Förster resonance energy transfer (FRET) is a phenomenon widely utilized in biomedical research of macromolecular interactions. In FRET energy is transferred between two fluorophores, the donor and the acceptor. Herein we describe a novel approach utilizing time-resolved FRET (TR-FRET) for the detection of antibodies not only in a solution-phase homogenous assay but also in single- and two-step solid-phase assays. Our method is based on the principle that the Y-shaped immunoglobulin G molecule is able to simultaneously bind two identical antigen molecules. Hence, if a specific IgG is mixed with donor- and acceptor-labeled antigens, the binding of antigens can be measured by TR-FRET. Using donor- and acceptor-labeled streptavidins (SAs) in conjunction with a polyclonal and a monoclonal anti-SA antibody we demonstrate that this approach is fully functional. In addition we characterize the immune complexes responsible for the TR-FRET signal using density gradient ultracentrifugation and solid-phase immunoassays. The homogenous TR-FRET assay described provides a rapid and robust tool for antibody detection, with a wide potential in medical diagnostics.

Chelated lanthanides such as europium (Eu) have uniquely long fluorescence emission half-lives permitting their use in time-resolved fluorescence (TRF) assays. In Förster resonance energy transfer (FRET) a donor fluorophore transfers its emission energy to an acceptor fluorophore if in sufficiently close proximity. The use of time-resolved (TR) FRET minimizes the autofluorescence of molecules present in biological samples. In this report, we describe a homogenous immunoassay prototype utilizing TR-FRET for detection of antibodies in solution. The assay is based on labeled protein L, a bacterial protein that binds to immunoglobulin (Ig) light chain, and labeled antigen, which upon association with the same Ig molecule produce a TR-FRET active complex. We show that the approach is functional and can be utilized for both mono- and polyvalent antigens. We also compare the assay performance to that of another homogenous TR-FRET immunoassay reported earlier. This novel assay may have wide utility in infectious disease point-of-care diagnostics.

P arvoviruses (family Parvoviridae) are small (18-to 26-nm), nonenveloped, icosahedral viruses encapsidating a linear single-stranded DNA genome of approximately 5,000 nucleotides (1). Parvovirus B19, currently designated B19V, is a member of the family Parvoviridae, genus Erythrovirus (2). The B19V viral capsid has an icosahedral structure and is comprised of two proteins, VP1 (84 kDa) and VP2 (58 kDa). VP2 is the major protein (95% of the capsid composition) and contains receptor-and coreceptor-binding domains which together with self-assembly domains lead to the formation of highly stable particles (3). The minor capsid protein, VP1, differs from VP2 only in an N-terminal unique region (VP1u) composed of an additional 227 amino acids and has phospholipase A 2 (PLA 2 ) activity (4). VP1u elicits a dominant immune response (5).B19V infection in many instances is subclinical or presents as the mild and self-limiting fifth disease, or erythema infectiosum, typically manifesting as a "slapped cheeks" rash (6). Infection by B19V during pregnancy can result in several serious complications in the foetus, such as anemia, nonimmune hydrops, and ultimately intrauterine death (7). B19V is a potent inhibitor of hematopoiesis because it lytically infects erythroid progenitor cells (8), and in immunodeficient individuals, chronic B19V infection can result in chronic anemia (9). In individuals with underlying chronic hemolytic disorders, transient aplastic crisis following B19V infection can be a life-threatening complication (10). Finally, there is some evidence to suggest that B19V may infect other cell types, suggesting a possible involvement in the pathogenesis of a broad range of medical conditions, including idiopathic arthritis, vasculitis, meningoencephalitis, hepatitis, and myocarditis (11).Initially, the laboratory diagnosis of B19V infection was hampered by the lack of a cell culture system to grow the virus, necessitating the use of diagnostic tests lacking in sensitivity (12). Recently, recombinant DNA technology using prokaryotic (e.g., Escherichia coli) or eukaryotic (e.g., insect cells) expression systems has been applied to produce selected B19V antigens, in particular, VP1 and VP2. Prokaryotically expressed VP1/VP2 antigens undergo denaturation, and the antigen epitopes expressed are linear, whereas eukaryotic expression using baculovirus vectors generates empty capsids that are antigenically analogous to the native virus and include conformational epitopes. Assays using linear and those using conformational B19V VP1/VP2 can produce different results, leading to the general conclusion that conformational epitopes should be used for diagnostic purposes (13). Such assays need to be designed so that the presentation of conformational epitopes is optimized (14). Antibodies to linear VP2 epitopes are found mostly during acute infections and early convalescence, whereas those to conformational epitopes persist

Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11-48K and U11/U12-65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5 0 splice sites (5 0 ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25-50 nt downstream from the target 3 0 splice site (3 0 ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11-35K protein for 3 0 ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5 0 ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5 0 ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.

In this study, we describe a competitive homogeneous immunoassay that makes use of Förster resonance energy transfer (FRET) in rapid detection of pathogen-specific antibodies. The assay principle is based on competition between a monoclonal antibody (MAb) and serum antibodies to a given antigen. In the assay, named competitive FRET immunoassay (CFRET-IA), the FRET signal is induced if MAb carrying a donor label binds to an acceptor-labeled antigen. Specific antibodies in serum compete for antigen binding, resulting in reduced FRET signal. The proof-of-principle for the assay was obtained using donor-labeled Puumala virus nucleocapsid protein (PUUV-N) and acceptor-labeled anti-PUUV-N MAb. The assay was evaluated by analyzing 329 clinical samples comprising 101 from individuals with acute PUUV infection, 42 from individuals with past infection, and 186 from individuals with PUUV-seronegative sera, and the results were compared to those of reference tests. The rapid serodiagnostic test we introduced herein performed with 100% sensitivity and 99% specificity for diagnosing acute hantavirus disease.