Generation and characterization of chimeric antibodies against NS3, NS4, NS5, and core antigens of hepatitis C virus.

Mouse-human chimeric antibodies (cAbs) against hepatitis C virus (HCV) core, NS3 (nonstructural), NS4, and NS5 antigens were developed as quality control (QC) reagents to replace the use of human sera/plasma for Abbott HCV immunoassays. The cAb retains the mouse monoclonal antibody (MAb) specificity and affinity but still reacts in the existing HCV assay format, which measures human anti-HCV immunoglobulin. Mouse heavy-chain (V(H)) and light-chain (V(L)) variable regions of anti-HCV core, NS3, NS4, and NS5 antigens were PCR amplified from hybridoma lines and then cloned with human IgG1 heavy-chain (C(H)) and light-chain (C(L)) constant regions, respectively. A single mammalian expression plasmid containing both heavy-chain and light-chain immunoglobulin genes was constructed and transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells. The transfected CHO cells were selected using hypoxanthine- and thymidine-free medium and screened by an enzyme immunoassay (EIA). The clone secreting the highest level of antibody was isolated from the CHO transfectants and further subcloned. Each cAb-expressing CHO cell line was weaned into serum-free medium, and the cAb was purified by protein A affinity chromatography. The levels of cAb production for the various CHO cell lines varied from 10 to 20 mg/liter. Purified anti-HCV cAbs were tested with Abbott HCV immunoassays and showed reactivity. Moreover, yeast surface display combined with alanine-scanning mutagenesis was used to map the epitope at the individual amino acid level. Our results suggest that these HCV cAbs are ideal controls, calibrators, and/or QC reagents for HCV assay standardization.

Interactions of two monoclonal antibodies with BNP: high resolution epitope mapping using fluorescence correlation spectroscopy.

Structure-function studies of antibody-antigen systems include the identification of amino acid residues in the antigen that interact with an antibody and elucidation of their individual contributions to binding affinity. We used fluorescence correlation spectroscopy (FCS) and alanine-scanning mutagenesis to characterize the interactions of brain natriuretic peptide (BNP) with two monoclonal antibodies. Human BNP is a 32 amino acid residue long cyclic polypeptide with the ring structure confined between cysteines in positions 10 and 26. It is an important cardiovascular hormone and a valuable diagnostic cardiac marker. We compare the binding strength of the N-terminus Alexa488-labeled BNP, native cyclic BNP, BNP alanine-substituted mutants, linear BNP, and its short fragments to determine the individual contributions of amino acid residues included in the continuous antigenic epitopes that are recognized by two different monoclonal antibodies raised toward BNP. Implementation of FCS for these studies offers all of the advantages of solution phase measurements, including high sensitivity, simplicity of manipulation with reagents, and elimination of solid phase interferences or separation steps. Significant differences in the molecular masses of the free and antibody bound BNP results in a substantial ( approximately 2.5-times) increase in the diffusion rates. Determination of the binding constants and inhibition effects by measuring the diffusion rates of the ligand at the single molecule level introduces the ultimate opportunity for researching systems where the fluorescence intensity and/or fluorescence anisotropy do not change upon interaction of the ligand with the protein. Monoclonal antibodies 106.3 and BC203 demonstrate high affinities to BNP and bind two distant epitopes forming robust antibody sandwiches. Both antibodies are used in Abbott diagnostic assays on AxSYM, IMx, and Architect platforms.