Synthesis of a unique high-performance poly-horseradish peroxidase complex to enhance sensitivity of immunodetection systems

Biotechnol Appl Biochem. 2012 Jan-Feb;59(1):45-9. doi: 10.1002/bab.58. Epub 2012 Jan 10.

Abstract

Because early detection is the first step in successful therapy, increasing the sensitivity of detection systems has always been considered as one of the major trends in development of these technologies. Therefore, we have fabricated a high-performance poly-horseradish peroxidase (HRP) complex and analyzed it in different formats of immunodetection systems. To construct this complex, dextran-aldehyde was prepared by oxidation of dextran in the presence of sodium periodate. Activated polymer was then coupled to lysine amino acids and accomplishment of the process was evaluated with trinitrobenzenesulfonic acid. Following conjugation of HRP to free amino groups of lysine, the stage's accuracy and the rate of conjugation were demonstrated by SDS-PAGE. Then, conjugation of poly-HRP complex to streptavidin by biotin was performed. The results of a series of experiments confirmed the complete synthesis of streptavidin-poly-HRP complex by this procedure. Finally, we compared our harvested complex with the golden standard complex available for ELISA and immunohistochemistry (IHC). The results showed the high efficiency of the synthesized complex. Consequently, this complex can be applicable in highly sensitive detection technologies. Conjugating this complex to any antibody by using biotin-streptavidin bridging and preparing poly-HRP-labeled antibodies will be a valuable multifold approach to increase the sensitivity of detection systems, which can be applicable in ELISA, immunocytochemistry, and IHC methods.

MeSH terms

  • Biotin / chemistry
  • Dextrans / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Horseradish Peroxidase / metabolism*
  • Immunohistochemistry / methods*
  • Streptavidin / chemistry

Substances

  • Dextrans
  • Biotin
  • Streptavidin
  • Horseradish Peroxidase