The use of antibodies for characterization and quantification of a recombinant protein.
In this study, we characterized proteinase A secreted by recombinant Saccharomyces cerevisiae bearing a multicopy plasmid containing the encoding gene (PEP4). Polyclonal and monoclonal antibodies were raised to study the product heterogeneity. Characterization of proteinase A was performed by immunoelectrophoresis and immunoblotting techniques. None of the monoclonal antibodies raised against proteinase A was found to react with the glycosyl side chains; thus cross-reaction with other glycosylated proteins (e.g. carboxypeptidase Y) was very low. This study allowed us to develop an ELISA method for the quantification of proteinase A in culture supernatants as well as the evaluation of monoclonal antibodies for their use in immunoaffinity chromatography.
Sabater
M
M
Department of Biotechnology, Technical University of Denmark, Lyngby, Denmark.
Heilmann
S
S
Frøkiaer
H
H
Biedermann
K
K
Emborg
C
C
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D016282:Aspartic Acid Endopeptidases / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods; D003429:Cross Reactions; D003488:Cyanogen Bromide; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005800:Genes, Fungal; D006031:Glycosylation; D015151:Immunoblotting; D007123:Immunoelectrophoresis, Two-Dimensional; D007525:Isoelectric Focusing; D051379:Mice; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics; D014305:Triose-Phosphate Isomerase / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry
D000906:Antibodies; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C022442:aspergillopepsin II; D014305:Triose-Phosphate Isomerase; D003488:Cyanogen Bromide