Tatiana González

Novel and sensitive ELISA for the rapid quantification of recombinant p64K protein

The antigenic P64k protein from the pathogenic bacterium Neisseria meningitidis has been used as an immunological carrier in several conjugated vaccines. The aim of this report was to develop and validate a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of recombinant p64k protein, to perform both manufacturing process and identification in different vaccine preparations. Validation studies were performed according to the guidelines of the International Conference of Harmonization (ICH). The reference curve showed to be precise and accurate over the entire linear range of 1.25 and 20ng/mL with a limit of quantification validated to 1.25ng/mL. The intra- and inter-assay coefficient of variation ranged from 0.35 to 6.65% and 4.70 to 10.63%, respectively. The ANOVA test used in the specificity/interference study revealed parallelism among curves (p>0.1), which indicates the lack of interference in the working range. Recovery obtained from the accuracy test, using three concentration levels, varied between 94 and 111%, confirming the assays reliability. The short-term study shown the P64k is stable to -20°C up to 1-week. This ELISA was fully used to assess its manufacturing process and molecular interaction issues in several vaccine preparations. Thus, this immunoassay could be an excellent analytical choice to characterize the quality of that recombinant protein in several contexts as manufacturing process and molecular conjugates.

Production of a monoclonal antibody by ascites, hollow fiber system, and transgenic plants for vaccine production using CB.Hep-1 mAb as a study case

Monoclonal antibody (mAb) production methods (ascites, in vitro technologies, transgenic animals, and dicot or monocot transgenic plants; moss, algae) have been improved since they were first developed in 1975. In this study, we illustrate a summary of a study case in which mice, a hollow fiber system, and tobacco transgenic plants were assessed for the production of mAb for vaccine manufacturing and vaccine production.

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.