Establishment of anti-HBcAg Monoclonal Antibodies for Sandwich ELISA Application by Iliac Method Utilizing Incomplete Adjuvant

Endah Puji Septisetyani, Nina Herlina, Arizah Kusumawati, Pekik Wiji Prasetyaningrum, Anika Prastyowati, Adi Santoso, Apon Zaenal Mustopa


The prevalence of hepatitis B virus (HBV) infection in Indonesia was moderate in 2013. This makes an appropriate action has to be taken immediately. In an attempt to evaluate our candidate HBV vaccine efficacy, we generated monoclonal antibodies specific to HBcAg for sandwich ELISA application for research purposes. Monoclonal antibodies are generally developed using hybridoma technology by isolating B cells from spleen or lymph nodes followed by fusion with myeloma cells. This study generated hybridoma by modifying the iliac lymph node method and used incomplete Freund’s adjuvant to emulsify the antigen. For this purpose, an eleven-week-old BALB/c mouse was immunized with a single shot recombinant HBcAg water in oil emulsion intramuscularly at the mouse tail base. After one month, besides enlargement of the medial iliac lymph nodes, we also found that the sub-iliac lymph nodes were enlarged. In two different fusion attempts, the single B cells were then isolated from each lymph node and fused with SP2/0—Ag14 mouse myeloma cells. Hybridoma cells derived from both lymph nodes were screened by ELISA coated with HBcAg peptide. As a result, we obtained two positive wells of hybridoma polyclones from each medial iliac and sub-iliac-derived B lymphocyte. After monoclonalization, we obtained candidate anti-HBcAg monoclonal antibodies as either capture or detection antibodies. Furthermore, we successfully retrieved hybridoma clones 9.3, 9.4, and 9.5, which could produce monoclonal antibodies for sandwich ELISA application.


HBcAg; iliac method; incomplete adjuvant; monoclonal antibodies; sandwich ELISA.

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