Prolonged stability of the plantibody HB-01 directed against the hepatitis B surface antigen in cryo-preserved tobacco leaves.

Since 1983, several recombinant antibodies have been expressed in important agronomic plant species. However, to date no evaluation has been published about prolonged antibody stability within plant tissues under cryo-preservation conditions. This current report presents an approach to the KDEL-plantibody HB-01 (PHB-01) stability in frozen tobacco leaves by presenting scientific evidence about the stability of a plantibody to a prolonged low temperature exposure in this biological source. Results clearly show that the PHB-01 amount is maintained during the storage of tobacco leaves at -20 degrees C for 90 days. The PHB-01 recovery was not affected by any irreversible physical and/or chemical change produced in tobacco leaves after this cryo-preservation time. The amount of total soluble proteins in the clarified extract decreased in proportion with the storage time and the PHB-01 molecules isolated from frozen leaf extracts were highly pure, >95%, according to an SDS-PAGE assessment under reducing conditions. Low temperature exposure of tobacco leaves did not reveal visible changes in frozen leaves, which is essential for the further extractability of proteins. The PHB-01 is stable in tobacco leaves at -20 degrees C during 90 days, which offers the possibility to overcome problems associated with detrimental climate conditions and optimize purification capabilities.

A combinatorial strategy of a new monoclonal ELISA and immunoaffinity chromatography using sodium deoxycholate to increase the recovery of multimeric proteins like r-HBsAg.

In this work, a sandwich monoclonal-based ELISA for quantifying the HBsAg obtained from yeast cells was standardized and validated. The monoclonal antibody employed in this assay reacts uniformly with different molecular isoforms of r-HBsAg. Immunoassay allowed the r-HBsAg quantification in an analytical range 11.9-191.7 ng/mL. Inter- and intra-assay precision variation coefficients were between 0.77-3.43% and 1.95-8.89%, respectively, and the recovery ranged 98.2-100.8%; which confirms its reliability. r-HBsAg is a complex of carbohydrates, proteins and lipids assembled into spherical particles with an average diameter of 24 nm. Many host contaminants accompany this protein during purification process, which can interfere the antigen recognition by the immunoaffinity matrix. To solve this problem, the effect of several detergents in the quantification and purification of r-HBsAg were studied. The addition of the surfactant sodium deoxycholate (NaDoc) at 0.1% in this ELISA improved the recognition and quantification of r-HBsAg by 2.4-fold higher than untreated samples. Similar results were observed in the immunoaffinity chromatography where a 1.5-fold increasing recovery values was shown. The application of NaDoc allows to reduce the inhibitory effect upon the antigen-antibody recognition, increasing the quantification and immunoaffinity chromatography efficiency. This analytical combination could be applied to multimeric proteins like r-HBsAg of HB vaccine.

Comparison of different ligand densities in immunoaffinity chromatography of the plantibody HB-01 coupled to Sepharose CL-4B to purify the rHBsAg.

This paper evaluates the immunopurification behavior of a plantibody HBsAg specific plantibody coupled to Sepharose CL-4B at different ligand densities. Results show no significant differences in the adsorption and elution capacities, and rHBsAg recovery of immunosorbents at 3.43, 4.45, and 5.31 mg/mL of ligand densities compared to its mouse-derived mAb counterpart consistently used in the rHBsAg purification process. Therefore, plantibody ligand densities higher than 3.43 mg/mL do not improve the immunopurification behavior of this immunosorbent, but increase the antibody consumption and the Hepatitis B vaccine cost. Immunosorbent of 2.23 mg/mL of ligand density demonstrated a poor performance. The IgG leached detectable level never exceeded the approved limit (3 ng IgG/microg rHBsAg). Values close to this limit were only observed at the ligand density of 5.31 and 2.27 mg/mL. In the case of the ligand density of 2.23 mg/mL the IgG leached value was high (2.90 ng IgG/microg rHBsAg) due to a low level of eluted antigen. In conclusion, it supports feasibility of using this plantibody at 3.43 mg/mL of ligand density for large-scale immunopurification of rHBsAg for human use, avoiding the biosafety and ethical concerns of the massive use of animals for this purpose.