A high-throughput microfluidic nanoimmunoassay for detecting anti-SARS-CoV-2 antibodies in serum or ultralow-volume blood samples.

Novel technologies are needed to facilitate large-scale detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies and vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nanoimmunoassay (NIA) for the detection of anti-SARS-CoV-2 IgG antibodies in 1,024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultralow-volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 µL of whole blood easily obtainable from a simple finger prick. The NIA platform achieves high throughput, high sensitivity, and specificity based on the analysis of 289 human serum samples, and negligible reagent consumption. We furthermore demonstrate the possibility to combine NIA with decentralized and simple approaches to blood sample collection. We expect this technology to be applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker analysis in general.

Rapid and accurate Bayesian diagnosis of heparin-induced thrombocytopenia.

Prompt diagnostic evaluation of suspected heparin-induced thrombocytopenia (HIT) is critical for guiding initial patient management. We assessed the performance of 3 immunoassays detecting anti-platelet factor 4 (PF4)/heparin antibodies, derived a diagnostic algorithm with a short analytical turnaround time (TAT), and prospectively validated the algorithm. Plasma samples were analyzed by Zymutest-HIA-IgG, HemosIL-AcuStar-HIT-IgG, and ID-H/PF4-PaGIA in retrospective (n = 221) and prospective (n = 305) derivation cohorts. We calculated likelihood ratios of result intervals and cutoff values with 100% negative (NPV) and positive (PPV) predictive values for a positive gold standard functional assay (heparin-induced platelet activation [HIPA]). A diagnostic algorithm was established based on the Bayesian combination of pretest probability and likelihood ratios of first- and second-line immunoassays. Cutoffs with 100% PPV for positive HIPA were >3.0 U/mL (HemosIL-AcuStar-HIT-IgG) and titer ≥16 (ID-H/PF4-PaGIA); cutoffs with 100% NPV were <0.13 U/mL and ≤1, respectively. During the prospective validation of the derived algorithm (n = 687), HemosIL-AcuStar-HIT-IgG was used as unique testing in 566 (82.4%) of 687 cases (analytical TAT, 30 minutes). In 121 (17.6%) of 687 unresolved cases, ID-H/PF4-PaGIA was used as second-line testing (additional TAT, 30 minutes). The algorithm accurately predicted HIT in 51 (7.4%) of 687 patients and excluded it in 604 (87.9%) of 687 patients, leaving only 20 (2.9%) cases unresolved. We also identified 12 (1.7%) of 687 positive predictions not confirmed by HIPA: 10 patients with probable HIT despite negative HIPA and 2 possible false-positive algorithm predictions. The combination of pretest probability with first- and second-line immunoassays for anti-PF4/heparin antibodies is accurate for ruling in or out HIT in ≥95% of cases within 60 minutes. This diagnostic approach improves initial management of patients with suspected HIT.

Automated liquid handling robot for rapid lateral flow assay development.

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.

Time scale performance of rapid antigen testing for SARS-CoV-2: Evaluation of 10 rapid antigen assays.

There is a great demand for more rapid tests for SARS-CoV-2 detection to reduce waiting time, boost public health strategies for combating disease, decrease costs, and prevent overwhelming laboratory capacities. This study was conducted to assess the performance of 10 lateral flow device viral antigen immunoassays for the detection of SARS-CoV-2 in nasopharyngeal swab specimens. We analyzed 231 nasopharyngeal samples collected from October 2020 to December 2020, from suspected COVID-19 cases and contacts of positive cases at Biotechnology Research Center laboratories, Tripoli, Libya. The performance of 10 COVID-19 Antigen (Ag) rapid test devices for the detection of SARS-CoV-2 antigen was compared to a quantitative reverse transcription-polymerase chain reaction (RT-qPCR). In this study, 161 cases had symptoms consistent with COVID-19. The mean duration from symptom onset was 6.6 ± 4.3 days. The median cycle threshold (Ct ) of positive samples was 25. Among the 108 positive samples detected by RT-qPCR, the COVID-19 antigen (Ag) tests detected 83 cases correctly. All rapid Ag test devices used in this study showed 100% specificity. While tests from six manufacturers had an overall sensitivity range from 75% to 100%, the remaining four tests had a sensitivity of 50%-71.43%. Sensitivity during the first 6 days of symptoms and in samples with high viral loads (Ct < 25), was 100% in all but two of the test platforms. False-negative samples had a median Ct of 34 and an average duration of onset of symptoms of 11.3 days (range = 5-20 days). Antigen test diagnosis has high sensitivity and specificity in early disease when patients present less than 7 days of symptom onset. Patients are encouraged to test as soon as they get COVID-19-related symptoms within 1 week and to seek medical advice within 24 h if they develop disturbed smell/taste. The use of rapid antigen tests is important for controlling the COVID-19 pandemic and reducing the burden on molecular diagnostic laboratories.

Performance of 30 commercial SARS-CoV-2 serology assays in testing symptomatic COVID-19 patients.

We report evaluation of 30 assays' (17 rapid tests (RDTs) and 13 automated/manual ELISA/CLIA assay (IAs)) clinical performances with 2594 sera collected from symptomatic patients with positive SARS-CoV-2 rRT-PCR on a respiratory sample, and 1996 pre-epidemic serum samples expected to be negative. Only 4 RDT and 3 IAs fitted both specificity (> 98%) and sensitivity (> 90%) criteria according to French recommendations. Serology may offer valuable information during COVID-19 pandemic, but inconsistent performances observed among the 30 commercial assays evaluated, which underlines the importance of independent evaluation before clinical implementation.

Colloidal gold-based lateral flow immunoassay with inline cleanup for rapid on-site screening of carbendazim in functional foods.

In this study, for the first time, we propose a sensitive colloidal gold-based lateral flow immunoassay (LFIA) that can be used to detect carbendazim residues in functional foods. The adoption of inline cleanup LFIA strips effectively improved background interference to reduce misjudgment of results. First, the hapten 2-(methylamino)-1H-benzo[d]imidazole-5-carboxylic acid was used to establish the carbendazim immunoassay method. Subsequently, colloidal gold-mAb preparation and LFIA detection conditions were systematically optimized. For root and fruit samples (ginseng, ginger, jujube, and Chinese wolfberry), the designed strips had a cutoff value of 8 ng/mL. For flower and seed samples (chrysanthemum, coix seed, and malt), the cutoff value was 12 ng/mL. Even in a complex matrix, the established LFIA method demonstrates satisfactory sensitivity and anti-interference ability. This method was successfully applied in detection of carbendazim residues in complex functional foods, and the assay results are consistent with those obtained via liquid chromatography-tandem mass spectrometry. In short, the proposed method is fast and sensitive and has strong anti-interference ability. Furthermore, it provides a new technical method highly relevant to the on-site rapid detection of carbendazim residues in complex sample matrix.

Rapid, simultaneous detection of mycotoxins with smartphone recognition-based immune microspheres.

How to achieve simultaneous and rapid detection of various mycotoxins in food has important practical significance in the field of food processing and safety. In this paper, a smartphone immunoassay system based on hydrogel microspheres has been constructed to quickly detect two mycotoxins at the same time. The rapid detection system was reflected in the following three processes: (1) rapid separation of free matter after direct competition reaction based on hydrogel solid-phase carrier particles; (2) rapid detection process based on efficient catalytic function of enzymes; (3) fast capture and analysis of images based on smartphone software. Ochratoxin A (OTA) and zearalenone (ZEN) are secondary toxic metabolites of fungi that can contaminate a wide range of foods and feeds. OTA and ZEN were used as detection model molecules to verify the feasibility of the intelligent rapid detection system. The entire detection process was within 30 min, and the results were analyzed in only 10 s. Detection limits of mycotoxins OTA and ZEN are 0.7711 ng L-1 and 1.0391 ng L-1. The recoveries of both mycotoxins ranged from 76.72 to 122.05%. This study provides a universal rapid detection method for on-site application of large-scale food security testing. Schematic diagram of the construction of the smartphone detection system: The system is divided into three parts: detection, image capture and analysis, and result.

One-pot high-yield synthesis of Pd nanocubes for Pd-Ir nanocube-based immunoassay of nucleocapsid protein from SARS-CoV-2.

Pd-Ir nanocubes are promising peroxidase-mimicking nanozymes for immunoassays, enabled by their excellent stability, relatively high catalytic activity, and reproducible performance. A key step involved in the preparation of Pd-Ir nanocubes is the synthesis of Pd nanocubes. However, the traditional method to synthesize Pd nanocubes requires sophisticated and expensive equipment to precisely control the reaction temperature and highly skilled technicians to achieve satisfactory and reproducible product yields. Herein, we report a simple, cost-effective, high-yield (> 99%) and one-pot strategy to synthesize Pd nanocubes with sizes of 7, 18, and 51 nm for the preparation of Pd-Ir nanocubes. The resulting 18 nm Pd-Ir nanocubes display three orders of magnitude higher peroxidase activity compared to horseradish peroxidase, leading to a significantly increased detection sensitivity when applied in the immunoassay of nucleocapsid protein from SARS-CoV-2. Due to the simplicity in both material synthesis and assaying procedures and the excellent detection sensitivity, our method should allow for the generalized application of Pd-Ir nanocube-based immunoassays for the diagnosis of human diseases.

