title: Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA.
abstract: Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1 μg of biotinylated protein.
authors.affiliation: Sanquin Research, Amsterdam, The Netherlands; Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands. Electronic address: t.rispens@sanquin.nl.
authors.forename: Theo
authors.initials: T
authors.lastname: Rispens
authors.affiliation: Sanquin Research, Amsterdam, The Netherlands; Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
authors.forename: Pleuni
authors.initials: P
authors.lastname: Ooijevaar-de Heer
mesh_terms: D000906:Antibodies; D001710:Biotin / Q000276:immunology; D019921:Biotinylation; D019286:Endopeptidase K / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019809:Streptavidin
chemical_list: D000906:Antibodies; D011506:Proteins; D001710:Biotin; D019809:Streptavidin; D019286:Endopeptidase K
keywords: Anti-biotin antibody; Biotin; Degree of biotinylation; Inhibition assay