Indirect ELISA-based detection of histatin 3 and cystatin D for the forensic identification of human saliva

Analyzing forensically relevant body fluids contributes to proving criminal acts, and saliva is often left on the scene, especially in sexual assault cases. Currently, saliva is presumptively identified using its salivary α-amylase activity as an indicator. However, the specificity of saliva presumptive tests is low, and therefore, they cannot confidently prove the presence of saliva. This study aimed to develop and validate an indirect ELISA-based saliva confirmatory test using the novel human salivary protein markers histatin 3 and cystatin D. Histatin 3 and cystatin D are known to be uniquely expressed in human salivary glands, and the molecular evolution of both genes occurred after the divergence of hominoids; thus, the high specificity of human saliva for both markers was expected. As a result of the validation experiments, both histatin 3 and cystatin D markers were able to distinguish human saliva from other human body fluids and animal saliva using appropriate thresholds of the quantitative absorbance values. Our results indicate that both histatin 3 and cystatin D markers are human saliva-specific; thus, we consider that this method will be useful and practical in identifying human saliva.