Simple workflow to repurpose SARS-CoV-2 swab/serum samples for the isolation of cost-effective antibody/antigens for proteotyping applications and diagnosis.

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.

A cost of illness comparison for toxigenic Clostridioides difficile diagnosis algorithms in developing countries.

BACKGROUND: Availability of several commercial tests with different Clostridioides difficile targets contributes to uncertainty and controversies around the optimal diagnostic algorithm. While numerous studies have estimated the financial impact of C. difficile infection, models to guide testing strategies decisions in developing countries, where economic value significantly impacts clinical practice, are currently not available. AIM: To determine the cost of illness of different C. difficile infection (CDI) diagnostic strategies in developing countries. METHODS: Cost-comparison analysis was performed to compare eleven different algorithms of CDI diagnosis. The basis of calculation was a hypothetical cohort of 1000 adult inpatients suspected of CDI. We analyzed turnaround time of test results (i.e., time from taking sample to results emission), test performance (i.e., sensitivity and specificity) and testing costs. Patients were divided in true positive, false positive, true negative and false negative in order to integrate test performance and economics effects. Additional medical costs were calculated: costs of hygiene, medication, length of stay and intensive care unit costs, based on a Brazilian University Hospital costs. CDI prevalence was considered 22.64%. FINDINGS: From laboratory-assisted tests, simultaneous glutamate dehydrogenase (GDH) and toxin A/B rapid immunoassay arbitrated by nucleic acid amplification test (NAAT) presented the lowest cost of illness (450,038.70 USD), whereas standalone NAAT had the highest (523,709.55 USD). Empirical diagnosis only presented the highest overall cost (809,605.44 USD). CONCLUSION: The two-step algorithm with simultaneous GDH and toxin A/B rapid immunoassay arbitrated by NAAT seems to be the best strategy for CDI diagnosis in developing countries.

An immunoassay cassette with a handheld reader for HIV urine testing in point-of-care diagnostics.

Currently, most HIV tests are performed with blood samples, or alternatively saliva samples are used for HIV testing. Simple HIV tests need to be performed in hospitals or other medical agencies instead of more invasive HIV blood tests. To enable point-of-care (POC) HIV diagnostics, based on a recently developed lateral flow strip for HIV urine testing, a microfluidic immunoassay cassette with a handheld optical reader is developed. Based on lateral flow strip with gold colloid reporter, the integrated immunoassay cassette can perform sample introduction, metering, discharging, applying and detection which simplifies HIV testing. An indicator is incorporated into the cassette to guide sample introduction based on color change, and further, the excess test sample is stored inside the sealed cassette to avoid any contamination. The low-cost handheld optical reader can provide a test result within a few seconds, which is useful for simple, sensitive and affordable HIV onsite detection. Instead of using normal white LEDs, a customized back light module embedded with green LEDs is adopted to illuminate the lateral flow strip with an appropriate working current to achieve optimal performance. Compared to the standard lateral flow strips using a benchtop reader, with the disposable immunoassay cassette assisted by the handheld optical reader, more convenient, easier-to-operate, and more affordable HIV urine testing can be achieved in POC diagnostics.

Cost-Effective Respiratory Virus Testing.

The timely and accurate diagnosis of respiratory virus infections has the potential to optimize downstream (posttesting) use of limited health care resources, including antibiotics, antivirals, ancillary testing, and inpatient and emergency department beds. Cost-effective algorithms for respiratory virus testing must take into consideration numerous factors, including which patients should be tested, what testing should be performed (for example, antigen testing versus reverse transcription-PCR testing or influenza A/B testing versus testing with a comprehensive respiratory virus panel), and the turnaround time necessary to achieve the desired posttesting outcomes. Despite the clinical impact of respiratory virus infections, the cost-effectiveness of respiratory virus testing is incompletely understood. In this article, we review the literature pertaining to the cost-effectiveness of respiratory virus testing in pediatric and adult patient populations, in emergency department, outpatient, and inpatient clinical settings. Furthermore, we consider the cost-effectiveness of a variety of testing methods, including rapid antigen tests, direct fluorescent antibody assays, and nucleic acid amplification tests.

