SYNTHESIS OF HUMAN IGG ANTIBODY AGAINTS HBSAG SUBTYPE ADR IN CHINESE HAMSTER LUNG (CHL-YN) CELLS

• Main Author: Diva Hakim, Meutia
• Format: Theses
• Language: Indonesia
• Subjects: Teknologi
• Online Access: https://digilib.itb.ac.id/gdl/view/55146
• Institution: Institut Teknologi Bandung

Hepatitis B is a disease caused by infection with the hepatitis B virus (HBV). HBV infection can cause acute and chronic illness. Chronic infection from HBV can cause other serious diseases such as cirrhosis and liver cancer. To prevent hepatitis B progression, prevention and treatment is very important. One of the treatments for hepatitis B that can be done is by using therapeutic antibodies, namely Hepatitis B Immunoglobulin (HBIG). In order to maximize protection from hepatitis B reinfection after liver transplantation, the patients are treated with HBIG daily for one week. HBIG treatment is also carried out together with the hepatitis B vaccine for infants born from the hepatitis B positive mothers. In addition, HBIG is also known to be effective for HBV patients infected through sexual contact with someone who is hepatitis B positive. HBIG is usually isolated from hepatitis B positive donor serum with high production standards, which causes high production costs. Therefore, it is necessary to find an alternative production of these therapeutic antibodies. Mammalian cells are commonly used for the production of recombinant proteins approved by the FDA. Therefore, this study aims to construct plasmid that will be inserted by human IgG antibody encoded gene and to synthesize the IgG1 human monoclonal antibody in vitro using CHL-YN cells. DNA sequences for heavy chain and light chain from monoclonal antibody against HBsAg can be accessed on GenBank with access numbers JF700211 and JF970210. Gene encoding heavy chain of antibody with size of 1422 bp was inserted in the pEHX1.2 plasmid and the gene encoding light gene with size of 729 bp was inserted in the pELX2.2 plasmid. Furthermore, both of plasmids are combined to produce the pELC2 + HC plasmid which is inserted with the heavy chain and light chain encoding gene. The pELC2 + HC?containing gene encoding heavy and light chain plasmid that were successfully constructed were amplified in E. coli cultured with 100 ml of liquid LB medium. Prior to transfection, endotoxin in the pELC2 + HC? containing gene encoding heavy and light chains plasmid was eliminated using the MiraCLEAN® Endotoxin Removal kit. pELC2 + HC? containing gene encoding heavy and light chains plasmid were transiently transfected by electroporation method into CHL-YN cells. 1 µg plasmid was transfected into 1 x 106 CHL-YN cells cultured in 6 well plate. As mock control, cell was transfected with only plasmid without gene insertion. Human IgG antibodies were successfully expressed and secreted on transfected CHL-YN cell culture medium. The results of Western blotting showed the presence of two bands with size of ±55 kDa (antibody heavy chain) and ±25 kDa (antibody light chain) in culture medium of transfected CHL-YN cells taken at 48 and 72 hours after transfection process. ELISA test results showed that the human IgG antibody produced had an affinity or ability to recognize and bind to HBsAg subtype adr. This was indicated by the absorbance value in all samples which was higher than mock (transfected with plasmid without gene insertion) and negative control (not transfected) at 48 and 72 hours after transfection process with absorbance values respectively (48 hour: 0,514; 0,531; 0,611; 0,649) and (72 hour: 0,789; 0,815; 0,721; 0,789). Based on these results, it can be concluded that plasmid was successfully constructed and human IgG antibodies that recognize HBsAg were succesfully developed in vitro by CHL-YN cells.