Antigen recognition characteristics and comparative performance in immunoaffinity purification of two monoclonal antibodies specific for the hepatitis B virus surface antigen.
In this paper we describe the antigen recognition characteristics, variable region base and amino acid sequence, and performance as immunoaffinity chromatography ligands of two MAb specific to the alpha determinant of the HBsAg, derived from the same fusion. We show that the epitope recognized by CB-Hep.0 (IgM) is probably associated to an intrachain disulfide bond in the antigen. On the other hand, CB-Hep.1 (IgG2b) recognizes a heat-resistant non-conformation dependent antigenic determinant on HBsAg. PCR-cloning and sequencing of the variable regions of these two MAb indicated that both heavy chain variable regions were originated from the usage of the same germinal V and J genes. However, the outstanding differences in the size of the VH CDR3, and the absolute difference in the light chain sequences, suggest that the hybridomas were originated from different precursor B lymphocytes. With respect to their use as immunoaffinity chromatography ligands for the purification of a recombinant HBsAg, we found that the IgM immunogel exhibited increased performance with respect to amount of eluted antigen, and final recovery. This difference in overall performance could be attributed to a series of factors: the higher valence number of IgM, a dissimilar distribution of IgM and IgG in the activated gel particles, and differences in antigen recognition between both MAb. Our results suggest that IgM antibodies may be useful in immunopurification, particularly if the antigen is structurally complex and has a high density of repeating epitopes.
Fernández de Cossío
M E
ME
Division of Immunotechnology and Diagnostics, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.
Díaz
T
T
Galván
A
A
Valdéz
R
R
González
E
E
Ayala
M
M
Díaz
J
J
Bestagno
M
M
Burrone
O
O
Gavilondo
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018604:Epitope Mapping; D006510:Hepatitis B Antibodies / Q000276:immunology / Q000302:isolation & purification*; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006825:Hybridomas; D007075:Immunoglobulin M / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D011994:Recombinant Proteins