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.
Leyva
Alberto
A
Process Control Department, Center for Genetic Engineering and Biotechnology, 31st Ave /158 and 190, P.O. Box 6162, Havana 10600, Cuba. alberto.leyva@cigb.edu.cu
Sánchez
Julio C
JC
Valdes
Rodolfo
R
Font
Milagros
M
López
Lissette
L
Hernández
Neyda
N
Ferro
Williams
W
Medina
Yenisley
Y
D000911:Antibodies, Monoclonal / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods*; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D003840:Deoxycholic Acid; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006514:Hepatitis B Surface Antigens / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D017325:Hepatitis B Vaccines; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D003902:Detergents; D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D011994:Recombinant Proteins; D003840:Deoxycholic Acid