title: A rapid method to improve protein detection by indirect ELISA.
abstract: The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measurable substrate. Numerous incarnations of the ELISA have resulted in its commercialization for sensitive diagnostic applications using a variety of detection platforms. Many of these applications require a pair of antibodies necessary for the capture and detection of a specific antigen (cELISA) in defined substrates. However, the availability of cELISA for target antigens is limited and thus restricts the use of this technique for quantitative measure of antigens during discovery. Alternatively, the indirect ELISA (iELISA) requires only a single antibody directed against a target antigen that has been immobilized to a surface. Unlike the cELISA, which uses an immobilized capture antibody that can bind a native antigen in solution followed by a detector antibody that binds captured antigen, the iELISA uses an antibody the binds directly to an immobilized antigen for detection. Although the iELISA may lack the sensitivity of a cELISA, its requirement of only a single antigen specific antibody makes it a simple technique for evaluating the relative difference in the level of target protein expression between samples. However, many antibodies that work effectively to detect protein antigens in other immunoassays such as Western blotting or immunohistochemistry fail to work in microplate based iELISA. Although these alternate immunoassay methods are useful for qualitative determination of target antigens, they provide limited quantitative information, limiting the assessment of sample specific differences in protein expression. We hypothesized that protein conformation following adsorption on the plastic surface of microplates impedes antibody epitope binding and this restriction could be overcome by a short chemical denaturation step. In this report we define a rapid method to assess the utility of an antibody for iELISA application and demonstrate a significant improvement in both qualitative and quantitative protein detection after chemical denaturation using defined assay conditions.
authors.affiliation: 800 Buchanan St., USDA-ARS, Western Regional Research Center, Albany, CA 94710, USA. Robert.Hnasko@ars.usda.gov
authors.forename: Robert
authors.initials: R
authors.lastname: Hnasko
authors.forename: Alice
authors.initials: A
authors.lastname: Lin
authors.forename: Jeffery A
authors.initials: JA
authors.lastname: McGarvey
authors.forename: Larry H
authors.initials: LH
authors.lastname: Stanker
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology; D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry; D005974:Glutamate-Ammonia Ligase / Q000032:analysis / Q000737:chemistry; D051379:Mice; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
chemical_list: D002021:Buffers; D004800:Enzymes, Immobilized; D011506:Proteins; D005974:Glutamate-Ammonia Ligase