Simultaneous detection of Human Immunodeficiency Virus 1 and Hepatitis B virus infections using a dual-label time-resolved fluorometric assay

Abstract

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.

Lanthanide-based time-resolved luminescence immunoassays

“…)T4–BSA–BTBCT 23 –Eu5.8 nmol L −1 –[42]T4 (comp. )T4–BSA–B, SA–BCTOT–Eu6.7 nmol L −1 –[40]Nanoparticle-basedPSASA-coated Eu(β-NTA) 3 -doped polystyrene NPs1.6 ng L −1 4[56]PSAIgG-coated Eu(β-NTA) 3 -doped polystyrene NPs0.21 ng L −1 4[57]AdenovirusIgG-coated Eu(β-NTA) 3 -doped polystyrene NPs5650000 virus particles L −1 4[58]Hepatitis B surface antigen (HBsAg)IgG-coated Eu(β-NTA) 3 -doped polystyrene NPs28 ng L −1 4[59] Listeria spp.IgG-coated Eu(β-NTA) 3 -doped polystyrene NPs20000 CFU L −1 4[60]Anthrax PASA-coated polystyrene NPs, biotinylated anti-SA, SA–Eu10 ng L −1 4[61]Hepatitis B surface antigen (HBsAg)F(ab) 2 -coated Eu(β-NTA) 3 -doped polystyrene NPs0.013 ng L −1 4[62]Anti-HIV-1 IgGPyridine-based Tb chelate-labeled HIV-1 antigenn.d.–Enzyme-amplified lanthanide luminescenceα-fetoproteinIgG–ALP1.4 ng L −1 >2[67]Salicylphosphate/salicylic acid/Tb–EDTAα-fetoproteinBt–pAb/SA–ALP0.15 ng L −1 >2[68]5-fluorosalicyl phosphate/5-fluorosalicylic acid/Tb–EDTAα-fetoproteinIgG–ALP3 ng L −1 >2[66]Diflunisal phosphate/diflunisal/Tb–EDTAInterleukin 6SA–ALP0.5 ng L −1 (serum)–[69]Diflunisal phosphate/diflunisal/Tb–EDTAPSASA–ALP2 ng L −1 –[70]Diflunisal phosphate/diflunisal/Tb–EDTA10 ng L −1 (serum)comp. = c...…”

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

“…Terbium is an excellent lanthanide in view of multiplexing as it has evenly spread emission bands along the emission spectrum (compared to, for example, europium), enabling the use of multiple acceptors in an energy transfer application (Kokko et al, 2009;Mathis and Bazin, 2011). To name just a few examples, multiplexing has been applied to the simultaneous detection of human immunodeficiency virus and hepatitis B virus infections using Tb 3+ -and Eu 3+ -chelates (Myyryläinen et al, 2010), to the simultaneous detection of three model analytes (prostate-specific antigen, thyroid-stimulating hormone, and luteinizing hormone) using UCP reporters in a microarray format (Päkkilä et al, 2012), and to an ultrasensitive lung cancer immunoassay in human serum using a commercial luminescent terbium complex (Geissler et al, 2012).…”

A verified multiplexed immunoassay for detecting antibodies against infectious pathogens

“…Для такого обследования может быть особо востребована мультиплексная тест-система, обеспечивающая надежное, оперативное и недорогое тестирование [3,8,10]. Попытки создания таких систем включали: микрожидкостные устройства [8]; иммунохроматографические системы с боковым потоком [4]; флюориметрический анализ с двойными метками [9]; суспензионные иммуночипы [15]; микроматрицы, напечатанные на поверхности дна ячеек микротитровального планшета [13]; а также иммуноочипы на плоских слайдах [3]. Однако применение большинства из перечисленных тестов в клинической диагности-ке остается проблематичным из-за сложности процессов их изготовления, значительных затрат и необходимости использования сложных устройств [5,6,10,14].…”