Preliminary Study on the Purification of Anti-Envelope Protein Domain III (ED3) IgY Antibody for the DENV1 Virus using Silica Modified with Propyl Diethylenetriamine-Glutaraldehyde-ED3
Felicia (a), Yovin Sugijo (a), Fernita Puspasari (a), Ihsanawati (a), Wyanda Arnafia(c), Dessy Natalia (a), Handajaya Rusli (b), Fifi Fitriyah Masduki (a)

(a) Biochemistry Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology Jalan Ganesha 10, Bandung 40132, Indonesia
(b) Analytical Chemistry Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology Jalan Ganesha 10, Bandung 40132, Indonesia
(c) PT Tekad Mandiri Citra, Jalan Raya Kawaluyaan No.20A Bandung 40286, Indonesia

Abstract

The 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 fevers caused by Dengue virus infection from other infections, the development of diagnostic tools that are accurate, fast, and economical is needed, such that dengue fever treatment can be carried out appropriately and quickly. One diagnosis method that meets the above requirements is Rapid Test Diagnosis (RDT) of antigens to diagnose Dengue virus in patient blood samples. One of the candidate biomarkers of the Dengue virus is blood Envelope Protein Domain III (ED3). A necessary component in the development of Dengue antigen RDT is the anti-ED3 antibody. Anti-ED3 antibodies in RDTs can interact specifically with viral antigens in blood samples so that dengue virus infection can be confirmed. Previous studies have successfully produced anti-ED3 IgY antibodies produced in chickens. To obtain anti-ED3 IgY antibodies that are usable in the development of Dengue antigen RDTs, it is necessary to carry out purification steps. Therefore, the purpose of this study was to purify anti-ED3 antibodies using propyl diethylenetriamine-glutaraldehyde-modified silica. The first stage of 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 IgY antibodies using the ElliPro. The second stage in this study involves (1) the synthesis of propyl diethylenetriamine-glutaraldehyde modified silica using the Strober method, (2) expression of recombinant DENV1-ED3 protein, (3) immobilization of the ED3 protein on modified silica and (4) purification of the anti-ED3 antibodies. The results of the in silico studies showed that glutaraldehyde was reactive toward amino acid side chain residues containing secondary amino, and hydroxyl groups. Epitope prediction results show that epitopes exist on the surface of ED3 which can be easily accessed by anti-ED3 IgY antibodies. The success of modified silica synthesis can be seen from absorption bands in the FTIR spectrum, namely in 1109 cm-1 from Si-O-Si vibrations, 810-939 cm-1 from Si-C vibrations, and 1639 cm-1 from C=O vibrations. C-N bond vibrations at 136 cm-1 indicate that ED3 has been immobilized on modified silica. Furthermore, antibody purification was carried out using 0.5 M NaCl and 1 M NaCl as elution buffer. Although this anti-ED3 IgY antibody purification system still needs to be optimized, it has the potential to be further developed as a method for purifying other antibodies.

Keywords: immobilization, silica, propyldiethylentriamine, glutaraldehyde, expression, ED3 protein, the anti-ED3 IgY antibody

Topic: Biokimia Medis