title: Luminometry: a novel bioluminescent immunoassay enhances the quantitation of mucosal and systemic antibody responses.
abstract: We have directly compared enzyme-linked immunoassays (ELISAs) with bioluminescent immunoassays employing derivatives of the bioluminescent molecule aequorin, and have shown that detection of mucosal and serum antibodies is considerably more sensitive when detected by luminometry. Luminometry is based upon counting photons of light via phototubes and is generally similar to scintillation spectrometry. Current commercial luminometric technology employs a phototube which is most efficient for light emission in the 400-420 nm wavelength range. For this reason, we have chosen the bioluminescent molecule, aequorin, which upon the addition of Ca2+ undergoes a conformational change resulting in the emission of blue light at 469 nm. The high quantum yield is reflected by the fact that addition of Ca2+ to 1 ng of recombinant streptaequorin, a covalent conjugate of streptavidin and aequorin, resulted in the production of 7 x 10(8) relative light units. In this study, we show the superior sensitivity of biotin-streptaequorin when directly compared with biotin-streptavidin linked horseradish peroxidase commonly used for ELISA. For example, mice orally immunized once with cholera toxin (CT) did not exhibit detectable fecal IgA antibodies as determined by ELISA, whereas use of streptaequorin and the bioluminescent immunoassay revealed fecal IgA anti-CT-B subunit antibody titers of 1:24 500. In addition, no detectable anti-CT-B antibodies were noted in saliva samples by ELISA 7 days following oral immunization with CT, while IgA endpoint titers could be extrapolated to 1:393 000. The 21 day fecal IgA anti-CT-B titers were 1:512 by ELISA, whereas titers determined by luminometry reached 1:10(7) when Neutralite avidin and biotinylated aequorin were employed. In general, the bioluminescent immunoassay was > 10(4)-fold more sensitive when compared with ELISA for detection of mucosal and serum antigen- and isotype-specific antibody responses. Thus, the bioluminescent immunoassay is a more sensitive assay for detection of antibodies in dilute external secretions.
authors.affiliation: Immunobiology Vaccine Center, University of Alabama at Birmingham, Medical Center, AL 35294, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Jackson
authors.forename: K
authors.initials: K
authors.lastname: Fujihashi
authors.forename: H
authors.initials: H
authors.lastname: Kiyono
authors.forename: J R
authors.initials: JR
authors.lastname: McGhee
mesh_terms: D000284:Administration, Oral; D000331:Aequorin; D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis / Q000096:biosynthesis / Q000097:blood; D000917:Antibody Formation; D002772:Cholera Toxin / Q000008:administration & dosage / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D007114:Immunization; D007118:Immunoassay / Q000379:methods* / Q000706:statistics & numerical data; D007070:Immunoglobulin A / Q000096:biosynthesis; D007413:Intestinal Mucosa / Q000276:immunology; D008163:Luminescent Measurements; D051379:Mice; D008810:Mice, Inbred C57BL; D012680:Sensitivity and Specificity
chemical_list: D000907:Antibodies, Bacterial; D007070:Immunoglobulin A; D000331:Aequorin; D002772:Cholera Toxin