QUANTITATIVE HEPATITIS B DIAGNOSTIC KIT DEVELOPMENT BASED ON ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

• Main Author: Rahmadini, Hafiza
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/76277
• Institution: Institut Teknologi Bandung

Hepatitis B is an inflammatory disease that affects the liver and is one of the top ten global health problems. WHO data shows that many people worldwide are infected with the Hepatitis B virus, including Indonesia, which has a prevalence rate of approximately 7.1% for Hepatitis B infection. Currently, patients infected with the Hepatitis B virus are treated with antiviral drugs, and during the therapy process, the Hepatitis B virus titer in the blood is monitored to assess the progress of the treatment's success. The monitoring of the Hepatitis B virus titer is done using a quantitative Hepatitis B kit to determine the titer of the virus in the blood. In the year 2022, ITB researchers have successfully developed an Enzyme-Linked Immunosorbent Assay (ELISA) based Hepatitis B kit. However, this diagnostic kit cannot be used to monitor the Hepatitis B virus titer effectively because the developed diagnostic kit is qualitative in nature. This study was conducted to further develop the kit into a quantitative diagnostic kit for Hepatitis B based on ELISA. The scope of this research includes determining the quantification method, selecting the chromogen, selecting the wash buffer, determining the kit linearity, determining the washing method, and validating the kit. The objective of this research is to obtain a quantitative diagnostic kit for Hepatitis B based on ELISA that can determine the Hepatitis B virus content in blood plasma samples. Based on the conducted research, it is concluded that quantification based on the kinetics of the absorption rate is ideal for measuring the titer of the Hepatitis B virus in the blood. Moreover, it is found that ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) can be used as a chromogenic substrate because it has a reaction rate that can be well monitored. This research also successfully demonstrates that the PBST (Phosphate Buffer Saline-Tween) wash buffer is superior to SBT (Sodium Borate- Tween) as a wash buffer, and therefore, it is selected as the wash buffer for this quantitative Hepatitis B kit. Furthermore, this wash buffer enables single and manual wash of the diagnostic kit. This is crucial as it saves time and effort, and the developed diagnostic kit can be operated in clinical laboratories that are not equipped with an ELISA washer. Furthermore, the validation test results using 19 negative samples and 20 positive samples showed that the developed quantitative Hepatitis B diagnostic kit based on ELISA has 100% sensitivity and 100% specificity, with a detection limit of 1.05 ?g/mL HBsAg. Although the research has demonstrated that the kit performs well, it is important to conduct independent durability testing and performance verification of the kit before commercialization.