ABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Hepatitis B Antibodies/isolation & purification , Hepatitis B Surface Antigens/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Base Sequence , Binding, Competitive , Blotting, Western , Chromatography, Affinity , Cloning, Molecular , Epitope Mapping , Hepatitis B Antibodies/immunology , Hepatitis B Surface Antigens/chemistry , Hepatitis B Surface Antigens/genetics , Hybridomas , Immunoglobulin M/immunology , Immunoglobulin M/isolation & purification , Immunoglobulin Variable Region/chemistry , Mice , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sequence Homology, Nucleic AcidABSTRACT
Immobilized metal affinity chromatography (IMAC) has been recently applied to the purification of of recombinant proteins bearing multi-histidine domains at their N or C terminus. We have now used this procedure for the single-step purification of an anti-Hepatitis B virus surface antigen (HBsAg) single-chain Fv (scFv) antibody fragment. Adjusting the metal ion (Cu+2 or Ni+2) and elution conditions (pH or imidazole), we efficiently separated active scFv forms from inactive molecules. Achieved purity was 93%, with a 20% yield with respect to the scFv content in the initial material. The pure scFv was coupled to CNBr-activated Sepharose 4B and compared the original monoclonal antibody (MAb) CB-Hep.1 in the immunoaffinity purification of a vaccine recombinant HBsAg (r-HBsAg). Results indicate that eluted antigen per mg of coupled ligand was similar for the scFv and the MAb when pure r-HBsAg was used as starting material. Preliminary results with unpurified starting material are also encouraging.
Subject(s)
Hepatitis B Surface Antigens/immunology , Hepatitis B Surface Antigens/isolation & purification , Immunoglobulin Fragments/isolation & purification , Antibodies, Monoclonal , Copper , Cyanogen Bromide , Enzyme-Linked Immunosorbent Assay , Hydrogen-Ion Concentration , Imidazoles , Immunosorbent Techniques , Nickel , Recombinant ProteinsABSTRACT
Nucleotide sequences for the variable regions of both the heavy and light chains for two human monoclonal antibodies have been determined. Both antibodies are directed against an outer membrane protein of Neisseria meningitidis, and their genes show a low resemblance to germline sequences.
Subject(s)
Antibodies, Bacterial/genetics , Antibodies, Monoclonal/genetics , Genes, Immunoglobulin , Immunoglobulin Variable Region/genetics , Neisseria meningitidis/immunology , Amino Acid Sequence , Base Sequence , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Molecular Sequence DataABSTRACT
Neisseria meningitidis is a causative agent of meningitis. Despite vaccination programs, it still causes a large number of deaths in young children. Early diagnosis followed by passive immunization with human monoclonal antibodies could be an approach to effective therapy. Peripheral blood lymphocytes from normal, healthy blood donors and from vaccinated individuals were immunized in vitro, using outer membrane proteins purified from N. meningitidis B:4:P1.15. The immunized human B cells were Epstein-Barr virus transformed and fused to a heteromyeloma. Several stable human hybridoma cell lines were established and two, secreting antibodies against the 31-kDa class 5c outer membrane protein, were characterized further. The human antibodies were of IgG1 and IgG3 isotypes, with kappa light chains. The recognized epitope was commonly found among pathogenic strains of N. meningitidis; thus, these human monoclonal antibodies may be important in the evaluation of N. meningitidis infections.
