Large amounts of biological samples are usually required to measure multiple components by the
enzyme-linked immunosorbant assay. However, the amounts of many
tissue extracts and fluids, including gingival crevicular fluid (GCF), are generally extremely small. The aim of this study was, therefore, to develop and validate a novel multiplex bead assay (MBA) to simultaneously measure a profile of healing-related mediators in the GCF of treated periodontal
wounds. An MBA was developed and validated by assessment of assay selectivity, recovery, precision and sensitivity, using eight recombinant human growth mediators as assay standards. GCF samples were collected on paper strips from healing wound (test) and healthy unaffected (control) sites of 15 patients with
periodontitis, seven days post-periodontal surgery. Each GCF sample was eluted and the levels of the mediators measured using the MBA and antibody pairs specific for
angiopoietin-1,
vascular endothelial growth-factor, bone morphogenetic protein-2,
osteoprotegerin, tissue inhibitor of metalloprotease-1 (TIMP-1),
basic fibroblast growth-factor,
keratinocyte growth-factor, and
platelet derived growth-factor. Less than 1.8% of cross-reactivity was observed between
antibodies and the eight different analytes, for which the recovery was more than 85%. Mean intra- and inter-assay precision were within the acceptance criteria of 20% and 25%, respectively. Detection of all mediators was highly sensitive (<or=70 ng/L) except for
TIMP-1 (215 ng/L). Angiogenic factors were the most highly secreted in the GCF seven days post-surgery. This new MBA can simultaneously measure small amounts of eight different growth mediators in the GCF of healing periodontal
wounds. It might also be a valuable tool for evaluating the components of
wound fluids as a prognostic
indicator of the success of therapeutic intervention.