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Japanese encephalitis (JE) is a viral disease in human, causes serious inflammation of the brain, which may lead to permanent brain damage. There is no specific treatment for this disease. Vaccination is the only effective option in preventing infection. In Thailand, Govement Pharmaceutical Organization has produced and imported vaccine from JE Beijing 14-14-2 strain. Antigen content is one of the key QC item used in identification and quantitation of the bulk vaccine in the manufacturing process. Preparation of HRP conjugated IgG specific to JE virus often prepares for this in-house assay. ELISA is the most widely used technique to quantify the antigen content. In this study, we aim to optimized the conditions for antibody conjugation. Our results show that pH and molar ratio affect on conjugation efficiency. High pH (9.4) and molar ratio (Ab:HRP;1:0.5) are the most optimal parameters tested in our study. In conclusion, we have successfully identified two parameters that have a great effect to the reaction. Further studies on other parameters are needed to improve more efficient reaction condition.