Immuno-quantification of Human Statherin in Major Salivary Secretions

Statherin is a 43-residue phosphoprotein of unusual composition and structure found mainly in human glandular salivary secretions. It is the only salivary protein shown to inhibit both primary calcium phosphate precipitation (spontaneous precipitation) and secondary calcium phosphate precipitation (crystal growth). Objective: To quantify statherin in individual salivary secretions using a statherin capture enzyme linked immunosorbent assay (ELISA). Methods: Two anti-statherin monoclonal antibodies (mAbs), JL 23 and JL 35, obtained upon immunization of mice with in vivo formed human acquired enamel pellicle, were shown to recognize different epitopes in statherin in a competitive ELISA. Using these mAbs, a statherin capture ELISA was developed for the measurement of statherin levels in salivary secretions collected from 10 individuals. Results: ELISA standards curve with statherin dilutions ranging from 0.5 to 2000.0 ng were obtained by plotting statherin concentrations (log₁₀) versus absorbance at 490 nm. These plots showed a linear relationship with an R²-value of 0.99. When purified statherin was added to 1:50 diluted salivary samples, added statherin was quantified with an experimental error of 7.8%, 1.9% and 8.5% in whole saliva supernatants (WS), parotid saliva (PS) and submandibular/sublingual secretions (SM/SL), respectively. The concentration range of statherin was 0.6 – 52.2 µg/ml (median = 6.2 µg/ml) in unstimulated WS and was 0.8 – 33.6 µg/ml (median = 4.7 µg/ml) in stimulated WS. The concentration range of statherin in stimulated PS and SM/SL were 6.0 – 227.1 µg/ml (median = 66.2 µg/ml) and 13.4 – 140.3 µg/ml (median = 75.8 µg/ml), respectively. Conclusions: The data indicate that this statherin capture ELISA is suitable for the measurement of statherin in saliva samples and will enable us to elucidate the role of statherin in oral health and disease. Supported by NIH/NIDCR grants DE05672 and DE07652.