Time-Resolved Förster Resonance Energy Transfer for Rapid Infectious Disease Serodiagnosis‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

Novel molecular tools for infectious disease diagnostics are constantly under development to reduce the time between onset of symptoms and diagnosis. Not only is it important to receive appropriate treatment, but also to avoid unnecessary use of antibiotics. The availability of rapid diagnostics is also important when epidemics or pandemics emerge. The purpose of this project was to examine the applicability of Förster resonance energy transfer (FRET) in homogeneous immunoassays, and to develop new diagnostic approaches. FRET has widely been applied in proximity-based assays, such as those measuring antigen-antibody binding. In FRET, energy is transferred between two chromophores, the donor and the acceptor, when in close proximity. Utilizing FRET as detection method for immunoassays enables the development of wash-free (homogeneous) simple workflow assays. In this thesis, of the three FRET-based rapid immunoassays that were set up, two served in clinical diagnosis. Study I (I) examined the possibility of detecting antibodies by FRET-pair forming fluorophore-labeled antigens, which upon binding to an antibody would induce a FRET signal. This homogeneous immunoassay, designated FRET-bridge, was successfully optimized for streptavidin (SA). By combining donor-labeled and acceptor-labeled SAs with anti-SA antibodies, FRET signals were recorded with high signal-to-noise ratios. When molecular determinants behind the FRET signals were examined, most of the FRET activity originated from fairly large immunocomplexes rather than from one IgG and two antigens. At the moment, SA represents the only antigen fully functioning in the FRET-bridge assay. This is most likely due to the multivalent nature of the antigen, which seems beneficial in FRET signal formation. Next, we introduced another homogeneous immunoassay (II), the LFRET assay. Here, a FRET pair forming fluorophore-labeled antigen and fluorophore-labeled protein L induce a signal if bound to the Fab (fragment antigen-binding) region of an immunoglobulin. To demonstrate the usefulness of the assay, SA served as test antigen. The assay was next optimized for virus diagnostics by use of Puumala virus (PUUV) nucleocapsid protein as antigen (III). In all, 211 serum samples underwent examination by the LFRET assay, representing acute (n=61) or past PUUV infection (n=27), and seronegative (n=123) individuals. With a simple workflow and an assay time of 30 minutes, the LFRET assay, compared to the reference tests, identified acute PUUV infection at 100% specificity and 95% sensitivity. The fourth study (IV) involved a competitive homogeneous immunoassay for the detection of PUUV antibodies from clinical samples. This assay, CFRET, is based on competition between fluorophore-labeled monoclonal antibodies (MAbs) and serum antibodies. Here, a donor-labeled antigen and an acceptor-labeled MAb form the FRET pair. If the clinical sample contains antibodies against the labeled antigen, they compete with the MAb for antigen binding, resulting in FRET signal decrease. Analysis of assay performance included a panel of 329 samples representing acute (n=101) or past (n=42) infection, and negative samples (n=186). The one-step CFRET assay performed at 99% specificity and 100% sensitivity in diagnosis of hantavirus disease compared to the reference tests, and with a rapid assay time of 30 minutes. The three rapid diagnostic approaches introduced herein represent simplicity, and show that diagnostics need not be time-consuming. Although the assays were optimized for accurate diagnosis of acute infection, both assays also recognized life-long immunity, albeit with lower sensitivity. By optimization, the assays could be developed towards more sensitive detection of past infection as well. The LFRET and CFRET assays thus represent innovative tools for rapid antibody detection, and their potential in serodiagnosis of diverse microbial infections and possibly even in detection of auto- and anti-allergen antibodies calls for further exploration.  