Point-of-care screening for sickle cell disease in low-resource settings: A multi-center evaluation of HemoTypeSC, a novel rapid test.

Sickle cell disease (SCD) is a common, life-threatening genetic disorder that is best managed when diagnosed early by newborn screening. However, SCD is most prevalent in low-resource regions of the world where newborn screening is rare and diagnosis at the point-of-care is challenging. In many such regions, the majority of affected children die, undiagnosed, before the age of 5 years. A rapid and affordable point-of-care test for SCD is needed. The diagnostic accuracy of HemoTypeSC, a point-of-care immunoassay, for SCD was evaluated in individuals who had SCD, hemoglobin C disease, the related carrier (trait) states, or a normal hemoglobin phenotype. Children and adults participated in low-, medium- and high-resource environments (Ghana [n = 383], Martinique [n = 46], and USA [n = 158]). Paired blood specimens were obtained for HemoTypeSC and a reference diagnostic assay. HemoTypeSC testing was performed at the site of blood collection, and the reference test was performed in a laboratory at each site. In 587 participants, across all study sites, HemoTypeSC had an overall sensitivity of 99.5% and specificity of 99.9% across all hemoglobin phenotypes. The test had 100% sensitivity and specificity for sickle cell anemia. Sensitivity and specificity for detection of normal and trait states were >99%. HemoTypeSC is an inexpensive (<$2 per test), accurate, and rapid point-of-care test that can be used in resource-limited regions with a high prevalence of SCD to provide timely diagnosis and support newborn screening programs.

Rapid detection of trace Salmonella in milk and chicken by immunomagnetic separation in combination with a chemiluminescence microparticle immunoassay.

Rapid detection of trace Salmonella is urgently needed to ensure food safety. We present an innovative pretreatment strategy, based on a two-step enrichment culture and immunomagnetic separation, combined with a chemiluminescence microparticle immunoassay to detect at least one proliferative Salmonella cell in 25 mL (25 g) food. The capture performance of immunomagnetic beads (IMBs) of sizes for Salmonella was investigated, and the IMBs of size 2.8 µm showed a high capture efficiency of 60.7% in 25 mL milk and 74.5% in 25 mL chicken culture filtrate, which ensured the successful capture of trace Salmonella after 2.5 h in situ enrichment even from only one Salmonella cell. The separated Salmonella cells, reaching an amount of 103 colony-forming units (CFU) by a secondary enrichment for 3 h, were detected by a horseradish peroxidase chemiluminescence reaction with 4-(1-imidazolyl)phenol as an enhancer, which evidenced a linear response for Salmonella concentrations ranging from 2.3 × 102 to 7.8 × 104 CFU/mL. The entire detection process was completed within 8 h, with a very low detection limit of 1 CFU/25 mL (25 g), which was verified by colony counting, and a small degree of interference of 0.17-1.06%. Trace Salmonella from five different serovars in milk and chicken was successfully detected without false negative or false positive results. Furthermore, this study provides a basis to develop a fully automated instrument based on IMBs that includes all steps from sample preparation to chemiluminescence microparticle immunoassay for high-throughput screening of foodborne pathogens. Graphical abstract.

Succinylated Jeffamine ED-2003 coated polycarbonate chips for low-cost analytical microarrays.

Analytical microarrays feature great capabilities for simultaneous detection and quantification of multiple analytes in a single measurement. In this work, we present a rapid and simple method for bulk preparation of microarrays on polycarbonate sheets. Succinylated Jeffamine® ED-2003 was screen printed on polycarbonate sheets to create a polyfunctional shielding layer by baking at 100 °C. After microdispension of capture probes (antibodies, oligonucleotides, or small molecules) in a microarray format, chips were assembled with a flow cell from double-sided tape. It was shown that the shielding layer was firmly coated and suppressed unspecific binding of proteins. Universal applicability was demonstrated by transferring established flow-based chemiluminescence microarray measurement principles from glass slides to polycarbonate chips without loss of analytical performance. Higher chemiluminescence signals could be generated by performing heterogeneous asymmetric recombinase polymerase amplification on polycarbonate chips. Similar results could be shown for sandwich microarray immunoassays. Beyond that, lower inter- and intra-assay variances could be measured for the analysis of Legionella pneumophila Serogroup 1, strain Bellingham-1. Even surface regeneration of indirect competitive immunoassays was possible, achieving a limit of detection of 0.35 ng L-1 for enrofloxacin with polycarbonate microarray chips. Succinylated Jeffamine ED-2003 coated polycarbonate chips have great potential to replace microtiter plates by flow-based chemiluminescence microarrays for rapid analysis. Therefore, it helps analytical microarrays to advance into routine analysis and diagnostics. Graphical abstract ᅟ.

Advances in immunosensors for clinical applications.

Immunoassay technique performs a fast, simple, reliable, and sensitive analysis of different compounds, being applied in several areas of interest such as clinical analysis for medical diagnosis, as well as in environmental analysis, and food quality control. The latest research activities in this field are represented by the attempts to achieve a low limit of detection by developing of new signal amplification strategies, eliminate the interferences, and decrease the cost of analysis.

A Low-Cost Micro-Volume Nephelometric System for Quantitative Immunoagglutination Assays.

Immunoassays have been widely used in scientific research and clinical diagnosis due to their versatile detection capability and high specificity. Immunoagglutination assays are kinds of immunoassay, which can simply and rapidly measure the concentration of analytes. In this work, we developed a low-cost micro-volume nephelometric system for quantitative immunoagglutination assays. We used off-the-shelf components to build the system, and the total cost of key components is only about 20 US dollars. The total detection volume in our system was as low as 3 µL, which could significantly reduce the reagent cost and required sample volume. We further evaluated the system performance via the immunoagglutination assay to measure the concentration of C-reactive protein, a plasma protein with levels rising in response to inflammation. The results demonstrated that our system could measure the concentration of analytes with relatively high sensitivity and precision within four minutes, and has high potential to be applied for clinical diagnostic tests.

A filtration-based rapid test using a partially purified third-stage larval antigen to detect specific antibodies for the diagnosis of gnathostomiasis.

Human gnathostomiasis is an emerging food-borne parasitic disease caused by nematodes of the genus Gnathostoma. Currently, serological tests are commonly applied to support clinical diagnosis. In the present study, a simple and rapid filtration-based test, dot immune-gold filtration assay (DIGFA) was developed using a partially purified antigen of Gnathostoma third-stage larvae (L3). A total of 180 serum samples were tested to evaluate the diagnostic potential of DIGFA for gnathostomiasis. The diagnostic sensitivity and specificity were 96.7% (29/30) and 100% (25/25), respectively. The cross-reactivity with sera from other helminthiasis patients ranged from 0 to 4%, with an average of 1.6% (2/125). DIGFA using a partially purified L3 antigen was not only simple and rapid, but also more accurate than standard assays for the diagnosis of human gnathostomiasis. DIGFA may represent a promising tool for application in laboratories or in the field, without requiring any instrumentation.

Design Principles for Enhancing Sensitivity in Paper-Based Diagnostics via Large-Volume Processing.

In this work, we characterize the impact of large-volume processing upon the analytical sensitivity of flow-through paper-based immunoassays. Larger sample volumes feature greater molar quantities of available analyte, but the assay design principles which would enable the rapid collection of this dilute target are ill-defined. We developed a finite-element model to explore the operating conditions under which processing large sample volumes via pressure-driven convective flow would yield an improved binding signal. Our simulation results underscore the importance of establishing a high local concentration of the analyte-binding species within the porous substrate. This elevated abundance serves to enhance the binding kinetics, matching the time scale of target capture to the period during which the sample is in contact with the test zone (i.e., the effective residence time). These findings were experimentally validated using the rcSso7d-cellulose-binding domain (CBD) fusion construct, a bifunctional binding protein which adsorbs to cellulose in high abundance. As predicted by our modeling efforts, the local concentration achieved using the rcSso7d-CBD species is uniquely enabling for sensitivity enhancement through large-volume processing. The rapid analyte depletion which occurs at this high surface density also permits the processing of large sample volumes within practical time scales and flow regimes. Using these findings, we present guidance for the optimal means of processing large sample volumes for enhanced assay sensitivity.

Antibody-Mediated Small Molecule Detection Using Programmable DNA-Switches.

The development of rapid, cost-effective, and single-step methods for the detection of small molecules is crucial for improving the quality and efficiency of many applications ranging from life science to environmental analysis. Unfortunately, current methodologies still require multiple complex, time-consuming washing and incubation steps, which limit their applicability. In this work we present a competitive DNA-based platform that makes use of both programmable DNA-switches and antibodies to detect small target molecules. The strategy exploits both the advantages of proximity-based methods and structure-switching DNA-probes. The platform is modular and versatile and it can potentially be applied for the detection of any small target molecule that can be conjugated to a nucleic acid sequence. Here the rational design of programmable DNA-switches is discussed, and the sensitive, rapid, and single-step detection of different environmentally relevant small target molecules is demonstrated.