Quantification molecular weight of human beta defensin-2 and human beta defensin-3 from saliva of caries patient

Abstract

Background: Dental caries is the most common multifactorial disease in the world. In saliva of caries patients, occur an increase in innate immunity, such as human beta defensin-2 (HBD-3) and human beta defensin-2 (HBD-3). Research on quantification the molecular weight of HBD-2 and HBD-3 in saliva of caries patients has not done yet. Objective: Determine the molecular weight from HBD-2 and HBD-3 from saliva of caries patients using the Western Blot method. Method: This study was approved by the Health Research Ethical Clearance Commission of Faculty of Dental Medicine Airlangga University No.156/HRECC.FODM/IV/2019. After every individual agreed, saliva sample from 16 children with caries and caries-free in the Dental Hospital Airlangga University was collected in June to July 2019. Male/female aged 9-12 years and DMF-t > 5, taken 5 ml of saliva between 8-10 o'clock, passive-drool method without stimulation. Salivary samples were centrifuged for 20 minutes, 4ºC, with 10.000 rpm. Total protein was calculated using bicinchoninic acid (BCA) Assays before loading into Sodium Dodecyl Sulfate PolyAcrilamide Gel Electrophoresis (SDS-PAGE) to evaluate protein separation based on molecular weight. Furthermore, Western Blot is performed to ensure the protein that appears is HBD-2 and HBD-3 specific protein using primary and secondary antibodies. Results: THE molecular weight of HBD-2 and HBD-3 in the saliva of caries patients is 43 kDa, while the salivary free-caries band in the gel is unreadable, thus the molecular weight cannot be determined. Conclusion: The molecular weight of HBD-2 and HBD-3 in the saliva of caries patients calculated using the Western Blot method was 43 kDa.