Production of Monoclonal Antibody Anti-NS1 Induced by DENV3 Indonesian Clinical Isolate to Detect Native NS1 Antigen of all DENV Serotypes KC05-KC08
Dr. Sabar Pambudi,
Center of Pharmaceutical and Medical Technology, Agency for the Assessment and Application of Technology (BPPT),
Tangerang Selatan, Banten, Indonesia.
Introduction: Dengue Virus (DENV) infection is a mosquito-borne disease, the most severe manifestation of the infection could lead to a lethal Dengue Haemorrhagic Fever (DHF) and/or Dengue Shock Syndrome (DSS). The currently licensed DENV vaccine has been launched in the market with some limitation applications in the field. Therefore, accurate diagnosis system is needed to identify effective treatment of DENV-infected patients and prevent the fatal outcomes caused by the severe complications. Non-Structural-1 (NS1) protein of DENV has been known as a biomarker for dengue diagnosis since the protein is abundantly circulating in the blood during the acute phase of the disease.
Aim: To produce and characterise mouse monoclonal Antibodies (mAbs) anti-DENV NS1 antigen.
Materials and Methods: This was an experimental study. Hybridoma mAb-producing cells were obtained by the fusion between PAI mouse myeloma cells and B cells from an immunized mice with lysed Chinese Hamster Ovary (CHO-K1) cells expressing NS1 protein. Monoclonal antibodies were tested against native NS1 antigen from DENV1, DENV2, DENV3, and DENV4 respectively, by ELISA, IFA and Western Blot analysis.
Results: Hybridoma selection with indirect ELISA showed 16 clones that potentially produce anti-NS1 antibodies. Six out of 16 clones showed positive results by IFA analysis against NS1 transfected CHO-K1 cells. Three mAb clones 4-2D, 4-4F, and 2-7A were able to recognise the specific NS1 antigen for all DENV serotypes and did not react against other DENV proteins. Moreover, clone 4-4F showed dissociation constant (Kd) approximately 1.25+0.47 nM indicating the antibody has a strong binding affinity to NS1 antigen.
Conclusion: The monoclonal antibodies anti-NS1 induced by DENV3 Indonesian clinical isolate are potentially used as the main reagent for development of the DENV NS1 diagnostic test.