Tartuntatautien diagnostiikkaan kehitetään jatkuvasti uusia pikatestejä, jotta diagnoosi saataisiin mahdollisimman nopeasti oireiden ilmaannuttua. Nopea diagnoosi varmistaa sen, että oikea hoito voidaan aloittaa mahdollisimman pian, ja samalla vältytään turhilta antibiooteilta. Diagnostiset pikatestit ovat myös tärkeitä epidemioiden tai pandemioiden hallinnassa. Tämän projektin tarkoituksena oli hyödyntää Förster resonance energy transfer (FRET) ilmiötä homogeenisten vasta-ainetestien kehittämisessä. Tutkimuksen tuloksena pystytettiin kolme erityyppistä vasta-ainetestiä, joista kahta sovellettiin kliiniseen diagnostiikkaan. Ensimmäisessä osajulkaisussa (I) tutkittiin, voisiko vasta-aineita tunnistaa sellaisten fluorofori-leimattujen antigeenien avulla, jotka yhteen liittyessään muodostaisivat FRET-parin. Tutkimusasetelma perustui oletukseen, jonka mukaan näiden fluorofori-leimattujen antigeenien sitoutuessa samaan vasta-aineeseen, muodostuu FRET-signaali. Tämä testi, FRET-bridge, kehitettiin alun perin streptavidiini (SA) -malliantigeenille. FRET-signaali muodostui, kun luovuttaja- ja vastaanottajafluoroforileimatut SA:t yhdistyivät SA-vasta-aineiden toimesta. Työssä tutkittiin muodostuvia FRET-signaaleja molekyylitasolla ja huomattiin, että suurin osa signaaleista oli peräisin isoista immuunikomplekseista, eikä niinkään kahdesta antigeenista ja yhdestä vasta-aineesta. Toistaiseksi SA on ainoa antigeeni, joka tässä testissä toimii kunnolla. Tämä johtuu todennäköisesti SA:n multivalentista luonteesta, mikä vaikuttaisi edistävän FRET-signaalin muodostusta. Toisessa ja kolmannessa osajulkaisussa (II, III) tutkittiin homogeenisen vasta-ainetestin soveltuvuutta tartuntatautien diagnostiikkaan. Kyseinen testi, nimeltään LFRET, perustuu fluoroforileimatun antigeenin sekä fluoroforileimatun proteiini L:n interaktioon niiden sitoutuessa saman vasta-aineen Fab (fragment antigen-binding) -osaan. Myös tämä testi optimoitiin käyttäen SA:ia, jonka jälkeen ensimmäisenä virusantigeenina käytettiin Puumala-viruksen (PUUV) nukleokapsidiproteiinia. Työssä testattiin kaikkiaan 211 seeruminäytettä, jotka edustivat akuuttia vaihetta (n=61), vanhaa immuniteettia (n=27) ja PUUV:lle negatiivisia näytteitä (n=123). LFRET-testi tunnisti akuuttia myyräkuumetta 100% tarkkuudella ja 95% herkkyydellä verrattuna referenssitestien tuloksiin. Neljännessä osajulkaisussa (IV) pystytettiin kilpaileva homogeeninen vasta-ainetesti nimeltään CFRET. Tämä testi perustuu kilpailuun antigeeniin sitoutumisesta fluoroforileimatun monoklonaalisen vasta-aineen (MAb) ja seerumin vasta-aineiden välillä. Tässä testissä MAb sekä antigeeni muodostavat FRET-parin. Jos seeruminäyte sisältää vasta-aineita, jotka sitovat kyseistä antigeenia, syntyy kilpailutilanne vasta-aineiden välille, mikä puolestaan johtaa FRET-signaalin laskuun. Työssä testattiin yhteensä 329 näytettä, jotka edustivat akuuttia vaihetta (n=101), vanhaa immuniteettia (n=42) ja PUUV:lle negatiivisia näytteitä (n=186). CFRET tunnisti akuuttia myyräkuumetta 99% tarkkuudella ja 100% herkkyydellä verrattuna referenssitestien tuloksiin. Edellä kuvatut kolme pikatestiä ovat yksinkertaisia ja ne todistavat, että diagnostisen testin ei tarvitse olla aikaavievä. Vaikka sekä LFRET- että CFRET-testi kehitettiin tunnistamaan akuuttia infektiota, molemmat testit tunnistivat myös vanhaa immuniteettia, joskaan ei yhtä herkästi kuin akuuttia infektiota. Hienosäädön myötä on mahdollista, että kyseistä ominaisuutta voitaisiin parantaa entistä herkemmäksi. LFRET- ja CFRET-testit tarjoavat uraauurtavia mahdollisuuksia vasta-aineiden pikadiagnostiikkaan, ja niiden soveltuvuutta useiden eri taudinaiheuttajien diagnostiikkaan sekä auto- että anti-allergeeni vasta-aineiden tunnistamiseen tulisi tutkia lisää

