Detection of hepatitis B virus in plasma using flow cytometric analyses of polymerase chain reaction-amplified DNA incorporating digoxigenin-11- dUTP

Yang, Gang; Ulrich, Paul P.; Aiyer, Ramani A.; Rawal, B. D.; Vyas, Girish N.

doi:10.1182/blood.v81.4.1083.1083

Cited by 25 publications

(12 citation statements)

References 11 publications

(1 reference statement)

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“…Another HBV DNA detection system that uses nested PCR and fluorescence detection of the amplicon achieved a detection limit with Eurohep reference plasma sample 1 40 times lower than that of our PCR-coupled ECL assay (13). Although numerous other nonisotopic labels and hybridization formats have been described for the detection of PCR products (8,19,21,31,33), a comparison of these studies is hampered by the fact that standardized calibrators have not yet been used.…”

Section: Discussionmentioning

confidence: 97%

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence

et al. 1996

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We have developed a sensitive and quantitative assay for hepatitis B virus (HBV) DNA in serum or plasma in which PCR and then microtiter hybridization analysis are used. Assay of HBV DNA in serum or plasma is important for demonstrating viral replication, indicating and monitoring antiviral therapy, determining the infectivities of virus carriers, and ensuring the safety of blood products. Under optimum conditions PCR can amplify one HBV DNA molecule to 10 8 copies, but detection of this amount of DNA still requires hybridization with labelled probes or a nested PCR. We labelled one strand of the PCR product with a biotinylated primer. The double-stranded amplicon was incubated in streptavidin-coated microplate wells. The nonlabelled strand was removed after denaturation of the double-stranded DNA with alkali, and the bound strand was hybridized with a peroxidase-coupled single-stranded probe. The amount of bound peroxidase was measured in a luminometer. Four picograms of amplicon was detectable in this system, whereas conventional ethidium bromide staining requires a 1,000 times higher amplicon concentration. The performance of the new assay was compared with those of nested PCR and a PCR system that uses a digoxigenin label, hybridization to a solid-phase adsorbed probe, and colorimetric detection. The chemiluminescence assay was found to be almost as sensitive as nested PCR and approximately five times more sensitive than the colorimetric test.

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Section: Discussionmentioning

confidence: 97%

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence

et al. 1996

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“…It is possible that the method we used for HBV DNA detection was insufficiently sensitive to screen out all potentially infectious donors. Its sensitivity of 60 virions per millilitre of serum is comparable with the results of other investigations (Keneko et af., Yang et al, 1993) but still too low to match the 100 particle threshold of infectivity (Prince et af., 1983). Even with a sensitive method.…”

Section: Discussionmentioning

confidence: 99%

Feasibility and usefulness of an efficient anti‐HBc screening programme in blood donors

Allain

Reeves²,

Kitchen³

et al. 1995

Transfusion Medicine

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Post-transfusion hepatitis B remains a risk for recipients of hepatitis B surface antigen (HBsAg) screened blood. Anti-hepatitis B core antibody (anti-HBc) screening may help reduce this risk. To evaluate its usefulness, 9,238 East Anglian blood donors were screened for anti-HBc. Those with isolated anti-HBc were identified with two confirmatory anti-HBc and anti-HB surface antibody (anti-HBs) assays. The prevalence of anti-HBc reactions in screening and confirmatory assays was 1.29% and 0.35%, respectively. The level of reactivity was significantly higher when two anti-HBc assays gave concordant results or, being concordant, were anti-HBs positive. All isolated anti-HBc-positive units (0.04%) were negative for additional HBV markers including DNA tested with nested polymerase chain reaction (PCR). A 0.31% prevalence of past HBV infection was found in this population, all carrying both anti-HBc and anti-HBs antibody, most above the protective level (0.1 IU/ml). The proposed screening schemes would limit the number of deferred donors and discarded units and keep the testing time within the remit of routine blood banking practices for an additional cost of approximately 1 pound per unit. However, no evidence was found in this donor population to suggest that anti-HBc screening would significantly reduce the incidence of post-transfusion hepatitis B.

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“…To demonstrate the potential of their assay system to detect low concentrations of toxins or soluble antigens, the same authors used IMS to capture FITC‐conjugated rabbit anti‐goat IgG antibodies, which served as a model antigen (21). Other studies describe exclusively the application of paramagnetic beads for the binding of polymerase chain reaction (PCR) ‐amplified viral DNA followed by flow cytometric detection (22, 23).…”

Section: Discussionmentioning

confidence: 99%

Serodiagnosis of human plague by a combination of immunomagnetic separation and flow cytometry

et al. 2003

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Background Plague is a severe, highly communicable bacterial disease caused by Yersinia pestis. It is still endemic in more than 20 countries worldwide. Although known as a devastating disease for centuries, laboratory confirmation of clinical suspected cases is still problematic. No standardized and internationally approved test system is commercially available. The aim of this study was the introduction and evaluation of a combination of immunomagnetic separation and flow cytometry for the serodiagnosis of human plague. Methods Paramagnetic polystyrene beads were coated with purified F1 capsular antigen (F1 CA) and reacted with sera from plague patients, from 26 laboratory personnel vaccinated against plague and from 102 healthy blood donors (HBD). After incubation with fluorescein isothiocyanate–conjugated anti‐human rabbit IgG, particle‐associated fluorescence was detected by flow cytometry. Results Anti‐F1 CA antibodies could be demonstrated in all patients with bacteriologically confirmed plague and in 22 sera (84.6%) from vaccinees. Only one serum in the HBD group showed a weakly positive reaction. The total assay time was less than 2 h. Conclusions Compared with a recently published combination of an anti‐F1 CA enzyme‐linked immunosorbent assay (ELISA) and immunoblot, the new assay showed the same sensitivity as the ELISA and almost the same specificity (99.0 versus 100%) as the immunoblot. Allowing a rapid, reliable, and quantitative analysis, immunomagnetic separation combined with flow cytometry might replace both conventional immunoassays. Cytometry Part A 53A:88–96, 2003. © 2003 Wiley‐Liss, Inc.

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Detection of hepatitis B virus in plasma using flow cytometric analyses of polymerase chain reaction-amplified DNA incorporating digoxigenin-11- dUTP

Cited by 25 publications

References 11 publications

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence

Feasibility and usefulness of an efficient anti‐HBc screening programme in blood donors

Serodiagnosis of human plague by a combination of immunomagnetic separation and flow cytometry

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