Development of a highly sensitive immunoassay by use of DNA-encapsulated liposomes and PCR

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15560683
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Kurume National College of Technology
• Principal Investigator: TOMIOKA Kanji Kurume National college of Technology, Biochemistry and Applied Chemistry, Associate Professor, 生物応用化学科, 助教授 (30332084)
• Project Period (FY): 2003 – 2004
• Keywords: liposomes / antigen / antibody / ELISA / PCR

Research Abstract

We proposed a liposome immnunosorbent assay (LISA) for competitive measurement of an antigen by use of antigen-coupled liposomes encapsulating fluorescent marker carboxyfluorescein (CF) and clarified the effects of measuring conditions on their assay characteristics. With increase in the spacer length of hetero-bifunctional reagents used for cross-linking reaction, the amount of CF released from liposomes on coupling of antigen molecules with activated liposomes increased. The sensitivity in the assay by LISA was al acted by the amount of the antigen coupled on the liposomes and the concentration of the antigen coexisting in assay solutions. This competitive LISA could detect two orders of magnitude lower concentration than that by the conventional ELISA and could measure over five orders of the antigen concentration. Since the washing step was needed only once for B/F separation in this competitive LISA, this competitive LISA was useful for rapid and easy measurement of antigen with high sensitivity.
We also proposed PCR-LISA for competitive measurement of an antigen by use of antigen-coupled liposomes encapsulating plasmid DNA instead of fluorescent marker carboxyfluorescein (CF). After B/F separation the liposomes was broken by detergent reagent β galactopyranoside and the plasmid DNA was copied by PCR and stained with DNA measuring dye, Hoechst 33258, to measure DNA concentrations. This competitive PCR-LISA could detect three orders of magnitude lower concentration than that by conventional LISA. PCR-LISA offers more sensitive detection systems that could be applied to very small amount of target proteins in many kinds of specimens.
Both have the potential to broaden the applications of ELISA to clinical, environmental, food diagnostics with high sensitivity.