Accurately identifying saliva from samples mixed with citric acid‐containing beverages by indirect <scp>ELISA</scp> ‐based detection of cystatin D

In forensic saliva testing, false negatives of the Phadebas® and false positives of the RSID™-saliva methods generated by citric acid are critical issues for interpreting the results. Citric acid-containing beverages, which are typically mixed with forensic saliva samples, may produce indistinguishable results in two commercially available saliva tests in the presence and absence of saliva. To solve this issue, this study aimed to evaluate the usefulness of the indirect enzyme-linked immunosorbent assay (ELISA)-based method of detecting cystatin D to identify saliva in citric acid-containing beverage-mixed samples. Four commercially available beverages were mixed with human saliva or ultrapure water, and the mixed stain samples were examined by the aforementioned three saliva tests. Two conventional saliva tests could not distinguish between the samples with and without saliva. Although the positive detection rates of the ELISA method decreased when mixed with beverages, no saliva-free beverage samples produced false positives. Our results indicate that the detection of cystatin D by indirect ELISA would be useful in identifying saliva from samples potentially mixed with citric acid-containing beverages.