Urine and Free Immunoglobulin Light Chains as Analytes for Serodiagnosis of Hantavirus Infection

Rapid point-of-care testing is a megatrend in infectious disease diagnosis. We have introduced a homogeneous immunoassay concept, which is based on the simultaneous binding of antigen and protein L to a given immunoglobulin molecule. The complex formation is detected utilizing time-resolved Förster resonance energy transfer between antigen-attached donor and acceptor-labeled protein L, hence the name LFRET. Here, we demonstrate that urine can be used as a sample matrix in LFRET-based serodiagnostics. We studied urine samples collected during the hospitalization and recovery of patients with acute Puumala orthohantavirus (PUUV) infection. We compared PUUV antibody-specific LFRET signals in urine to those in plasma, and found excellent correlation in the test outcomes The LFRET test from urine was positive in 40/40 patients with acute PUUV infection. PUUV causes a mild form of hemorrhagic fever with renal syndrome, characterized by acute kidney injury and proteinuria. Immunofluorescence and western blotting demonstrated PUUV-IgG and -IgA in urine, however, the presence of intact immunoglobulins did not fully explain the LFRET signals. We purified free light chains (FLCs) from both urine and serum of healthy volunteers and patients with acute PUUV infection, and verified the presence of antigen-specific FLCs. Antigen-specific FLCs provide a new means for non-invasive antibody detection and disease diagnosis

Hantavirus infection-induced B cell activation elevates free light chains levels in circulation

In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients' peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro, and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings. Author summary Orthohantaviruses are globally spread zoonotic pathogens, which can cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) with significant burden to human health. The pathogenesis mechanisms of orthohantavirus-caused diseases are not known in detail; however, excessive immune response towards the virus with concomitant pathological effects against host tissues appears to be a contributing factor. Here we report an increase of free immunoglobulin (Ig) light chains (FLCs), components required to make complete Ig molecules, in blood of acute HFRS and HPS. Samples collected during acute HFRS demonstrated increased FLCs levels in the urine and blood of patients hospitalized due the disease. Furthermore, the FLC levels positively correlated with markers of acute kidney injury. In addition, our results show that orthohantaviruses can infect and activate B cells to produce FLCs as well as whole Igs, which provides a mechanistic explanation of the increased FLC levels in patients. Taken together, our results suggest that aberrant antibody responses might play a role in the pathogenesis of orthantavirus infections.Peer reviewe

LFRET, a novel rapid assay for anti-tissue transglutaminase antibody detection

The diagnosis of celiac disease (CD) is currently based on serology and intestinal biopsy, with detection of anti-tissue transglutaminase (tTG) IgA antibodies recommended as the first-line test. Emphasizing the increasing importance of serological testing, new guidelines and evidence suggest basing the diagnosis solely on serology without confirmatory biopsy. Enzyme immunoassays (EIAs) are the established approach for anti-tTG antibody detection, with the existing point-of-care (POC) tests lacking sensitivity and/or specificity. Improved POC methods could help reduce the underdiagnosis and diagnostic delay of CD. We have previously developed rapid homogenous immunoassays based on time-resolved Förster resonance energy transfer (TR-FRET), and demonstrated their suitability in serodiagnostics with hanta- and Zika virus infections as models. In this study, we set out to establish a protein L -based TR-FRET assay (LFRET) for the detection of anti-tTG antibodies. We studied 74 patients with biopsy-confirmed CD and 70 healthy controls, with 1) the new tTG-LFRET assay, and for reference 2) a well-established EIA and 3) an existing commercial POC test. IgG depletion was employed to differentiate between anti-tTG IgA and IgG positivity. The sensitivity and specificity of the first-generation tTG-LFRET POC assay in detection of CD were 87.8% and 94.3%, respectively, in line with those of the reference POC test. The sensitivity and specificity of EIA were 95.9% and 91.9%, respectively. This study demonstrates the applicability of LFRET to serological diagnosis of autoimmune diseases in general and of CD in particular.Peer reviewe

LFRET, a novel rapid assay for anti-tissue transglutaminase antibody detection

The diagnosis of celiac disease (CD) is currently based on serology and intestinal biopsy, with detection of anti-tissue transglutaminase (tTG) IgA antibodies recommended as the first-line test. Emphasizing the increasing importance of serological testing, new guidelines and evidence suggest basing the diagnosis solely on serology without confirmatory biopsy. Enzyme immunoassays (EIAs) are the established approach for anti-tTG antibody detection, with the existing point-of-care (POC) tests lacking sensitivity and/or specificity. Improved POC methods could help reduce the underdiagnosis and diagnostic delay of CD. We have previously developed rapid homogenous immunoassays based on time-resolved Förster resonance energy transfer (TR-FRET), and demonstrated their suitability in serodiagnostics with hanta- and Zika virus infections as models. In this study, we set out to establish a protein L -based TR-FRET assay (LFRET) for the detection of anti-tTG antibodies. We studied 74 patients with biopsy-confirmed CD and 70 healthy controls, with 1) the new tTG-LFRET assay, and for reference 2) a well-established EIA and 3) an existing commercial POC test. IgG depletion was employed to differentiate between anti-tTG IgA and IgG positivity. The sensitivity and specificity of the first-generation tTG-LFRET POC assay in detection of CD were 87.8% and 94.3%, respectively, in line with those of the reference POC test. The sensitivity and specificity of EIA were 95.9% and 91.9%, respectively. This study demonstrates the applicability of LFRET to serological diagnosis of autoimmune diseases in general and of CD in particular

Immunoassay for serodiagnosis of Zika virus infection based on time-resolved Förster resonance energy transfer

Zika virus (ZIKV) is a mosquito-borne pathogen causing a febrile illness with arthralgia, conjunctivitis and rash. The complications include Guillain-Barré syndrome, congenital brain and other abnormalities and miscarriage. The serodiagnosis of ZIKV infection is hampered by cross-reactivity with other members of the Flavivirus family, notably dengue (DENV). This report describes a novel serological platform for the diagnosis of ZIKV infection. The approach utilizes time-resolved Förster resonance energy transfer (TR-FRET) elicited by two chromophore-labeled proteins (a ZIKV antigen and a super-antigen) simultaneously binding to a given antibody molecule. The antigen used in the assay is ZIKV non-structural protein 1 (NS1) and the super-antigen is bacterial protein L. Three assay variants were developed: the first measuring all anti-ZIKV-NS1 antibodies (LFRET), the second measuring IgM and IgA (acute-LFRET) and the third measuring IgG (immunity-LFRET). The assays were evaluated with a panel of samples from clinical ZIKV cases in travelers (n = 25) and seronegative (n = 24) samples. DENV (n = 38), yellow fever (n = 16) and tick-borne-encephalitis (n = 20) seropositive samples were examined for assessment of flavivirus cross-reactivity. The diagnostic sensitivities of the respective LFRET assays were 92%, 100% and 83%, and the diagnostic specificities 88%, 95% and 100% for LFRET, acute-LFRET and immunity-LFRET. Furthermore, we evaluated the assays against a widely-used commercial ELISA. In conclusion, the new FRET-based serological approaches based on NS1 protein are applicable to diagnosing zika virus infections in travelers and differentiating them from other flavivirus infections.Peer reviewe

Challenge of paediatric compounding to solid dosage forms sachets and hard capsules - Finnish perspective

Objectives The study evaluated the quality of compounded sachets and hard gelatine capsules and their feasibility in paediatric drug administration. Methods Commercial tablets were compounded to sachets and capsules in hospital environment, and the uniformity of content and simulated drug dose were determined. Key findings Compounded formulations were successfully obtained for a range of drug substances; dipyridamole, spironolactone, warfarin and sotalol formulations were within acceptable limits for uniformity of content, in most cases. However, some loss of drug was seen. The type and amount of excipients were found to affect uniformity of content; good conformity of capsules was obtained using lactose monohydrate as filler, whereas microcrystalline cellulose was a better choice in sachets. In capsules, content uniformity was obtained for a range of drug doses. If the drug is aimed to be administered through a nasogastric tube, solubility of the drug and excipients should be considered, as they were found to affect the simulated drug dose in administration. Conclusions Compounded sachets and capsules fulfilled the quality requirements in most cases. In compounding, the choice of excipients should be considered as they can affect conformity of the dosage form or its usability in practice. Quality assurance of compounded formulations should be taken into consideration in hospital pharmacies.Peer reviewe

Puumala Hantavirus Infections Show Extensive Variation in Clinical Outcome

The clinical outcome of Puumala hantavirus (PUUV) infection shows extensive variation, ranging from inapparent subclinical infection (70–80%) to severe hemorrhagic fever with renal syndrome (HFRS), with about 0.1% of cases being fatal. Most hospitalized patients experience acute kidney injury (AKI), histologically known as acute hemorrhagic tubulointerstitial nephritis. Why this variation? There is no evidence that there would be more virulent and less virulent variants infecting humans, although this has not been extensively studied. Individuals with the human leukocyte antigen (HLA) alleles B*08 and DRB1*0301 are likely to have a severe form of the PUUV infection, and those with B*27 are likely to have a benign clinical course. Other genetic factors, related to the tumor necrosis factor (TNF) gene and the C4A component of the complement system, may be involved. Various autoimmune phenomena and Epstein-Barr virus infection are associated with PUUV infection, but hantavirus-neutralizing antibodies are not associated with lower disease severity in PUUV HFRS. Wide individual differences occur in ocular and central nervous system (CNS) manifestations and in the long-term consequences of nephropathia epidemica (NE). Numerous biomarkers have been detected, and some are clinically used to assess and predict the severity of PUUV infection. A new addition is the plasma glucose concentration associated with the severity of both capillary leakage, thrombocytopenia, inflammation, and AKI in PUUV infection. Our question, “Why this variation?” remains largely unanswered

Puumala Hantavirus Infections Show Extensive Variation in Clinical Outcome

The clinical outcome of Puumala hantavirus (PUUV) infection shows extensive variation, ranging from inapparent subclinical infection (70–80%) to severe hemorrhagic fever with renal syndrome (HFRS), with about 0.1% of cases being fatal. Most hospitalized patients experience acute kidney injury (AKI), histologically known as acute hemorrhagic tubulointerstitial nephritis. Why this variation? There is no evidence that there would be more virulent and less virulent variants infecting humans, although this has not been extensively studied. Individuals with the human leukocyte antigen (HLA) alleles B*08 and DRB1*0301 are likely to have a severe form of the PUUV infection, and those with B*27 are likely to have a benign clinical course. Other genetic factors, related to the tumor necrosis factor (TNF) gene and the C4A component of the complement system, may be involved. Various autoimmune phenomena and Epstein-Barr virus infection are associated with PUUV infection, but hantavirus-neutralizing antibodies are not associated with lower disease severity in PUUV HFRS. Wide individual differences occur in ocular and central nervous system (CNS) manifestations and in the long-term consequences of nephropathia epidemica (NE). Numerous biomarkers have been detected, and some are clinically used to assess and predict the severity of PUUV infection. A new addition is the plasma glucose concentration associated with the severity of both capillary leakage, thrombocytopenia, inflammation, and AKI in PUUV infection. Our question, “Why this variation?” remains largely unanswered

A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection—SARS-CoV-2 as a Proof of Concept

Peer reviewe