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Enhanced Detection of Low Abundant Molecules using Polymerized HRP Conjugates
Author(s) -
Ganapathy Ramesh,
Wolf Brian,
Nlend Marie,
Stanaitis Mike,
Schofield Mark,
Kilmer Greg,
Desai Surbhi
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.539.5
Subject(s) - horseradish peroxidase , blot , chemistry , conjugate , microbiology and biotechnology , streptavidin , immunohistochemistry , peroxidase , detection limit , biochemistry , enzyme , chromatography , biology , biotin , immunology , mathematical analysis , mathematics , gene
Enzyme conjugated secondary detection reagents are routinely used for amplified detection of specific antigens in immunoassays. Horseradish peroxidase (HRP) is the most widely used enzyme in ELISA, Western blotting, immunohistochemistry, Northern and Southern blotting applications and hybridization assays, because it is economical, robust and compatible with both insoluble and soluble substrates. We have developed a series of Streptavidin, Goat anti‐mouse (GAM) and Goat anti‐rabbit (GAR) Poly HRP for the detection of low‐abundant molecules. The new polymerized HRP conjugates are purified to remove unconjugated HRP, secondary detection reagents and monomer HRP conjugates. We have demonstrated increased sensitivity and signal intensity with the Thermo Scientific Pierce Poly HRP conjugates in ELISA, Western blotting and immunohistochemistry (IHC) applications. Human IFNγ and Bovine IL4 ELISAs using the Pierce Poly HRP conjugates displayed significant signal amplification over non‐polymerized and polymerized HRP conjugates. Western blotting and IHC detection of low abundant proteins displayed similar signal enhancement with Thermo Scientific Pierce Poly HRP conjugates.