Microsphere-Based IgM and IgG Avidity Assays for Human Parvovirus B19, Human Cytomegalovirus, and Toxoplasma gondii

Human parvovirus B19 (here B19), human cytomegalovirus (HCMV), and Toxoplasma gondii infections during pregnancy can lead to severe complications. While traditional diagnosis of infections is mostly confined to one pathogen at a time, a multiplex array is a feasible alternative to improve diagnostic management and cost-efficiency. In the present study, for these three pathogens, we developed microsphere-based suspension immunoassays (SIAs) in multiplex and monoplex formats for the detection of antimicrobial IgM antibodies as well as corresponding chaotrope-based IgG avidity SIAs. We determined the diagnostic performances of the SIAs versus in-house and commercial reference assays using a panel of 318 serum samples from well-characterized clinical cohorts. All the newly developed assays exhibited excellent performance compared to the corresponding high-quality reference methods. The positive and negative percent agreements of the IgM SIAs in comparison with reference methods were 95 to 100% and 98 to 100%, and those of the IgG avidity SIAs were 92 to 100% and 95 to 100%, respectively. Kappa efficiency values between the SIAs and the corresponding reference assays were 0.91 to 1. Furthermore, with another panel comprising 391 clinical samples from individuals with primary infection by B19, HCMV, or T. gondii, the IgM SIAs were highly sensitive for the detection of acute infections, and the IgG avidity SIAs were highly specific for the separation of primary infections from past immunity. Altogether, the strategy of IgM multiplex screening followed by IgG avidity reflex testing can provide high-throughput and accurate means for the detection and stage determination of B19, HCMV, and T. gondii infections.IMPORTANCE Human parvovirus B19, human cytomegalovirus, and Toxoplasma gondii are ubiquitous pathogens. Their infections are often asymptomatic or mild in the general population yet may be transmitted from mother to fetus during pregnancy. Maternal infections by these pathogens can cause severe complications to the fetus or congenital abnormalities. As a rule, the risk of maternal transmission is critically related to the infection time; hence, it is important to determine when a pregnant woman has acquired the infection. In this study, we developed new diagnostic approaches for the timing of infections by three pathogens. All the new assays appeared to be highly sensitive and specific, providing powerful tools for medical diagnosis.Peer reviewe

Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7, and B 1.351 Isolates in Microneutralization Assays

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection

Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7, and B 1.351 Isolates in Microneutralization Assays

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection

Blood donation screening for hepatitis B virus core antibodies: The importance of confirmatory testing and initial implication for rare blood donor groups

Background and ObjectivesExclusion of blood donors with hepatitis B virus (HBV) core antibodies (anti-HBc) prevents transfusion-transmitted HBV infection but can lead to significant donor loss. As isolated anti-HBc positivity does not always indicate true past HBV infection, we have investigated the effectiveness of confirmatory anti-HBc testing and the representation of rare blood groups in anti-HBc-positive donors.Materials and MethodsThree hundred ninety-seven HBV surface antigen-negative and anti-HBc initially reactive blood donor samples were tested by five different anti-HBc assays.ResultsEighty percentage of samples reactive in Architect anti-HBc assay were positive by the Murex assay and anti-HBc neutralization. Eleven out of 397 samples showed discordant results in supplementary testing from the Murex confirmatory test result, and five remained undetermined following extensive serological testing. Thirty-eight percentage of anti-HBc-positive donors identified as minority ethnic groups compared with 11% representation in anti-HBc-negative donors (p < 0.0001); the frequency of the Ro blood group in anti-HBc-positive donors was 18 times higher in non-white ethnic groups.ConclusionUsing two anti-HBc assays effectively enabled the identification of HBV-exposed and potentially infectious donors, their deferral and potential clinical follow-up. However, the exclusion of confirmed anti-HBc-positive donors will still impact the supply of rare blood such as Ro

A Protein L -Based Immunodiagnostic Approach Utilizing Time-Resolved Forster Resonance Energy Transfer

Peer reviewe

Time-Resolved FRET -Based Approach for Antibody Detection - A New Serodiagnostic Concept

Peer reviewe

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

<div><p>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′ splice sites (5′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′ splice site (3′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′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′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′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.</p></div

Alien predation in wetlands : the Raccoon dog and waterbird breeding success

Alien predators are known to potentially strongly affect their prey populations. We studied the impact of raccoon dogs (Nyctereutes procyonoides) on waterbird breeding success in eight semi-urban wetlands in Finland. We manipulated raccoon dog density in two wetlands by removing individuals (2002 protection year, 2003 and 2004 removal years). We additionally performed nest predation experiments. We monitored raccoon dog density, estimated hunting bag size and observed waterbird breeding success. Our hypothesis predicts that the omnivorous raccoon dog plays a role in waterbird breeding success by depredating nests. Our experiments shown that the raccoon dog hunting bag in eutrophic wetlands may be large, as we removed 8.6–20.0 animals per km2. Both our nest predation experiment and field data indicated that raccoon dogs affect the breeding success of waterbirds. We found a significant relationship between raccoon dog density index and predation rate of the artificial nests, but not between red fox (Vulpes vulpes) density and predation on artificial nests. We did not find an association between raccoon dog abundance and the breeding success of mallards (Anas platyrhynchos) and great crested grebes (Podiceps cristatus). However, our study shows that birds species with different breeding strategies – e.g. great crested grebe, mute swan (Cygnus olor), mallard, Eurasian wigeon (Mareca penelope), coot (Fulica atra), lapwing (Vanellus vanellus) and marsh harrier (Circus aeruginosus) – when considered together showed higher breeding success both in 2003 and 2004 when compared to breeding success before removal. There was, however, variation in how strongly the species responded to raccoon dog removal. Our results indicate that the removal of alien raccoon dogs can be an important tool in wetland management

Antibody titration assays using AF-labeled or Eu-labeled protein L.

<p><b>A</b>) Anti-GST antibody titrated against Eu-labeled GST-VP1u and AF-labeled protein L. <b>B</b>) Anti-SA antibody titrated against Eu-labeled SA and AF-labeled protein L. <b>C</b>) Anti-SA antibody titrated against AF-labeled SA and Eu-labeled protein L. <b>D</b>) Anti-GST antibody titrated against AF-labeled GST-VP1u and Eu-labeled protein L. In all setups antibody concentrations were from 3.1 nM to 50 nM, and the antigen and protein concentration was constant (20 nM). Anti-GST antibody was used as a control for SA assays, and anti-SA for GST assays. The third line represents a background control with no antibody. The y-axis represents response counts obtained from Victor² fluorometer. The error bars represent ± standard deviation between parallel wells.</p

Antibody titration in ELISA.

<p>Monoclonal <b>A</b>) SA and <b>B</b>) GST antibodies were titrated in SA- or GST coated ELISA. The error bars represent ± standard deviation between parallel wells.</p