Antimicrobial peptides (AMPs) HNP1-3 and LL-37 may be useful biomarkers of periodontal disease. The aim of this investigation was to test the stability of salivary AMP samples during storage at room temperature and following repeated freeze-thaw cycles.
Method:
13 unstimulated saliva samples were collected from patients with periodontitis. Room temperature stability samples were covered, stored at a lab bench and subsequently placed frozen at 0, 6, 24, 48, 72 and 96 hours. Samples for freeze-thaw stability were frozen for at least 4 hours at -80°C before thawing at 37°C and being refrozen. Samples underwent seven cycles of freeze-thawing (C0 to C7). The samples were then tested by ELISA for HNP1-3 and LL-37. Percentage differences from baseline values for each sample set were calculated to determine changes in AMP concentration. Samples were analysed by Repeated Measures 1-way analysis of variance and Bonferoni Post-test.
Result:
HNP concentration remained largely stable in freeze thaw cycles up to C5, and dropped to 55.54% of initial concentration by C7. LL-37 concentrations remained consistently stable through C1- C6 and increased 125.76 ± 46.5 % by C7. Following maintenance of samples at room temperature for prolonged periods, HNP1-3 concentrations dropped to 65.28 ± 42.5 % after 96 hours storage at room temperature. Again LL-37 showed stability through to 96h at room temperature with 106.42 ± 23.3% of baseline concentrations.
Conclusion:
Both HNP1-3 and LL-37 concentrations remained stable during prolonged periods of storage at room temperature. HNP1-3 showed some gradual decline in percentage concentration over 96 hours. Both AMPs also demonstrated relative stability throughout repeated cycles of freezing and thawing. In particular LL-37 concentrations remained highly stable throughout the experiments. The stability of salivary AMPs suggests that they may be useful for sampling in “field” conditions away from a dental surgery.