PRELIMINARY STUDY ONPURIFICATION OF DENVJ VIMSANTI­ ENVELOPE PROTEINDOMAIN M(ED3) LGY ANTIBODY USING SILICA MODIFIED WITHPROPYLDIETHYLENEDIAMINE-GLUTARALDEHYDE-ED3

• Main Author: Felicia
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/76114
• Institution: Institut Teknologi Bandung

WHO lists Indonesia as the country with the highest dengue hemorrhagic fever (DHF) cases in Southeast Asia. The cause of DHF is a Dengue virus infection that is transmitted to humans through the bite of the Aedes aegypti mosquito. High fever is the first symptom of the disease. To distinguish fever caused by Dengue virus infection from other infections, an accurate, rapid and economical diagnostic tool is needed, so that dengue fever treatment can be carried out appropriately and quickly. One of the diagnosis methods that meet the above requirements is Rapid Test Diagnosis (RDT) to diagnose Dengue virus in patient blood samples. One of the candidate biomarkers of Dengue virus is Envelope Protein Domain III(ED3). An important component in the development of Dengue Antigen RDT kit is a biomarker which is specific for DENY virus. The anti-ED3 antibody can be used as biomarker in RDT and it can interact specifically with DENY virus antigens in blood samples so that the presence of Dengue virus in the patient's blood can be detected. Previous studies have successfully produced anti-ED3 IgY antibodies produced in chickens. Anti-ED3 IgY antibody with high purity is needed in the development of Dengue Antigen RDT kit. Therefore, the aim of this study was to develop a purification technique for anti-ED3 antibodies using propyl diethylenetriamine­ glutaraldehyde modified silica. The first stage in this research is to conduct an in silico study consisting of (1) analysis of amino acid side chains that can bind to glutaraldehyde and (2) analysis of epitopes on ED3 that can interact with anti-ED3 lgY antibodies using ElliPro. The second stage of this research includes (1) synthesis of propyl diethylenetriamine­ glutaraldehyde modified silica using the Strober method, (2) expression of recombinant DENY1-ED3 protein, (3) immobilization of ED3 protein on modified silica, and (4) purification of anti-ED3 antibody. The results of in silico studies showed that glutaraldehyde is reactive toward amino acid side chain residues contaiuing s-amino, a-amino, secondary amino, and hydroxyl groups. The epitope prediction results show that there are epitopes on the ED3 surface that can be easily accessed by anti-ED3 IgY antibodies. The successful synthesis of modified silica can be seen from the absorption bands in the FTIR spectrum, namely at 1109 cm-I from Si-0-Si vibrations, 810-939 cm·' from Si-C vibrations, and 1639 cm·' from C=O vibrations. The C-N bond vibration at 1436 cm·' indicates that ED3 has been immobilized on modified silica. Furthermore, antibody purification was performed using 0.5 M NaCl and I M NaCl as elution buffers. Although this anti-ED3 IgY antibody purification system still needs to be optimized, it has the potential to be further developed as another antibody purification method.