Expression of VLPs and total HBsAg in plants. a Expression of M-HBsAg in tobacco plants of 6- and 18-months of age, b expression of M-HBsAg in lettuce plants of T0 and T1 generation, c expression of L-HBsAg in tobacco plants of 6- and 18-months of age, d expression of L-HBsAg in lettuce plants of T0 and T1 generation. Plants were divided into three expression groups, depending on total HBsAg content calculated in μg/g of fresh weight (FW): low < 2 μg/g FW, medium 2–10 μg/g FW and high > 10 μg/g FW. Level of both VLP-assembled antigens was determined by monoclonal IMx® diagnostic kit (Abbott). Total contents of the antigens were determined by sandwich ELISA using anti-HBsAg rabbit polyclonal antibody (B65811R, Meridian Life Science) and monoclonal antibodies specific for S, preS2 or preS1 domains (respectively, C86123 M and C8A031 M, Meridian Life Science, and 5a-19, Institut Pasteur). Data show values calculated as the arithmetic mean and standard deviation (SD) from three assays