A new diagnostic method for periodontal diseases by determining hepatocyte growth factor in gingival cervical fluid with a high sensitive ELISA.

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The degree of periodontitis may reflect the concentration of hepatocyte growth factor (HGF) in gingival cervical fluid (GCF) because HGF is thought to be involved in regeneration of various tissues. However, sample volume of GCF is too little (several microliter) to determine the concentrations of HGP by current ELISA for HGF used in clinics (the sensitivity is 200 pg/ml sample). Therefore, we developed two other high sensitive sandwich ELISA systems for HGP as follows. 1. Using mouse anti-HGF monoclonal antibody as the first antibody and chicken anti-HGF IgY conjugated with biotin as the second antibody, we obtained the sensitivity of 20 pg/ml. 2. Using mouse anti-HGF monoclonal antibody as the first antibody and beta-galactosidase conjugated Fab' of rabbit anti-HGF lgG as the second antibody, we obtained the sensitivity of 2 pg/ml. Using the latter ELISA, we found that concentrations of HGF in GCF collected by periopaper from patients with periodontitis were 1-6 ng/ml, and that the concentrations were positively correlated with the depth of periodontal pocket (probing depth) and the gingival index. The concentrations of HGF in GCF in healthy controls were less than 1 ng/ml. Because the average concentration of HGF in serum of healthy subjects is 0.2 ng/ml, these findings suggest that HGP is synthesized and secreted in the inflamed gingival tissues and is involved in regeneration of the tissue. Our finding also suggest the high sensitive ELISA for HGF we developed in this project may be useful for diagnosis of periodontal diseases.