Open AccessSequential ELISA for Cytokine Levels in Limited Volumes of Biological Fluids
Author(s) -
M. J. Steffen,
Jeffrey L. Ebersole
Publication year - 1996
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/96213rr04
Subject(s) - polyclonal antibodies , cytokine , monoclonal antibody , microbiology and biotechnology , recombinant dna , alkaline phosphatase , antibody , biological fluids , chemistry , replicate , biology , immunology , enzyme , chromatography , biochemistry , gene , statistics , mathematics
We have developed an enzyme-linked immunosorbent assay (ELISA) for the sequential analysis of multiple cytokines in limited volumes of biological fluids, including gingival crevicular fluid (GCF) and fibroblast culture supernatants (CS). GCF and CS samples were assayed for multiple cytokines, including IL-1β, IL-6, IL-8, GMCSF and IFNg. Immulon 3 ® microplates were coated with a monoclonal antibody, and a rabbit polyclonal antibody was used to detect the cytokine of interest. Biological samples (200μL) were added to an anti-IL-1β-coated plate and incubated, and 175μL of each sample were replicate transferred to an anti-IFNγ-coated plate containing 25 μL/well of diluent. This was repeated in an identical fashion with sequential replicate transfers to an anti-IL-8-coated and finally an anti-IL-6-coated plate. The cytokine standard was a pooled combination of the recombinant human cytokines that were included in the sequence. The plates were developed using an alkaline phosphatase-conjugated goat anti-rabbit IgG and NPP as the substrate. Individual ELISAs ranged in sensitivity from 30 to 2 pg/0.2 mL, with cross-reactivity between these cytokines of 85% correlation between the two assays.