ABSTRACT
The aim of this study is to develop a novel method for the accurate diagnosis of the infection status of viral diseases, which requires discriminated and quantitative detection of different anti-virus immunoglubulin subtypes. Considering hepatitis A as a representative model disease, viral antigen nanoparticles (vAgNPs) were designed and synthesized by genetically presenting hepatitis A virus (HAV) antigens on the surface of human heavy chain ferritin (hFTH) nanoparticles to detect anti-HAV antibodies with discriminating immunoglobulin subtypes M and G (IgM and IgG, respectively). The vAgNPs also display multi-copies of hexa-histidine peptide (H6) on their surface to chemisorb gold ions (Au3+), which is vital for the autonomous generation of quantitatively meaningful detection signals. The quantitative level of anti-HAV IgM or IgG in 30 patient sera was successfully analyzed using the vAgNPs of HAV, which was performed through label-free one-step-immunoassay based on the self-enhancement of optical signals from gold nanoparticles clustered on the viral antigen nanoparticles. The diagnostic performance was compared with that of enzyme-linked immunosorbent assay (ELISA), which did not enable accurate quantitative assay due to the poor linearity between the antibody concentration and detection signal. Furthermore, these vAgNP-based immunoassays did not produce any false negative/positive signals, indicating 100% sensitivity and 100% specificity.
Subject(s)
Gold/chemistry , Hepatitis A Antibodies/blood , Hepatitis A Antigens/chemistry , Hepatitis A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Metal Nanoparticles/chemistry , Apoferritins/chemistry , Enzyme-Linked Immunosorbent Assay , Humans , Sensitivity and SpecificityABSTRACT
Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine.
Subject(s)
Epitopes/immunology , Hepatitis A Antigens/immunology , Hepatitis A virus/immunology , Hepatitis A/diagnosis , Hepatitis A/immunology , Recombinant Proteins/immunology , Viral Hepatitis Vaccines/immunology , Amino Acid Sequence , Enzyme-Linked Immunosorbent Assay , Gene Expression , Gene Order , Hepatitis A Antigens/chemistry , Hepatitis A Antigens/genetics , Hepatitis A Antigens/isolation & purification , Hepatitis A virus/genetics , Hepatitis Antibodies/blood , Hepatitis Antibodies/immunology , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Plasmids/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
A phage-displayed peptide approach was used to identify ligands mimicking antigenic determinants of hepatitis A virus (HAV) for the first time. Bacteriophages displaying HAV mimotopes were isolated from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Selected phage-peptides were screened for reactivity with sera from HAV infected patients and healthy controls. Four cloned peptides with different sequences were identified as mimotopes of HAV; three of them showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV. One clone was recognised by 92% of the positive sera. The phagotopes competed effectively with HAV for absorption of anti-HAV-specific antibodies in human sera, as determined by ELISA. The four phage clones induced neutralising anti-HAV antibodies in immunised mice. These results demonstrate the potential of this method to elucidate the disease related epitopes of HAV and to use these mimotopes in diagnostic applications or in the development of a mimotope-based hepatitis A vaccine without the necessity of manipulation of the virus.
