title: Modified Nanoantibodies Increase Sensitivity in Avidin-Biotin Immunohistochemistry.
abstract: Revealing the spatial arrangement of molecules within a tissue through immunohistochemistry (IHC) is an invaluable tool in biomedical research and clinical diagnostics. Choosing both the appropriate antibody and amplification system is paramount to the pathologic interpretation of the tissue at hand. The use of single domain VHH nanoantibodies (nAbs) promise more robust and consistent results in IHC, but are rarely used as an alternative to conventional immunoglobulin G (IgG) antibodies. nAbs are originally obtained from llamas and are the smallest antigen-binding fragments available. To determine whether the unique biophysical properties of nAbs give them an advantage in IHC, we first compared a basic fibroblast growth factor nAb to polyclonal IgG antibodies using tissue isolated from pancreatic adenocarcinoma. The nAb was extremely effective in antigen signal detection and allowed for a more streamlined and reproducible protocol. Furthermore, because nAbs are expressed in Escherichia coli from a single gene, they are quite amenable to genetic engineering. As such, we then covalently bound a highly biotinylated amplifier protein to basic fibroblast growth factor and p16 nAbs (termed nAb Plus), resulting in improved IHC sensitivity. The use of a biotinylated nAb Plus not only achieved local, covalent signal amplification, but also eliminated the need for a secondary antibody and subsequent amplification steps. These results highlight nAbs as valuable alternatives to conventional IgG antibodies, decreasing overall processing time and costs of reagents while increasing sensitivity and reproducibility across individual IHC assays.
authors.affiliation: Allele Biotechnology.
authors.forename: Anthony
authors.initials: A
authors.lastname: Wong
authors.affiliation: Allele Biotechnology.
authors.forename: Chelsea
authors.initials: C
authors.lastname: Sykora
authors.affiliation: Allele Biotechnology.
authors.forename: Lewis
authors.initials: L
authors.lastname: Rogers
authors.affiliation: Allele Biotechnology.|Scintillon Institute, San Diego, CA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Higginbotham
authors.affiliation: Allele Biotechnology.|Scintillon Institute, San Diego, CA.
authors.forename: Jiwu
authors.initials: J
authors.lastname: Wang
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000818:Animals; D000912:Antibodies, Neoplasm / Q000737:chemistry*; D001360:Avidin / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D007150:Immunohistochemistry / Q000379:methods; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology; D011817:Rabbits; D061905:Single-Domain Antibodies / Q000737:chemistry*
chemical_list: D000912:Antibodies, Neoplasm; C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D007074:Immunoglobulin G; D061905:Single-Domain Antibodies; D016222:Fibroblast Growth Factor 2; D001360:Avidin; D001710:Biotin