Evaluation of an Antigen-Antibody “Combination” Enzyme Linked Immunosorbent Assay for Diagnosis of Hepatitis C Virus Infections

Background: Development of “combination” assays detecting in parallel, within a single test, Hepatitis C Virus (HCV) antigens and antibodies, not only reduces the window period in HCV-infection but also costs. Reduction of costs is important for developing countries where money and personal resources are limited.Methods: We compared the Monolisa® HCV Antigen-Antibody Ultra (Bio-Rad Laboratories Limited, Marnes La Coquette, France) with the AXSYM HCV version 3.0 (Abbot Diagnostics, Germany)-the latter assay detecting only antibodies to HCV. Seventy three HCV-PCR positive and negative samples were tested.Results: Although the two assays showed comparable results, two samples from a bone marrow transplant (BMT) patient of viral loads 7.8 x 105 and 8.9 x 106 IU/mL could not be detected by the Monolisa® HCV Antigen-Antibody Ultra assay. Failure to detect the two samples with viral loads considered above threshold of detection for antigen proteins suggested a lack of sensitivity by this assay to discover viral capsid protein in patient samples. Genotyping of these samples revealed genotype 1b, a HCV-subtype which is widespread and should thus be easily detected.Conclusion: We conclude that although this assay depicts high sensitivity and specificity in detecting antibodies to HCV, it seems not to add further benefit in our study population to detect HCV infections by enhanced sensitivity due the potential contingency to trace viral capsid antigens.Keywords: Ag-Ab Combination assay, Hepatitis C Virus, ELISA, Monolisa HCV Ag-Ab Ultr

Rapid enzymatic test for phenotypic HIV protease drug resistance

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid onestep procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIVinfected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for highthroughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (K-i). The Michaelis constant (K-m) and the inhibition constant (K-i) of recombinant wildtype protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIVpositive patients scored from sensitive to highly resistant according to mutations detected by genotypic analysis. The measured K-i values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The noninfectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays

Rapid enzymatic test for phenotypic HIV protease drug resistance

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid onestep procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIVinfected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for highthroughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (K-i). The Michaelis constant (K-m) and the inhibition constant (K-i) of recombinant wildtype protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIVpositive patients scored from sensitive to highly resistant according to mutations detected by genotypic analysis. The measured K-i values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The noninfectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays

A systematic approach for the identification of novel, serologically reactive recombinant Varicella-Zoster Virus (VZV) antigens

<p>Abstract</p> <p>Background</p> <p>Varicella-Zoster virus causes chickenpox upon primary infection and shingles after reactivation. Currently available serological tests to detect VZV-specific antibodies are exclusively based on antigens derived from VZV-infected cells.</p> <p>Results</p> <p>We present a systematic approach for the identification of novel, serologically reactive VZV antigens. Therefore, all VZV open reading frames were cloned into a bacterial expression vector and checked for small scale recombinant protein expression. Serum profiling experiments using purified VZV proteins and clinically defined sera in a microarray revealed 5 putative antigens (ORFs 1, 4, 14, 49, and 68). These were rearranged in line format and validated with pre-characterized sera.</p> <p>Conclusions</p> <p>The line assay confirmed the seroreactivity of the identified antigens and revealed its suitability for VZV serodiagnostics comparable to commercially available VZV-ELISA. Recombinant ORF68 (gE) proved to be an antigen for high-confidence determination of VZV serostatus. Furthermore, our data suggest that a serological differentiation between chickenpox and herpes zoster may be possible by analysis of the IgM-portfolio against individual viral antigens.</p

HCMV Spread and Cell Tropism are Determined by Distinct Virus Populations

Human cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells

Evaluation of an Antigen-Antibody “Combination” Enzyme Linked Immunosorbent Assay for Diagnosis of Hepatitis C Virus Infections

Background: Development of “combination” assays detecting in parallel, within a single test, Hepatitis C Virus (HCV) antigens and antibodies, not only reduces the window period in HCV-infection but also costs. Reduction of costs is important for developing countries where money and personal resources are limited.Methods: We compared the Monolisa® HCV Antigen-Antibody Ultra (Bio-Rad Laboratories Limited, Marnes La Coquette, France) with the AXSYM HCV version 3.0 (Abbot Diagnostics, Germany)-the latter assay detecting only antibodies to HCV. Seventy three HCV-PCR positive and negative samples were tested.Results: Although the two assays showed comparable results, two samples from a bone marrow transplant (BMT) patient of viral loads 7.8 x 105 and 8.9 x 106 IU/mL could not be detected by the Monolisa® HCV Antigen-Antibody Ultra assay. Failure to detect the two samples with viral loads considered above threshold of detection for antigen proteins suggested a lack of sensitivity by this assay to discover viral capsid protein in patient samples. Genotyping of these samples revealed genotype 1b, a HCV-subtype which is widespread and should thus be easily detected.Conclusion: We conclude that although this assay depicts high sensitivity and specificity in detecting antibodies to HCV, it seems not to add further benefit in our study population to detect HCV infections by enhanced sensitivity due the potential contingency to trace viral capsid antigens.Keywords: Ag-Ab Combination assay, Hepatitis C Virus, ELISA, Monolisa HCV Ag-Ab Ultr