title: A chemiluminescent sandwich ELISA enhancement method using a chromium (III) coordination complex.
abstract: Enzyme linked immunosorbent assays (ELISAs) are employed for the detection and quantification of antigens from biological sources such as serum and cell culture media. A sandwich ELISA is dependent on the immobilization of a capture antibody, or antibody fragment, and the effective presentation of its antigen binding sites. Immobilization to common microtitre plates relies on non-specific interactions of the capture protein with a surface that may result in unfavourable orientation and conformation, compromising ELISA signal strength and performance. We have developed a wet chemical surface activation method that utilizes a chromium (III) solution to immobilize native, non-tagged, capture antibodies on commercially available microtitre plates. Antibodies captured by this method had increased antigen binding, particularly from dilute antibody solutions, relative to antibodies adsorbed directly to the plate surface. A variety of monoclonal antibodies with complementary antigen systems were used to demonstrate improvements in ELISA signal and reproducibility. The simplicity and versatility of this method should enable ELISA enhancement in assays where chemiluminescence is used as the detection method.
authors.affiliation: Centre for Materials and Surface Science, Department of Chemistry and Physics, La Trobe University, VIC 3086, Australia; CSIRO Manufacturing, Bayview Ave, Clayton, VIC 3168, Australia.
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Welch
authors.affiliation: CSIRO Manufacturing, Bayview Ave, Clayton, VIC 3168, Australia.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Easton
authors.affiliation: CSIRO Manufacturing, 343 Royal Pde, Parkville, VIC 3052, Australia.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Scoble
authors.affiliation: CSIRO Manufacturing, 343 Royal Pde, Parkville, VIC 3052, Australia.
authors.forename: Charlotte C
authors.initials: CC
authors.lastname: Williams
authors.affiliation: Centre for Materials and Surface Science, Department of Chemistry and Physics, La Trobe University, VIC 3086, Australia.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Pigram
authors.affiliation: CSIRO Manufacturing, Bayview Ave, Clayton, VIC 3168, Australia. Electronic address: Ben.Muir@csiro.au.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Muir
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D000941:Antigens / Q000032:analysis*; D002857:Chromium / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007128:Immunoglobulin Fragments; D008163:Luminescent Measurements; D051379:Mice; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D007128:Immunoglobulin Fragments; D002857:Chromium
keywords: Antibody immobilization; Chromium complex; ELISA; Surface modification