Development and evaluation of a rapid and sensitive homogeneous assay for haptoglobin measurements in saliva

摘要

Human saliva has gained interest in the last years as a diagnostic fluid and several proteomic studies have identified haptoglobin (Hp) as a possible salivary biomarker in different diseases. However, there are only a few reports that documented the measurement of Hp in human saliva. The objectives of this study were to develop and validate a method based on AlphaLISA (R) technology to measure Hp in human saliva and apply it to evaluate the changes in salivary Hp in different conditions such as inflammation, psychological and physical stress, and acid stimulation. Thus, an AlphaLISA (R) based method was developed and validated through calculation of precision, accuracy, limit of detection (LD) and lower limit of quantification (LLOQ). A number of 20 controls, 22 subjects with inflammation sampled once, and also 10 with psychological stress, 10 with physical stress, and 6 that had an acetic acid stimulation that were sampled before and at different times after the induction of the stressor, were used in the trials. CVs were lower than 20% and recovery rates were between 90 and 120% when the calibration material was used to spike a saliva sample, and between 86 and 97% when saliva samples were mixed. The LD and LLOQ were low enough to detect salivary Hp concentrations in healthy control patients. A significant increase of Hp was observed in case of inflammation and physical stress, and a decrease after acetic acid stimulation. The assay developed allowed the measurement of Hp in saliva in a rapid, sensitive and reproducible way. In addition, the method can detect changes in Hp salivary concentrations in the different situations tested. The possibility of use of saliva opens a new approach to measure the concentrations of Hp through non-invasive and non-stressful methodology.