, R18. This novel detection system is a modification of the original immuno-polymerase chain reaction (immuno-PCR), but oligonucleotide-labeled antibodies are not required in the assay. This method is performed with the usual ELISA protocol, using an RNA aptamer for rabbit IgG instead of the conventional secondary antibody. After the assay plate was washed, quantitative reverse transcription (RT)-PCR was performed. Ribonuclease (RNase) inhibitors are not needed for this method. The detection limit of the quantitative RT-PCR is over 100 times more sensitive than the original ELISA method, even with the same sandwich–antibody combination. Only 1 mg of aptamer is sufficient for more than 10 million assays. This aptamer-based quantitative PCR successfully detected 16 attomoles (16 × 10−18) of vascular endothelial growth factor (VEGF). This is a cost-effective and easy method to increase the sensitivity of the rabbit antibody-based ELISA systems. The new method is referred to as immuno-aptamer PCR (iaPCR), to distinguish it from the original immuno-PCR.

[1]  Satoshi Nishikawa,et al.  Rabbit antibody detection with RNA aptamers. , 2008, Analytical biochemistry.

[2]  E. Engvall,et al.  Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. , 1971, Immunochemistry.

[3]  Juewen Liu,et al.  Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticles. , 2005, Angewandte Chemie.

[4]  L. Gold,et al.  The use of aptamers in large arrays for molecular diagnostics. , 1999, Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology.

[5]  D. Drolet,et al.  An enzyme-linked oligonucleotide assay , 1996, Nature Biotechnology.

[6]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[7]  R. Fisher,et al.  Universal immuno-PCR for ultra-sensitive target protein detection. , 1993, Nucleic acids research.

[8]  Ioanis Katakis,et al.  Aptamers: molecular tools for analytical applications , 2008, Analytical and bioanalytical chemistry.

[9]  T. Mothes,et al.  Assay of gliadin by real-time immunopolymerase chain reaction. , 2003, Die Nahrung.

[10]  Christof M Niemeyer,et al.  Sensitivity by combination: immuno-PCR and related technologies. , 2008, The Analyst.

[11]  S. Mendoza,et al.  Treatment of childhood prednisone-resistant nephrotic syndrome and focal segmental glomerulosclerosis with intravenous methylprednisolone and oral alkylating agents. , 1987, Nephron.

[12]  E. Engvall,et al.  Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. , 1972, Journal of immunology.

[13]  J. Wands,et al.  Development of a two-site immuno-PCR assay for hepatitis B surface antigen. , 1995, Journal of virological methods.

[14]  H. Schluesener,et al.  Ultrasensitive detection of protein using an aptamer-based exonuclease protection assay. , 2004, Analytical chemistry.

[15]  C. Hales,et al.  Labelled Antibodies and Immunological Assay Systems , 1968, Nature.

[16]  W. Griswold.  Theoretical analysis of the reaction of multivalent antigen with heterogeneous antibody: a model of soluble phase antibody assays. , 1987, Journal of immunoassay.

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

[18]  U. Landegren,et al.  Protein detection using proximity-dependent DNA ligation assays , 2002, Nature Biotechnology.

[19]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[20]  Dr Ferdiye Taner,et al.  The enzyme-linked immunosorbent assay (ELISA). , 1976, Bulletin of the World Health Organization.

[21]  Jeong-O Lee,et al.  Aptamers as molecular recognition elements for electrical nanobiosensors , 2007, Analytical and bioanalytical chemistry.

[22]  Kouhei Tsumoto,et al.  Open sandwich ELISA: A novel immunoassay based on the interchain interaction of antibody variable region , 1996, Nature Biotechnology.

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

[24]  Sergey E. Ilyin,et al.  Nanoliter high throughput quantitative PCR , 2006, Nucleic acids research.

[25]  Jack W. Szostak,et al.  An RNA motif that binds ATP , 1993, Nature.

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

[27]  H. Saiki,et al.  An immunoassay for small analytes with theoretical detection limits. , 2001, Analytical Chemistry.

[28]  S A BERSON,et al.  Immunoassay of endogenous plasma insulin in man. , 1996, The Journal of clinical investigation.

[29]  Juewen Liu,et al.  A simple and sensitive "dipstick" test in serum based on lateral flow separation of aptamer-linked nanostructures. , 2006, Angewandte Chemie.