Simultaneous detection of Human Immunodeficiency Virus 1 and Hepatitis B virus infections using a dual-label time-resolved fluorometric assay
- Tiina Myyryläinen†1,
- Sheikh M Talha†2,
- Sathyamangalam Swaminathan2,
- Raija Vainionpää3,
- Tero Soukka1,
- Navin Khanna2 and
- Kim Pettersson1Email author
© Myyryläinen et al; licensee BioMed Central Ltd. 2010
Received: 11 August 2010
Accepted: 26 November 2010
Published: 26 November 2010
A highly specific and novel dual-label time-resolved immunofluorometric assay was developed exploiting the unique emission wavelengths of the intrinsically fluorescent terbium (Tb3+) and europium (Eu3+) tracers for the simultaneous detection of human immunodeficiency virus 1 (HIV-1) and hepatitis B virus (HBV) infections, respectively. HIV-1 infection was detected using a double antigen sandwich format wherein anti-HIV-1 antibodies were captured using an in vivo biotinylated version of a chimeric HIV-1 antigen and revealed using the same antigen labeled with Tb3+ chelate. Hepatitis B surface antigen (HBsAg), which served as the marker of HBV infection, was detected in a double antibody sandwich using two monoclonal antibodies (mAbs), one chemically biotinylated to capture, and the other labeled with Eu3+ nanoparticles, to reveal. The performance of the assay was evaluated using a collection (n = 60) of in-house and commercially available human sera panels. This evaluation showed the dual-label assay to possess high degrees of specificity and sensitivity, comparable to those of commercially available, single analyte-specific kits for the detection of HBsAg antigen and anti-HIV antibodies. This work demonstrates the feasibility of developing a potentially time- and resource-saving multiplex assay for screening serum samples for multiple infections in a blood bank setting.
The World Health Organization recommends screening for infections by human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Treponema pallidum (syphilis) for the provision of a safe blood supply . Currently these infections are detected using independent tests. In a step towards a multiplex assay for blood bank screening, we have explored the feasibility of developing an integrated dual-label assay designed to identify infections by HIV and HBV.
Evaluation of the dual-label TRF assay for simultaneous detection of HIV-1 and HBV infections
In-house sera panel
BBI co-infection panel
To examine the performance of the dual-label assay in the background of other infections, we tested it against a BBI viral co-infection panel PCA 201 (from Boston Biomedica Inc., now SeraCare Life Sciences Inc., Milford MA). This panel was characterized for HIV-1, HBV, HCV and HTLV infections using standard commercially available reference tests (see Additional file 1: Table S1). Twenty-three of the 25 panel members were available for this study. One member of this panel was seronegative for both HIV-1 and HBV infections (sample# 24). The dual-label assay identified this correctly as HIV-/HBV-. Of the remaining 22 serum samples, 16 and 19 samples were designated as HIV+ and HBV+, respectively, with 13 samples seropositive for both HIV-1 and HBV (Table 1). Out of these 13 HIV+/HBV+ serum samples, 6 were positive for HCV, and two for HTLV as well. The remaining three HIV+ serum samples were negative for HBV but positive for HCV and HTLV. The dual-label assay could identify 16 out of 16 HIV+ serum samples (100%). It is noteworthy that one borderline serum sample (sample# 20, S/Co ratio = 1.1) was also picked up unambiguously by the dual-label test (S/Co ratio = 14.8). This essentially is indicative of enhanced sensitivity of the dual-label test and is in agreement with the conclusions based on Figure 3B. Our data show that the dual-label assay is capable of identifying HIV+ serum samples regardless of the presence or absence of HBV, HCV and HTLV co-infections, with high sensitivity and specificity. However, it is to be noted that we have used r-HIV-1env as the antigen to detect anti-HIV-1 antibodies to obtain a technical proof-of-concept. Of the 19 HBV+ serum samples, 13 samples were also HIV+, as mentioned already, and the rest (n = 6) were HIV-. Many of these serum samples were co-infected with HCV, HTLV or both. The dual-label assay identified 16 of the 19 HBV+ serum samples, regardless of HIV, HCV or HTLV infection status. Of the 3 HBV+ serum samples that were missed by the dual-label assay, one was a borderline sample (sample# 9, S/Co ratio = 1). As with the in-house serum samples, these 3 members of panel PCA 201 also turned out to be false-negative in the single label HBV assay. This rules out the possibility that Tb3+ cross-talk may have masked Eu3+ signals and interfered with HBsAg detection. The data show that the concordance of the dual-label assay with regard to HBsAg detection using the reference assay (Abbott EIA) is 84%. This presumably stems from low sensitivity of the mAbs used for detection of HBsAg, despite the use of a tracer F(ab)2-Eu3+ nanoparticle for the detection of this analyte in the dual-label assay.
In conclusion, we have developed a lanthanide fluorescent reporter-based dual-label assay for the simultaneous detection of HIV-1 and HBV infections in donated blood samples. The high sensitivity of this approach derives from the temporal resolution of the long lifetime high intensity fluorescence of Eu3+ and Tb3+ lanthanide tracers measured by TRF. Qdots have emerged recently as highly efficient fluorescent probes. However, these have short-lived fluorescence. Therefore, TRF cannot be employed to measure their signals and their detection is limited by autofluorecence. Further, the Eu3+ and Tb3+ tracers used in this study are inherently fluorescent, obviating the need for additional signal development steps as in the DELFIA and LANFIA methods [2–5], and can be measured directly from the dry surface of the microtiter wells. The simultaneous detection of two analytes combined with a relatively simpler assay format eliminating the extra signal development step, will contribute to both cost and time saving.
To our knowledge, this work, which represents the first report of a dual-label HIV/HBV assay, demonstrates in principle, the feasibility of developing a multiplex assay for screening samples for multiple infections in a blood bank setting. However, a limitation is the potential for interference among the reporters, as illustrated by the Tb3+ cross-talk in Eu3+ measurements in this study. It may be a challenging task to eliminate the interference arising out of cross-talk among multiple fluorescent reporters. One way of circumventing the cross-talk problem in multiplexing would be to design an array-in well strategy in which multiple analyte-capturing reagents are spatially isolated in the same well and used in conjunction with a single reporter, for example Eu3+ nanoparticles. In this set up, analytes can be identified based on the specific locations from which signals are detected.
This work was supported by grants from the Department of Biotechnology, Government of India and Academy of Finland (grant #115524) under a joint Indo-Finnish collaborative research programme. SMT was the recipient of a research fellowship from the University Grants Commission, Government of India.
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