ELISA for anti-HCV antibody employing a shorter synthetic core region peptide

Nakagiri, Itsuhiro; Ichihara, Kiyoshi

doi:10.1016/0166-0934(94)00164-c

Cited by 8 publications

(4 citation statements)

References 14 publications

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“…Published data show that the 50-70 N-terminal amino acids of the HCV-core protein exhibit the highest antigenicity [Akatsuka et al 1993;Cerino et al, 1993;Nakagiri and Ichihara, 1995;Sallberg et al, 1994]. Some authors suggest that the C-terminal 70-191 aa is relatively insignificant for a humoral immune response [Goeser et al, 1994], while others report on the antigenicity of other core regions [Akatsuka et al, 1993;Sallberg et al, 1994].…”

Section: Discussionmentioning

confidence: 99%

Detection of hepatitis C virus core protein circulating within different virus particle populations

et al. 1998

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Progress in studying pathogenesis and increasing the reliability of hepatitis C diagnosis can be achieved by analysis of different forms of virus particles circulating in blood of both patients and infected persons. Detection of hepatitis C virus (HCV) proteins faces two basic difficulties: low concentration of HCV proteins, and their blocking by antibodies. The aim of this work was to develop a method for the detection of nucleocapsid (core) protein in the plasma of HCV-infected persons using monoclonal antibodies (MABs). Twenty-seven anti-HCV-positive donor plasmas were studied of which 21 contained HCV RNA and 6 were negative. The plasmas were centrifuged for 3 hr at 143,000 g and the antigenic activity of core-protein was studied in the pellets by EIA using four MABs able to recognize four nonoverlapping determinants, two at N-terminus and two at C-terminus of recombinant core (1-150 aa). The determinants detected were present in the natural core protein of at least two genotypes (1b and 3a). Maximal efficiency of recombinant protein detection was achieved with 2 MABs, whereas a combination of 4 MABs was necessary for optimal detection of natural core protein. This is indicative of different conformational structures of natural protein and its gene-engineered analog. The sensitivity of core detection by monoclonal sandwich assay was 1 ng/ml in the pellet or 5 pg/ml after normalization to the initial plasma volume. To dissociate immune complexes, the pellet was treated with 2.5 M KBr after first treating the pellet with the nonionic detergent Tween 80 to remove the virus lipid envelope. Using this treatment protocol, core protein was found in 19 of 21 RNA positive plasmas.

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Section: Discussionmentioning

confidence: 99%

Detection of hepatitis C virus core protein circulating within different virus particle populations

et al. 1998

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“…A marked correlation was found between the detection of IgG anti-HCV core and the presence of serum HCV RNA sequences in chronic hepatitis C patients [Okamoto et al, 1990]. Thus, HCV core is an important target for diagnosis of HCV infection by detecting either specific anticore antibodies or circulating viral antigen [Nakagiri et al, 1995;Park et al, 1995].…”

Section: Introductionmentioning

confidence: 96%

HCV core immunodominant region analysis using mouse monoclonal antibodies and human sera: Characterization of major epitopes useful for antigen detection

et al. 1998

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Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.

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“…The most commonly used method is based on the detection of antigen specific antibodies from serum samples by conventional ELISA assays. This format offers sensitivity, specificity and automation, and progress was made by replacing structural and non-structural viral proteins with shorter recombinant immunodominant domains [6]. A drawback of the ELISA is the lack of parallelity, i.e., sample multiplexing, which renders the method relatively expensive.…”

Section: Introductionmentioning

confidence: 99%

Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics

Andresen

Grötzinger

Zarse

et al. 2006

Sensors and Actuators B: Chemical

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Peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide-NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples.

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ELISA for anti-HCV antibody employing a shorter synthetic core region peptide

Cited by 8 publications

References 14 publications

Detection of hepatitis C virus core protein circulating within different virus particle populations

Detection of hepatitis C virus core protein circulating within different virus particle populations

HCV core immunodominant region analysis using mouse monoclonal antibodies and human sera: Characterization of major epitopes useful for antigen detection

Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics

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