Immunomagnetic quantitative immuno-PCR for detection of less than one HIV-1 virion

Barletta, Janet; Bartolome, Amelita; Constantine, Niel T.

doi:10.1016/j.jviromet.2008.12.013

Cited by 52 publications

(39 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The background signal has been attributed to nonspecific binding of the oligonucleotide-labeled secondary antibody, similar to the results observed for other immunodiagnostics (31). Although a number of approaches have been proposed to minimize the level of background amplification (32)(33)(34), no approach to date has proven successful at completely eliminating the background signal. For diagnosing Lyme disease, we propose that the iPCR background signal provides an intrinsic advantage due to the fact that a positive result is a relative measure above the established background threshold, thereby limiting the potential contribution of contamination, whereas a positive result for standard PCR is an absolute measure that can be highly sensitive to low-level laboratory contamination (7).…”

Section: Discussionsupporting

confidence: 56%

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

Halpern

Molins

Schriefer

et al. 2014

Clin. Vaccine Immunol.

View full text Add to dashboard Cite

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n ‫؍‬ 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n ‫؍‬ 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n ‫؍‬ 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n ‫؍‬ 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting hostgenerated antibodies against B. burgdorferi.

show abstract

Section: Discussionsupporting

confidence: 56%

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

Halpern

Molins

Schriefer

et al. 2014

Clin. Vaccine Immunol.

View full text Add to dashboard Cite

show abstract

“…Similar results were reported by Kwon et al (14), who evaluated fourth-generation (combination antigen-antibody) assays against the same panel of isolates. Furthermore, several anti-p24 antibodies that showed strong reactivity with the subtype B virus in our study have been reported to efficiently capture p24 in different assays (2,3,17), providing a foundation for the application of current HIV subtype B-specific antibodies in HIV diagnosis. However, variations in p24 monoclonal antibody reactivity among different subtypes have also been reported (31).…”

Section: Discussionmentioning

confidence: 68%

Characterization of Immune Responses to Capsid Protein p24 of Human Immunodeficiency Virus Type 1 and Implications for Detection

Tang

Zhao

Wang

et al. 2010

Clin Vaccine Immunol

View full text Add to dashboard Cite

To further refine our current nanoparticle-based HIV-1 p24 antigen assay, we investigated immune responses to p24 to identify diagnostically significant immune dominant epitopes (IDEs) in HIV-infected human sera, to address cross-reactivity of anti-p24 antibodies to different subtypes, and to identify new biomarkers that distinguish acute from chronic HIV infection for more accurate incidence estimation. We

show abstract

“…The total plasmid content of each inoculum was confirmed to be as expected (20). Whole-blood samples were collected from the three inoculated mice as well as one noninoculated mouse by submandibular bleed preinoculation and at days 1,3,4,7,9,11,14,16,18, and 21 postinfection. The coagulated blood was spun at 4,000 ϫ g for 9 min to prepare serum.…”

Section: Methodsmentioning

confidence: 95%

“…Using iPCR, a typical 100-to 10,000-fold improvement over the detection limit of the EIA has been obtained in almost all applications (8). iPCR has been used to detect viral antigens (9), bacterial antigens (10), prions (11), and bacterial toxins (12). There has also been a limited application of iPCR for antibody detection, such as the measurement of mumps virusspecific immunoglobulin G in human serum (13).…”

mentioning

confidence: 99%

Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR

Halpern¹,

Jain²,

Jewett³

2013

Clin. Vaccine Immunol.

View full text Add to dashboard Cite

Lyme disease is the fastest-growing zoonotic disease in North America. Current methods for detection of Borrelia burgdorferi infection are challenged by analysis subjectivity and standardization of antigen source. In the present study, we developed an immuno-PCR (iPCR)-based approach employing recombinant in vivo-expressed B. burgdorferi antigens for objective detection of a host immune response to B. burgdorferi infection. iPCR is a liquid-phase protein detection method that combines the sensitivity of PCR with the specificity and versatility of immunoassay-based protocols. Use of magnetic beads coated with intact spirochetes provided effective antigen presentation and allowed detection of host-generated antibodies in experimentally infected mice at day 11 postinoculation, whereas host-generated antibodies were detected at day 14 by enzyme-linked immunosorbent assay (ELISA) and day 21 by immunoblotting. Furthermore, magnetic beads coated with recombinant B. burgdorferi in vivoexpressed antigen OspC or BmpA demonstrated positive detection of host-generated antibodies in mice at day 7 postinoculation with markedly increased iPCR signals above the background, with the quantification cycle (C q ) value for each sample minus the mean background C q plus 3 standard deviations (⌬C q ) being 4 to 10, whereas ⌬C q was 2.5 for intact spirochete-coated beads. iPCR demonstrated a strong correlation (Spearman rank correlation ‫؍‬ 0.895, P < 0.0001) with a commercial ELISA for detection of host antibodies in human Lyme disease patient sera using the B. burgdorferi VlsE C6 peptide. In addition, iPCR showed potential applicability for direct detection of spirochetes in blood. The results presented here indicate that our iPCR assay has the potential to provide an objective format that can be used for sensitive detection of multiple host response antibodies and isotypes to B. burgdorferi infection.

show abstract

Immunomagnetic quantitative immuno-PCR for detection of less than one HIV-1 virion

Cited by 52 publications

References 47 publications

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

Characterization of Immune Responses to Capsid Protein p24 of Human Immunodeficiency Virus Type 1 and Implications for Detection

Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR

Contact Info

Product

Resources

About