title: Standardization and validation of an alkaline phosphatase-linked immunoassay to quantify a plant-derived antibody directed against the hepatitis B virus surface antigen.
abstract: Immunopurification is one of the most effective chromatography steps to purify the hepatitis B surface antigen, which have successfully been used as an active pharmaceutical ingredient of hepatitis B vaccines. Plant-derived antibodies could be an appropriated ligand for such purposes because plants are the most cost-effective production systems and have the additional advantage that plant viruses cannot infect humans. In this work, a polyclonal antibody alkaline phosphatase-linked immunoassay was standardized and validated to quantify a plant-derived antibody directed against the HBsAg. The validation of an immunoassay to quantify plantibodies is a relatively complex task due to the complexity of the plant extract, the low level of expression of this molecule, and the potential interferences of endogenous peroxidases contributed by plants. These results allow estimating the plant-derived antibody concentration up to 3.81 ng/mL with high specificity, precision, and repeatability. The working range of the standard curve was between 3.81 and 60 ng/mL, and the intra- and inter-variation coefficients were between 10% and 20% in a production process's sample dependent way. This enzyme-linked immunosorbent assay is considered valuable to improve the design of the purification process and also to obtain a better estimation of the antibody expression level and process's recovery.
authors.affiliation: Process Control Department, Center for Genetic Engineering and Biotechnology, Ave 31 be/ 158 and 190, Box 6162, Havana 106000, Cuba.
authors.forename: Alberto
authors.initials: A
authors.lastname: Leyva
authors.forename: Neyda
authors.initials: N
authors.lastname: Hernández
authors.forename: Tatiana
authors.initials: T
authors.lastname: González
authors.forename: Julio C
authors.initials: JC
authors.lastname: Sánchez
authors.forename: Abrisleida
authors.initials: A
authors.lastname: Franco
authors.forename: Iliana
authors.initials: I
authors.lastname: Delgado
authors.forename: Margela
authors.initials: M
authors.lastname: Montañez
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Valdés
mesh_terms: D000469:Alkaline Phosphatase; D002138:Calibration / Q000592:standards; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006510:Hepatitis B Antibodies / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D010936:Plant Extracts / Q000032:analysis*; D010940:Plant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D006510:Hepatitis B Antibodies; D010936:Plant Extracts; D010940:Plant Proteins; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase