Improved sensitivity and automation of a multi-step upconversion lateral flow immunoassay using a 3D-printed actuation mechanism.

The development of sensitive point-of-care (POC) assay platforms is of interest for reducing the cost and time of diagnostics. Lateral flow assays (LFAs) are the gold standard for POC systems, but their sensitivity as such is inadequate, for example, in the case of cardiac diagnostics. The performance can be improved by incorporating different steps, such as pre-incubation to prolong the interaction time between sample and reporter for immunocomplex formation, and washing steps for background reduction. However, for POC assays, manual steps by the assay conductor are not desired. In this research, upconverting nanoparticles (UCNPs) were coated with poly(acrylic acid) (PAA) and conjugated to anti-cTnI antibodies, yielding non-clustering particles with low non-specific binding. The performance of cTnI-LFA in the PAA-anti-cTnI-UCNPs was compared to the same UCNPs with a commercial carboxyl surface. A kitchen-timer mechanism was embedded in a 3D-printed housing to produce a low-cost actuator facilitating a timed pre-incubation step for reporter and sample, and a washing step, to enable a multi-step cTnI-LFA with minimized manual labour. PAA-UCNPs showed improved mobility on nitrocellulose compared to those with a commercial surface. The mechanical actuator system was shown to improve sensitivity compared to a labour-intensive multi-step dipstick method, despite pre-incubation occurring during shaking and heating in the dipstick method. The limit of detection decreased from 7.6 to 1.5 ng/L cTnI in human plasma. The presented actuator can be easily modified for sensitivity improvement in the LFA for different analytes via pre-incubation and washing steps.

Spectrally separated dual-label upconversion luminescence lateral flow assay for cancer-specific STn-glycosylation in CA125 and CA15-3.

Multiplexed lateral flow assays (LFAs) offer efficient on-site testing by simultaneously detecting multiple biomarkers from a single sample, reducing costs. In cancer diagnostics, where biomarkers can lack specificity, multiparameter detection provides more information at the point-of-care. Our research focuses on epithelial ovarian cancer (EOC), where STn-glycosylated forms of CA125 and CA15-3 antigens can better discriminate cancer from benign conditions. We have developed a dual-label LFA that detects both CA125-STn and CA15-3-STn within a single anti-STn antibody test line. This utilizes spectral separation of green (540 nm) and blue (450 nm) emitting erbium (NaYF4:Yb3+, Er3+)- and thulium (NaYF4: Yb3+, Tm3+)-doped upconverting nanoparticle (UCNP) reporters conjugated with antibodies against the protein epitopes in CA125 or CA15-3. This technology allows the simultaneous detection of different antigen variants from a single test line. The developed proof-of-concept dual-label LFA was able to distinguish between the ascites fluid samples from diagnosed ovarian cancer patients (n = 10) and liver cirrhosis ascites fluid samples (n = 3) used as a negative control. The analytical sensitivity of CA125-STn for the dual-label LFA was 1.8 U/ml in buffer and 3.6 U/ml in ascites fluid matrix. Here we demonstrate a novel approach of spectrally separated measurement of STn-glycosylated forms of two different cancer-associated protein biomarkers by using UCNP reporter technology.

Upconverting nanoparticle reporter-based highly sensitive rapid lateral flow immunoassay for hepatitis B virus surface antigen.

Detection of hepatitis B Virus surface antigen (HBsAg) is an established method for diagnosing both acute and chronic hepatitis B virus (HBV) infection. In addition to enzyme immunoassays (EIAs), rapid diagnostic tests (RDTs) are available for the detection of HBsAg in resource-poor settings. However, the available RDTs have inadequate sensitivity and therefore are not suitable for diagnosis of patients with low levels of HBsAg and for blood screening. To provide a high-sensitivity RDT, we developed a lateral flow immunoassay (LFIA) for HBsAg utilizing upconverting nanoparticle (UCNP) reporter. The UCNP-LFIA can use whole blood, serum, or plasma and the results can be read in 30 min using a reader device. When compared with a commercial conventional visually read LFIA, the developed UCNP-LFIA had a Limit of Detection (LoD) of 0.1 IU HBsAg/ml in spiked serum, whereas the LoD of the conventional LFIA was 3.2 IU HBsAg/ml. The developed UCNP-LFIA fulfills the WHO criterion for blood screening (LoD ≤ 0.13 IU HBsAg/ml) in terms of LoD. The UCNP-LFIA and conventional LFIA were evaluated with well-characterized sample panels. The UCNP-LFIA detected 20/24 HBsAg-positive samples within the HBsAg Performance Panel and 8/10 samples within the Mixed Titer Performance Panel, whereas the conventional LFIA detected 8/24 and 4/10 samples in these panels, respectively. The performance of the assays was further evaluated with HBsAg-positive (n = 108) and HBsAg-negative (n = 315) patient samples. In comparison with a central laboratory test, UCNP-LFIA showed 95.4% (95% CI: 89.5-98.5%) sensitivity whereas sensitivity of the conventional LFIA was 87.7% (95%CI: 79.9-93.3%).

Double-Antigen Lateral Flow Immunoassay for the Detection of Anti-HIV-1 and -2 Antibodies Using Upconverting Nanoparticle Reporters.

Rapid diagnostic tests (RDTs) are often used for the detection of anti-human immunodeficiency virus (HIV) antibodies in remote locations in low- and middle-income countries (LMIC) with low or limited access to central laboratories. The typical format of an RDT is a lateral flow assay (LFA) with visual interpretation prone to subjectivity. This risk of misinterpretation can be overcome with luminescent upconverting nanoparticle reporters (UCNPs) measured with a miniaturized easy-to-use reader instrument. An LFA with UCNPs for anti-HIV-1/2 antibodies was developed and the assay performance was evaluated extensively with challenging patient sample panels. Sensitivity (n = 145) of the UCNP-LFA was 96.6% (95% CI: 92.1-98.8%) and specificity (n = 309) was 98.7% (95% CI: 96.7-99.7%). Another set of samples (n = 200) was used for a comparison between the UCNP-LFA and a conventional visual RDT. In this comparison, the sensitivities for HIV-1 were 96.4% (95% CI: 89.8-99.3%) and 97.6% (95% CI: 91.6-99.7%), for the UCNP-LFA and conventional RDT, respectively. The specificity was 100% (95% CI: 96.4-100%) for both assays. The developed UCNP-LFA demonstrates the applicability of UCNPs for the detection of anti-HIV antibodies. The signal measurement is done by a reader instrument, which may facilitate automated result interpretation, archiving and transfer of data from de-centralized locations.

Ultrasensitive and Robust Point-of-Care Immunoassay for the Detection of Plasmodium falciparum Malaria.

Plasmodium falciparum malaria is widespread in the tropical and subtropical regions of the world. There is ongoing effort to eliminate malaria from endemic regions, and sensitive point-of-care (POC) diagnostic tests are required to support this effort. However, current POC tests are not sufficiently sensitive to detect P. falciparum in asymptomatic individuals. After extensive optimization, we have developed a highly sensitive and robust POC test for the detection of P. falciparum infection. The test is based on upconverting nanophosphor-based lateral flow (UCNP-LF) immunoassay. The developed UCNP-LF test was validated using whole blood reference panels containing samples at different parasite densities covering eight strains of P. falciparum from different geographical areas. The limit of detection was compared to a WHO-prequalified rapid diagnostic test (RDT). The UCNP-LF achieved a detection limit of 0.2-2 parasites/µL, depending on the strain, which is 50- to 250-fold improvement in analytical sensitivity over the conventional RDTs. The developed UCNP-LF is highly stable even at 40 °C for at least 5 months. The extensively optimized UCNP-LF assay is as simple as the conventional malaria RDTs and requires 5 µL of whole blood as sample. Results can be read after 20 min from sample addition, with a simple photoluminescence reader. In the absence of a reader device at the testing site, the strips after running the test can be transported and read at a central location with access to a reader. We have found that the test and control line signals are stable for at least 10 months after running the test. The UCNP-LF has potential for diagnostic testing of both symptomatic and asymptomatic individuals.

Microparticle-based platform for point-of-care immunoassays.

There is a need for quantitative and sensitive, yet simple point-of-care immunoassays. We have developed a point-of-care microparticle-based immunoassay platform which combines the performance of a microtiter well-based assay with the usability of a rapid assay. The platform contained a separate reaction and detection chambers and microparticles for the solid-phase. Photoluminescent up-converting nanoparticles (UCNPs) were used as labels. The platform was tested with a cardiac troponin I assay, and a limit of detection of 19.7 ng/L was obtained. This study demonstrates the feasibility of developing point-of-care assays on the new platform for various analytes of interests.

Lateral flow immunoassay with upconverting nanoparticle-based detection for indirect measurement of interferon response by the level of MxA.

Myxovirus resistance protein A (MxA) is a biomarker of interferon-induced gene expression state involved in many viral infections and some autoimmune disorders. It has a variety of potential utilities in clinical diagnostics, including distinguishing between bacterial and viral infections. Currently, MxA-assays are used for monitoring of IFN-ß therapy in multiple sclerosis (MS) patients. As a proof-of-concept for rapid quantitative measurement of interferon response, a lateral flow immunoassay (LFIA) with upconverting nanoparticle (UCNP) reporters was developed and evaluated with clinical whole blood samples to assess the potential for a rapid and user-friendly quantitative assay for MxA, since the currently available rapid test for MxA (FebriDX) produces only qualitative result. The high detection sensitivity enabled by the UCNP reporter technology allowed the sample pre-treatment with dilution of whole blood into lysis buffer at a detectable analyte concentration. The assay can be done within 2 hr and the results correlate with the reference MxA-ELISA, which requires an overnight incubation. With 36 samples, R2 for linear regression was 0.86. The assay detected 96% of the IFN-ß responders with 89% specificity using a cut-off level of 100 µg/L for an elevated MxA-concentration. J. Med. Virol. 89:598-605, 2017. © 2016 Wiley Periodicals, Inc.

Effects of blood sample anticoagulants on lateral flow assays using luminescent photon-upconverting and Eu(III) nanoparticle reporters.

Many quantitative and semiquantitative lateral flow (LF) assays have been introduced for clinical analytes such as biomarkers for cancer or acute myocardial infarction (AMI). Various detection technologies involving quantitative analyzing devices have been reported to have sufficient analytical sensitivity and quantification capability for clinical point-of-care tests. Fluorescence-based detection technologies such as quantum dots, Eu(III) nanoparticles, and photon-upconverting nanoparticles (UCNPs) have been introduced as promising solutions for point-of-care devices because of their high detectability by optical sensors. Lateral flow assays can be used for various sample types, e.g., urine, saliva, cerebrospinal fluid, and blood. This study focuses on the properties of serum and plasma because of their relevance in cancer and AMI diagnostics. The limit of detection was compared in LF assays having Eu(III) nanoparticles or UCNPs as reporters and the antibody configurations for two different analytes (prostate-specific antigen and cardiac troponin I (cTnI)). The results indicate a significant effect of anticoagulants in venipuncture tubes. The samples in K3EDTA tubes resulted in significant interference by decreased reporter particle mobility, and thus the limit of detection was up to eightfold less sensitive compared to serum samples. Despite the matrix interference in the cTnI assay with UCNP reporters, limits of detection of 41 ng/L with serum and 66 ng/L with the Li-heparin sample were obtained.

Performance of fluorescent europium(III) nanoparticles and colloidal gold reporters in lateral flow bioaffinity assay.

Lateral flow (LF) immunoassays (i.e., immunochromatographic assays) have traditionally been applied to analytes that do not require very high analytical sensitivity or quantitative results. The selection of potential analytes is often limited by the performance characteristics of the assay technology. Analytes with more demanding sensitivity requirements call for reporter systems enabling high analytical sensitivity. In this study, we systematically compared the performance of fluorescent europium(III) [Eu(III)] chelate dyed polystyrene nanoparticles and colloidal gold particles in lateral flow assays. The effect of time-resolved measurement mode was also studied. Because binder molecules used in immunoassays might not behave similarly when conjugated to different reporter particles, two model assays were constructed to provide reliable technical comparison of the two reporter systems. The comparative experiment demonstrated that the fluorescent nanoparticles yielded 7- and 300-fold better sensitivity compared with colloidal gold in the two test systems, respectively. Although the two reporter particles may induce variable effects using individual binders, overall the high specific activity of Eu(III) nanoparticles has superior potential over colloidal gold particles for the development of robust high-sensitivity bioaffinity assays.

