DEVELOPMENT OF ISOLATION TECHNIQUES AND USE OF WHITE RADISH PEROXIDASE (WRP) AS A SUBSTITUTE FOR HORSERADISH PEROXIDASE (HRP) ENZYME IN ELISA-BASED DIAGNOSTIC SYTEMS

• Main Author: Al Faizah, Nur
• Format: Theses
• Language: Indonesia
• Subjects: Teknologi
• Online Access: https://digilib.itb.ac.id/gdl/view/84503
• Institution: Institut Teknologi Bandung

With the advancement of medicine, biotechnology has become increasingly important in the development of diagnostic tools. An example of a commonly used diagnostic tool is the enzyme-linked immunosorbent assay (ELISA). Within ELISA diagnostic systems, horseradish peroxidase (HRP) is frequently utilized as a marker enzyme to detect an analyte on the sample. However, horseradish, the source plant for HRP, only grows in subtropical regions, which presents a challenge for its accessibility to Indonesia's dependency on importing commercial horseradish peroxidase (HRP) to fulfill industrial demand. This condition prompts the need for alternative peroxidase enzymes sourced from locally grown plants such as white radish (Raphanus sativus L.), which naturally contains white radish peroxidase (WRP). This study aims to develop a white radish peroxidase (WRP) isolation technique and to evaluate its viability as a substitute for HRP in ELISA-based diagnostic kits. This study adopted a method to extract WRP using the polyethylene glycol (PEG) precipitation method. The isolation process utilized a combination of PEG 400 20% (v/v) and PEG 6000 30% (w/v). This study compared one-step isolation using only PEG 6000 30% (w/v); and two-step isolation using PEG 400 20% (v/v) and PEG 6000 30% (w/v) sequentially. The result showed that the WRP yield from one-step and two-step isolation did not differ from one another in terms of concentration. However, the inclusion of PEG 400 20% at the beginning of the isolation process was able to eliminate protein impurities with a lower partition coefficient than WRP, thus resulted in crude WRP isolate which is higher in purity compared to the one-step extraction yield. Furthermore, adding PVP to the WRP preparation showed an increase in the solubility of the lyophilized WRP when dissolved. This study also showed that adding a lyoprotectant protects WRP during lyophilization. The experimental results to determine the potential of WRP as an HRP substitute showed that the crude WRP isolate can be conjugated with mouse anti-human HbsAg. However, although WRP was successfully conjugated, the ELISA test did not provide sufficient signals to confirm that the crude WRP isolate can replace HRP in the ELISA system. This condition is estimated to be due to the low purity level of crude WRP isolate causing impurities to be conjugated and resulted in relatively low signals from the conjugate. Based on this, further development is needed to improve the purity of the WRP isolate, so it can be used as an HRP substitute in the ELISA system.