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1.
Biopolymers ; 93(7): 657-67, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20526991

RESUMO

The recent remarkable rise in biomedical applications of antibodies and their recombinant constructs has shifted the interest in determination of antigenic epitopes in target proteins from the areas of protein science and molecular immunology to the vast fields of modern biotechnology. In this article, we demonstrated that measuring binding induced changes in two-dimensional NMR spectra enables rapid determination of antibody binding footprints on target protein antigens. Such epitopes recognized by six high-affinity monoclonal murine antibodies (mAbs) against human neutrophil gelatinase-associated lipocalin (NGAL) were determined by measuring chemical shifts or broadening of peaks in (1)H-(15)N-TROSY HSQC and (1)H-(13)C HSQC spectra of isotope-labeled NGAL occurring upon its binding to the antibodies. Locations of the epitopes defined by the NMR studies are in good agreement with the results of antibody binding pairing observed by dual-color fluorescence cross-correlation spectroscopy. In all six cases, the antibodies recognize conformational epitopes in regions of relatively rigid structure on the protein. None of the antibodies interact with the more flexible funnel-like opening of the NGAL calyx. All determined epitope areas in NGAL reflect the dimensions of respective antibody binding surface (paratopes) and contain amino acid residues that provide strong interactions. This NMR-based approach offers comprehensive information on antigenic epitopes and can be applied to numerous protein targets of diagnostic or therapeutic interest.


Assuntos
Proteínas de Fase Aguda/química , Anticorpos Monoclonais/química , Antígenos/química , Epitopos/química , Lipocalinas/química , Ressonância Magnética Nuclear Biomolecular , Proteínas Proto-Oncogênicas/química , Proteínas de Fase Aguda/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Epitopos/imunologia , Humanos , Lipocalina-2 , Lipocalinas/imunologia , Camundongos , Proteínas Proto-Oncogênicas/imunologia
2.
Clin Vaccine Immunol ; 17(6): 1040-7, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20427624

RESUMO

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.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas do Core Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Monoclonais Murinos/biossíntese , Anticorpos Monoclonais Murinos/genética , Células CHO , Cricetinae , Cricetulus , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/biossíntese , Anticorpos Anti-Hepatite C/genética , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética
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