Escherichia coli-expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen.

BACKGROUND: The Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r)-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies. METHODS: A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni(II)-affinity chromatography. Biotinylated and europium(III) chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n=131), that included an in-house panel and four commercially procured panels. RESULTS: In-frame deletion of three hydrophobic regions, spanning amino acid residues 1-43, 519-538 and 676-706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C, and human T cell leukemia viruses. CONCLUSIONS: This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion of hydrophobic regions, and its purification in reasonable yields. This underscores the potential for cost saving in antigen production. Evaluation of this antigen in a double antigen sandwich two-step assay showed it to be a highly sensitive and specific HIV-1 diagnostic reagent. The amenability of this assay to the one-step format suggests its potential utility in developing a rapid point-of-care HIV-1 diagnostic test.

High-sensitivity lateral flow immunoassay with a fluorescent lanthanide nanoparticle label.

Lateral flow (LF) immunoassays are commonly used for point-of-care testing and typically incorporate visually read reporters, such as gold particles. To improve sensitivity and develop quantitative LF immunoassays, visual reporters can be replaced by fluorescent reporters detected by an instrument. In this study, we used fluorescent europium(III) chelate doped nanoparticle (Eu-np) reporters to develop a quantitative high-sensitivity LF immunoassay for free prostate specific antigen (fPSA). Furthermore, we tested different simplified formats of the assay and the effect of different modifiable parameters on the detection limit of the assay: dynamic range, assay duration and number of assay steps. The molar detection limits of the different assay formats were compared with published detection limits of LF immunoassays with different reporters. The cutoff was calculated from 11 female serum samples. The detection limit of the sensitivity optimized fPSA assay with fPSA spiked into pooled female serum was 0.01 ng/ml, which is approximately 100-fold lower than the most sensitive gold particle LF assays and 10-fold lower than other Eu-np and carbon nanoparticle based LF immunoassays. Thus, Eu-np reporters can be used to develop highly sensitive and quantitative LF immunoassays.

A rapid lateral flow immunoassay for serological diagnosis of pertussis.

Current serological diagnosis of pertussis is usually done by ELISA. However, the ELISAs are often central-laboratory based, require trained staff and have long turnaround times. A rapid point-of-care (POC) assay for pertussis serology would aid in both diagnosis and surveillance of the disease. While lateral flow immunoassays (LFIA) are simple to use and ideal for point-of-care diagnostics, they were limited to qualitative assays until recently. In this study, we developed a quantitative LFIA with fluorescent Eu-nanoparticle reporters for the detection of anti-pertussis toxin (PT) IgG. The assay was evaluated by testing 198 serum samples with varying anti-PT IgG levels and the result was compared to those obtained with standardized anti-PT IgG ELISA. At the diagnostic cutoff of 100 IU/mL in ELISA, the LFIA had a concordance of 92% with the ELISA, with a specificity of 96% [95% confidence interval (CI): 89-99%] and a sensitivity of 88% [CI: 77-94%]. The developed LFIA has a turnaround time of one hour and requires only a simple manipulation by the user and an instrument for the quantitative detection of the signal. We conclude that the LFIA is specific and sensitive for serological diagnosis of pertussis and is suitable for a POC test.

Anti-HCV immunoassays based on a multiepitope antigen and fluorescent lanthanide chelate reporters.

There is a need for simple to produce immunoassays for hepatitis C virus (HCV) antibody capable of detecting all genotypes worldwide. Current commonly used third generation immunoassays use three to six separate recombinant proteins or synthetic peptides. We have developed and expressed in Escherichia coli a single recombinant antigen incorporating epitopes from different HCV proteins. This multiepitope protein (MEP) was used to develop two types of HCV antibody immunoassays: a traditional antibody immunoassay using a labeled secondary antibody (indirect assay) and a double-antigen assay with the same MEP used as capture binder and labeled binder. The secondary antibody assay was evaluated with 171 serum/plasma samples and double-antigen assay with 148 samples. These samples included an in-house patient sample panel, two panels of samples with different HCV genotypes and a seroconversion panel. The secondary antibody immunoassay showed 95.6% sensitivity and 100% specificity while the double-antigen assay showed 91.4% sensitivity and 100% specificity. Both assays detected samples from all six HCV genotypes. The results showed that combining a low-cost recombinant MEP binder antigen with a high sensitivity fluorescent lanthanide reporter can provide a sensitive and specific immunoassay for HCV serology. The results also showed that the sensitivity of HCV double-antigen assays may suffer from the low avidity immune response of acute infections.

Europium nanoparticle-based simple to perform dry-reagent immunoassay for the detection of hepatitis B surface antigen.

Hepatitis B infection, caused by hepatitis B virus (HBV), presents a huge global health burden. Serological diagnosis of HBV mainly relies on the detection of hepatitis B surface antigen (HBsAg). Although there are high sensitivity commercial HBsAg enzyme immunoassays (EIAs) available, many low-resource laboratories lacking trained technicians continue to use rapid point-of-care assays with low sensitivities for HBsAg detection, due to their simplicity to operate. We developed a time-resolved fluorometric dry-reagent HBsAg immunoassay which meets the detection limit of high sensitivity EIAs but is simple to operate. To develop the assay, anti-HBsAg monoclonal antibody coated on europium nanoparticles was dried atop of biotinylated anti-HBsAg polyclonal antibody immobilized on streptavidin-coated microtiter wells. To test a sample in dry-reagent assay, serum sample and assay buffer were added to the wells, incubated, washed and europium signals were measured. The assay showed a detection limit of 0.25 ng/ml using HBsAg spiked in serum sample. When evaluated with 24 HBV positive and 37 negative serum samples, assay showed 100% sensitivity and specificity. Assay wells are stable for at least 26 weeks when stored at 4°C, and can tolerate elevated temperatures of up to 35°C for two weeks. The developed assay has high potential to be used in low-resource laboratories.

All-in-one dry-reagent time-resolved immunofluorometric assay for the rapid detection of HIV-1 and -2 infections.

An all-in-one (AIO) dry-reagent time-resolved fluorometric immunoassay that requires minimal liquid handling was developed for the detection of anti-HIV-1 and -2 antibodies. To prepare the AIO wells, in vivo biotinylated capture antigens (r-Bio-HIV-1env and r-Bio-HIV-2env) were immobilized on streptavidin-coated microtitration wells and Eu(III) chelate labelled non-biotinylated tracer antigens [r-HIV-1env-Eu(III) and r-HIV-2env-Eu(III)] were dried in stable form in the same wells. The HIV AIO assay was evaluated with serum/plasma samples (n=148) from in-house and commercial panels at two different incubation times of 15 min and 1h. The overall sensitivity of the AIO assay was 98.6% and specificity was 100% for both the incubation times. The AIO assay can accept whole blood matrix. This assay is envisioned to fill the gap between the rapid point-of-care assays and traditional enzyme immunoassays (EIA) in terms of complexity and turnaround time, without compromising the performance.

A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and -2.

This study addresses the continuing need to develop human immunodeficiency virus-1 (HIV-1) and HIV-2 immunoassays with increased sensitivity. Two chimeric antigens, r-HIV-1env, incorporating immunoreactive regions of HIV-1 glycoprotein (gp) 120 and gp41, and r-HIV-2env, incorporating HIV-2 gp125 and gp36, and their corresponding in vivo biotinylated versions, r-Bio-HIV-1env and r-Bio-HIV-2env, were expressed in Escherichia coli and purified by single step affinity chromatography. These antigens were used to set up a bridge assay for the detection of anti-HIV antibodies. Anti-HIV-1 and HIV-2 antibodies in sera were captured using a mixture of the biotinylated antigens, immobilized on streptavidin-coated microtiter wells, and revealed using a mixture of the non-biotinylated antigens, labeled with either Eu(3+) chelate or with nanoparticles doped with the Eu(3+) chelate, followed by fluorescence measurement using time resolved fluorometry (TRF). The performance of this TRF immunoassay was compared to that of five commercial HIV ELISAs using well-characterized sera panels. The results show that the TRF immunoassay using either form of the label was in complete agreement with the commercial assays. The use of the Eu(3+) chelate label enhanced sensitivity significantly when used in the nanoparticle format as evidenced by the very high signal-to-cut-off ratios.

Inexpensive designer antigen for anti-HIV antibody detection with high sensitivity and specificity.

A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity.