Gold Standard Assay Research Articles

AptaShield: A Universal Signal-Transduction System for Fast and High-Throughput Optical Molecular Biosensing.

Biosensing technologies are often described to provide facile, sensitive, and minimally to noninvasive detection of molecular analytes across diverse scientific, environmental, and clinical diagnostic disciplines. However, commercialization has been very limited mostly due to the difficulty of biosensor reconfiguration for different analyte(s) and limited high-throughput capabilities. The immobilization of different biomolecular probes (e.g., antibodies, peptides, and aptamers) requires the sensor surface chemistry to be tailored to provide optimal probe coupling, orientation, and passivation and prevent nonspecific interactions. To overcome these challenges, here we report the development of a solution-phase biosensor consisting of an engineered aptamer, the AptaShield, capable of universally binding to any antigen recognition site (Fab') of fluorescently labeled immunoglobulins (IgG) produced in rabbits. The resulting AptaShield biosensor relies on a low affinity dynamic equilibrium between the fluorescently tagged aptamer and IgG to generate a specific Förster resonance energy transfer (FRET) signal. As the analyte binds to the IgG, the AptaShield DNA aptamer-IgG complex dissociates, leading to an analyte concentration-dependent decrease of the FRET signal. The biosensor demonstrates high selectivity, specificity, and reproducibility for analyte quantification in different biological fluids (e.g., urine and blood serum) in a one-step and low sample volume (0.5-6.25 μL) format. The AptaShield provides a universal signal transduction mechanism as it can be coupled to different rabbit antibodies without the need for aptamer modification, therefore representing a robust high-throughput solution-phase technology suitable for point-of-care applications, overcoming the current limitations of gold standard enzyme-linked immunosorbent assays (ELISA) for molecular profiling.

Ultrabright NIR AIEgen nanoparticles‐enhanced lateral flow immunoassay platform for accurate diagnostics of complex samples

AbstractAccurate and sensitive near‐infrared (NIR) luminescent lateral flow immunoassay (LFIA) has attracted considerable attention in the field of point‐of‐care testing (POCT). However, the detection accuracy and sensitivity are often compromised by the low fluorescence quantum efficiency (<10%) of the NIR fluorescent probe. Herein, ultrabright NIR AIEgen nanoparticles (PS@AIE830NPs) composed of polystyrene (PS) nanoparticles and NIR aggregation‐induced emission luminogen (AIEgen) with the maximum emission at 830 nm (AIE830) is reported, and its potential to promote an accurate and sensitive detection of complex samples by LFIA is described. The relative quantum yield (QY) of the PS@AIE830NPs was 14.76%, which was superior to that of the polymer embedding method and indocyanine green (ICG)‐based NIR nanoparticles. The PS@AIE830NPs immunolabeled‐LFIA combined with laboratory‐built NIR‐LFIA portable quantitative instruments (detected light range > 800 nm) completely eliminated background interference and allowed highly accurate and sensitive detection without any pre‐treatment steps. The limits of detection (LODs) for aflatoxin B1 (AFB1) in soy sauce, alpha hemolysin (Hla) of Staphylococcus aureus biomarker in joint fluid, and C‐reactive protein (CRP) in human haemolysed samples were 0.01 ng mL−1, 0.02 µg mL−1, and 0.156 mg L−1, respectively, commensurating with those of the corresponding gold standard assays and covering the detection range of interests. It is anticipated that the ultrabright NIR AIEgen nanoparticles will serve as a universally applicable signal probe for NIR‐LFIA diagnostics, promising to expand the range of applications for quantitative detection of complex samples.

Abstract 938: Screening the entire kinase-directed, FDA-approved pharmacopeia against the largest collection of wild-type and mutant kinases reveals many opportunities for drug repurposing and targeted therapy

Abstract We sought to screen all FDA approved, cancer-indicated, kinase targeting drugs using the gold standard radiometric biochemical activity assay (HotSpot) against the largest collection of kinases in the world (>100 drugs against 735 total kinases including 395 wild type and 340 mutants). Virtually every drug examined exhibits polypharmacology inhibiting many kinases. Rarely does the intended target represent the greatest inhibition observed from a drug. Furthermore, most drugs inhibit a select group of mutant kinases, including especially mutants of kinases distinct from the intended target. Within a given kinase, each cancer-identified mutation is often as distinct as the different wild-type kinases are from each other, in terms of inhibition profile. Considering mutants, then, the cancer-relevant druggable kinome is many thousand distinct targets, not 518 “plus a few”. Exposing this reality is a first step towards developing all the therapeutic tools necessary to ultimately defeat cancer. Here, we reveal vast and specific repurposing opportunities among the existing pharmacopeia for tumors that are driven by kinases not previously recognized as targets for certain drugs, and for tumors that have become chemotherapy-resistant through mutation of specific kinases. The most exciting and clinically relevant opportunities were confirmed in cell-based assays, results of which will also be presented. Citation Format: Christian M. Loch, Shuguang Liang, Jianghong Wu, Mia Eason, Alison Goupil, Anthony Acinapura, Jamie Purcell, Maia Dominguez. Screening the entire kinase-directed, FDA-approved pharmacopeia against the largest collection of wild-type and mutant kinases reveals many opportunities for drug repurposing and targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 938.

Abstract 6489: Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations

Abstract We sought to screen all FDA approved, cancer-indicated, kinase targeting drugs using the gold standard radiometric biochemical activity assay (HotSpot) against the largest collection of kinases in the world (>100 drugs against 735 total kinases including 395 wild type and 340 mutants). One of these drugs, mitapivat, is an activator of Pyruvate Kinase. Here, we show mitapivat-driven biochemical inhibition of several, but not all, mutants of c-KIT. We also observed the equal and opposite phenomenon whereby many ATP-competitive kinase inhibitors demonstrate biochemical activation of certain kinases, several of which seem particularly susceptible to the effect. Previous work by other investigators (at Genentech and the Rosen lab, MSKCC) demonstrated that PLX4720 (precursor to Vemurafenib) paradoxically activated wild type BRAF, but not BRAF mutants, by driving dimerization of the kinase in drug concentrations insufficient to inhibit both members of the dimerized pair. Our data suggests this phenomenon may be more widespread than previously appreciated and fully observable in vitro. Such paradoxical activation could be driving both toxicities and inefficacy against cancers expressing some of these activatable kinases. This information could suggest novel opportunities for combination therapy and improve outcomes by contraindicating certain medication from use based on tumor expression profile. Citation Format: Christian M. Loch, Mia Eason, Shuguang Liang, Alison Goupil, Anthony Acinapura, Jamie Purcell, Maia Domiguez. Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6489.

Capillary electrophoresis-mass spectrometry for creatinine analysis in residual clinical plasma samples and comparison with gold standard assay.

When hospitalized, infants, particularly preterm, are often subjected to multiple painful needle procedures to collect sufficient blood for metabolic screening or diagnostic purposes using standard clinical tests. For example, at least 100µL of whole blood is required to perform one creatinine plasma measurement with enzymatic colorimetric assays. As capillary electrophoresis-mass spectrometry (CE-MS) utilizing a sheathless porous tip interface only requires limited amounts of sample for in-depth metabolic profiling studies, the aim of this work was to assess the utility of this method for the determination of creatinine in low amounts of plasma using residual blood samples from adults and infants. By using a starting amount of 5µL of plasma and an injection volume of only 6.7nL, a detection limit (S/N=3) of 30nM could be obtained for creatinine, and intra- and interday precisions (for peak area ratios) were below 3.2%. To shorten the electrophoretic separation time, a multi-segment injection (MSI) strategy was employed to analyze up to seven samples in one electrophoretic run. The findings obtained by CE-MS for creatinine in pretreated plasma were compared with the values acquired by an enzymatic colorimetric assay typically used in clinical laboratories for this purpose. The comparison revealed that CE-MS could be used in a reliable way for the determination of creatinine in residual plasma samples from infants and adults. Nevertheless, to underscore the clinical efficacy of this method, a subsequent investigation employing an expanded pool of plasma samples is imperative. This will not only enhance the method's diagnostic utility but also contribute to minimizing both the amount and frequency of blood collection required for diagnostic purposes.

Electrochemical Biosensors for the Detection of Viruses: Must-Have Products or Just Science for Publication?

Electrochemical Biosensors for the Detection of Viruses: Must-Have Products or Just Science for Publication?

On-Site Melanoma Diagnosis Utilizing a Swellable Microneedle-Assisted Skin Interstitial Fluid Sampling and a Microfluidic Particle Dam for Visual Quantification of S100A1.

Malignant melanoma (MM) is the most aggressive form of skin cancer. The delay in treatment will induce metastasis, resulting in a poor prognosis and even death. Here, a two-step strategy for on-site diagnosis of MM is developed based on the extraction and direct visual quantification of S100A1, a biomarker for melanoma. First, a swellable microneedle is utilized to extract S100A1 in skin interstitial fluid (ISF) with minimal invasion. After elution, antibody-conjugated magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) are introduced. A high expression level of S100A1 gives rise to a robust binding between MMPs and PMPs and reduces the number of free PMPs. By loading the reacted solution into the device with a microfluidic particle dam, the quantity of free PMPs after magnetic separationis displayed with their accumulation length inversely proportional to S100A1 levels. A limit of detection of 18.7ngmL-1 for S100A1 is achieved. The animal experiment indicates that ISF-based S100A1 quantification using the proposed strategy exhibits a significantly higher sensitivity compared with conventional serum-based detection. In addition, the result is highly comparable with the gold standard enzyme-linked immunosorbent assay based on Lin's concordance correlation coefficient, suggesting the high practicality for routine monitoring of melanoma.

Investigation of NK cell function against two target hematological cell line using radioactive chromium assay

NK (Natural killer) cells are a special population of peripheral blood lymphocytes that kill virus-infected cells as well as tumor cells. For testing NK cell function, the classic gold standard assay has been used for a long time, determining the activity from target tumor cells using radioactive chromium in cell cultures for 4h. In this study two hematological cell lines K562 and MDS where used and target and results showed different sensitivity to killing by NK cells separated from healthy volunteers. Results have been shown that MDS release significantly more radioactive chromium indicating higher degree of necrosis during cell culture. In addition, K562 cell line is better target for NK killing in all different E:T ratio in comparison to MDS cell line previously described. Based on this, it is suggested that K562 cells be continues used in the future as better target for investigation NK killing.

Laboratory-based evaluation of the 4th-generation AlereTM HIV Combo rapid point-of-care test.

Mozambique is a high-prevalence country for HIV and early detection of new HIV infections is crucial for control of the epidemic. We aimed to evaluate the accuracy of the 4th-generation rapid diagnostic test (RDT) AlereTM HIV Combo in detecting acute and seroconverted HIV-infection, among sexually-active women attending three clinical health centers in Maputo, Mozambique. Women aged 14-55 years (n = 920) seeking care at the Mavalane Health Area, Maputo (February 2018-January 2019) were included, and blood specimens sampled. Sociodemographic and sexual behavior data were collected. Point-of-care HIV testing was performed using Alere DetermineTM HIV-1/2 and Uni-GoldTM HIV-1/2. All samples were also tested using Enzygnost® HIV Integral 4 and Innotest® HIV Antigen mAb in laboratory. The 4th-generation RDT AlereTM HIV Combo was evaluated on serum samples in the laboratory. Finally, Innotest® HIV Antigen mAb, Enzygnost® HIV Integral 4 (Ag/Ab), and HIV RNA quantification acted as gold standard assays in the evaluation of AlereTM HIV Combo test for HIV antigen detection (in clinical samples and in three HIV-1 seroconversion panels). The antibody component of the 4th generation AlereTM HIV Combo RDT demonstrated a sensitivity and specificity of 100% examining clinical samples. However, the test did not detect HIV p24 antigen in any clinical samples, while Innotest® HIV Antigen mAb, verified by Enzygnost® HIV Integral 4 (Ag/Ab) and/or HIV RNA quantification, detected HIV antigen in six clinical samples. Furthermore, the AlereTM HIV Combo RDT had a low sensitivity in the detection of HIV p24 antigen in seroconversion panels. The HIV prevalence among the examined women was 17.8%. The 4th-generation RDT AlereTM HIV Combo showed similar sensitivity to the 3rd-generation RDTs to detect seroconverted HIV-infections. However, the sensitivity for detection of HIV p24 antigen and diagnosing acute HIV infections, before seroconversion, was low. There is an urgent need to develop and evaluate simple and affordable POC tests with high sensitivity and specificity for diagnosing individuals with acute HIV infection in resource-limited settings with high HIV prevalence.

A novel high-throughput microwell outgrowth assay for HIV-infected cells.

Although antiretroviral therapy (ART) is effective at suppressing HIV replication, a viral reservoir persists that can reseed infection if ART is interrupted. Curing HIV will require elimination or containment of this reservoir, but the size of the HIV reservoir is highly variable between individuals. To evaluate the size of the HIV reservoir, several assays have been developed, including PCR-based assays for viral DNA, the intact proviral DNA assay, and the quantitative viral outgrowth assay (QVOA). QVOA is the gold standard assay for measuring inducible replication-competent proviruses, but this assay is technically challenging and time-consuming. To begin progress toward a more rapid and less laborious tool for quantifying cells infected with replication-competent HIV, we developed the Microwell Outgrowth Assay, in which infected CD4 T cells are co-cultured with an HIV-detecting reporter cell line in a polydimethylsiloxane (PDMS)/polystyrene array of nanoliter-sized wells. Transmission of HIV from infected cells to the reporter cell line induces fluorescent reporter protein expression that is detected by automated scanning across the array. Using this approach, we were able to detect HIV-infected cells from ART-naïve people with HIV (PWH) and from PWH on ART with large reservoirs. Furthermore, we demonstrate that infected cells can be recovered from individual rafts and used to analyze the diversity of viral sequences. Although additional development and optimization will be required for quantifying the reservoir in PWH with small latent reservoirs, this assay may be a useful prototype for microwell assays of infected cells.IMPORTANCEMeasuring the size of the HIV reservoir in people with HIV (PWH) will be important for determining the impact of HIV cure strategies. However, measuring this reservoir is challenging. We report a new method for quantifying HIV-infected cells that involves culturing cells from PWH in an array of microwells with a cell line that detects HIV infection. We show that this approach can detect rare HIV-infected cells and derive detailed virus sequence information for each infected cell.

Comparative study between gold standard real-time RT-PCR assay and rapid antigen test for detection of COVID-19 in Sylhet CMH

Background: The notorious pandemic coronavirus disease 2019 (COVID-19) has been spread all over the world. Its third pandemic wave has been completed and now a fourth wave is running over many countries. Almost 60 million people have been infected and more than 5.7 million people have died. Till now almost 1.2 million people have been infected and about 30000 people have died in Bangladesh from Covid-19. It has not only taken away human lives but also has put a tremendous impact on our society and economy. So early detection of this virus and isolating the patients have a significant role to control the disease. Henceforth there is an urgent need for simple, accurate and rapid identifications of C0VID -19. Rapid antigen tests can provide timely results, which is of particular importance in a primary setting. So, Performance of the Rapid antigen detection test (RAT) should be evaluated and compared with gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19. Methods: Specimens were collected from both naso and oropharyngeal region from 1000 patients who reported to the flu centre in CMH Sylhet with the complaints of fever, cough & headache for detection of COVID virus-2 RNA by rapid antigen (RAT) and RT-PCR test. We used real time RT-PCR kit (Sansure, Biotech china, gene RdRP & N) and COVID-19 Ag Kit (Wondfo, Republic of China) for detection of COVID-19 RNA. Results: Out of 1000 samples 158 were positive and 842 were negative by real-time RT-PCR assay where 154 samples were positive, and 846 cases were negative by rapid antigen test for severe acute respiratory syndrome (SARS) CoV-2. The duration from the onset of symptoms to laboratory test in all suspected cases ranged from 0 to 02 days (with a median of 01 days). The rapid SARS-CoV-2 antigen detection tests sensitivity and specificity were 97.29% (95% CI, 90.06–99.89%) and 97.94% (95% CI, 97.26–98.57%), respectively. Seven samples were found negative in RAT but were found positive in RT-PCR, other three samples were found positive in RAT while they were found negative in RT-PCR. Conclusions: It is observed that most rapid antigen tests for COVID-19 are significantly comparable with RT-PCR tests and had enough sensitivity and specificity for 158 individuals, infected with severe acute respiratory syndrome-CoV-2.

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification.

The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa, which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa. The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.

Reference spectrophotometric values for glucose-6-phosphate dehydrogenase activity in two-to six-month-old infants on the Thailand-Myanmar border.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency represents a barrier to the full deployment of anti-malarial drugs for vivax malaria elimination and of first-line antibiotics. Lack of established reference ranges for G6PD activity in breast-fed infants puts them at risk of drug-induced haemolysis and restricts access to safe treatment of their mothers. The present work was undertaken to establish age-specific G6PD normal values using the gold standard spectrophotometric assay to support the future clinical use of tafenoquine in lactating women and safer antibiotic treatment in infants. Spectrophotometric results collected at the Thai-Myanmar border from 78 healthy infants between the ages of 2 and 6 months showed a trend of decreased enzymatic activity with increasing age (which did not reach statistical significance when comparing 2-3 months old against 4-6 months old infants) and provided a reference normal value of 100% activity for infants 2-6 months old of 10.18IU/gHb. Normal reference G6PD activity in 2-6-month-old infants was approximately 140% of that observed in G6PD normal adults from the same population. Age specific G6PD activity thresholds should be used in paediatric populations to avoid drug-induced haemolysis.

Virulence evaluation of Israeli Marek's disease virus isolates from commercial poultry using their meq gene sequence.

Fifty-sevenGallid alphaherpesvirus 2 (GaHV-2) isolates, collected during a 30-year period (1990-2019)from commercial poultry flocks affected by Marek's disease (MD), were molecularly characterised. The GaHV-2 meq gene was amplified and sequenced to evaluate the virus virulence, based on the number of PPPPs within the proline-rich repeats (PRRs) of its transactivation domain. The present illustration of virus virulence evaluation on a large scale of field virus isolates by molecular analysisexemplifies the practical benefit and usefulness of the molecular marker in commercial GaVH-2 isolates. The alternative assay of GaVH-2 virulence pathotyping is the classical Gold Standard ADOL method, which is difficult and impossible to employ on a large scale using the Specific Pathogen Free (SPF) chicks of the ADOL strainskept in isolators for two months. The phylogenetic analysis performed in the present study showed that the meq gene amino acid sequences of the 57 Israeli strains divide into 16 phylogenetic branches. The virulence evaluation was performed in comparison with 36 GaHV-2 prototype strains, previously characterised by the in vivo Gold Standard ADOL assay. The results obtained revealed that the GaHV-2 strains circulating in Israel have evolved into a higher virulence potential during the years, as the four-proline stretches number in the meq gene decreased over the investigated period, typically of very virulent virus prototypes. The present study supports the meq gene molecular markers for the assessment of field GaVH-2 strains virulence.

Comparison of Xpert Xpress SARS-CoV-2 Assay Compared with Standard M nCoV Real-Time PCR: Prospective Study.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can be diagnosed using rapid real-time polymerase chain reaction (PCR), real-time reverse transcription PCR (rRT-PCR), or rapid antigen testing. Among these, rRT-PCR is considered the gold standard assay. The Xpert Xpress SARS-CoV-2 assay is a rapid, real-time PCR test; approved by the Korean Disease Control and Prevention Agency in 2020. Current performance of the Xpert assay (Xpert) with the STANDARD M nCoV Real-Time Detection kit (SD) were determined. All samples used by the SD test team were immediately transferred to the Xpert test team within 24 hours. Both tests were conducted between April 2023 and July 2023. Exclusion criteria were studies which show either inconclusive, invalid, or erroneous results. Positive rate, sensitivity, specificity, overall concordance rate, positive concordance rate, discordance rate, false-positive rate, and false-negative rates of the Xpert assay with the STANDARD M nCoV Real-Time Detection kit were determined. Samples from 347 patients (174 men and 173 women) with a median age of 60 years (range; 6 - 90 years) were included. Positive rate, sensitivity, specificity, overall concordance rate, positive concordance rate, discordance rate, false-positive rate, and false-negative rates of the Xpert assay were 11.2%, 82.1%, 95.0%, 93.9%, 6.6%, 6.1%, 41.0%, and 1.6%, respectively. COVID-19 results from Xpert should be confirmed through rRT-PCR because of low sensitivity (82.1%) and high false-positive rate (41.0%).

RT-RPA-PfAgo detection platform for one-tube simultaneous typing diagnosis of human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSVA) and B (HRSVB) of HRSV. Next, the Pyrococcus furiosus Argonaute (PfAgo) protein utilizes small 5'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/μL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSVA and HRSVB infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings.

Microfluidic particle counter visualizing mucosal antibodies against SARS-CoV-2 in the upper respiratory tract for rapid evaluation of immune protection.

Mucosal antibodies in the upper respiratory tract are the earliest and most critical responders to prevent respiratory infections, providing an indication for the rapid evaluation of immune protection. Here, we report a microfluidic particle counter that directly visualizes mucosal antibody levels in nasal mucus. The mucosal anti-SARS-CoV-2 spike receptor binding domain (RBD) antibodies in nasal secretions first react with magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are surface-modified to form a "MMPs-anti-spike RBD IgG-PMPs" complex when RBD is present. After magnetic separation and loading into the microfluidic particle counter, the free PMPs, which are reduced with increasing anti-spike RBD IgG antibody levels, are trapped by a microfluidic particle dam and accumulate in the trapping channel. A sensitive mode [limit of detection (LOD): 14.0 ng mL-1; sample-to-answer time: 70 min] and an equipment-free rapid mode (LOD: 37.4 ng mL-1; sample-to-answer time: 20 min) were achieved. Eighty-seven nasal secretion (NS) samples from vaccinees were analyzed using our microfluidic particle counter, and the results closely resemble those of the gold-standard enzyme-linked immunosorbent assay (ELISA). The analysis shows that higher antibody levels were found in convalescent volunteers compared to noninfected volunteers. Together, we demonstrate a rapid kit that directly indicates immune status, which can guide vaccine strategy for individuals and the government.

Comparison of different algorithms for lupus anticoagulant detection: a single-center experience

BackgroundThe laboratory tests for lupus anticoagulant (LA) detection comprise complex and multistep coagulation testing procedures. There is no established gold standard assay or direct comparison of algorithms as recommended by different guidelines. ObjectivesThis study aimed to evaluate and compare the LA detection performance of different laboratory algorithms suggested by the existing guidelines. MethodsThe routine LA test data of 1801 plasma samples, including 188 LA-positive and 1613 LA-negative samples, were re-evaluated by applying the algorithms recommended by existing guidelines and were interpreted using various methods. Diagnostic performance indices for each LA detection algorithm were compared with those of the other algorithms. The efficacies of the different interpretation methods were analyzed to determine a suitable interpretation methodology for each assay. ResultsThe diagnostic performance for detecting LA varied by the algorithm and method of interpretation used. All laboratory algorithms displayed exceptional diagnostic performance with all diagnostic parameters of >90.0%. Nearly perfect agreement was observed in all algorithms when compared to the Clinical and Laboratory Standards Institute 2014 guideline interpreted by normalized screen-to-confirm ratio (NSCR) and mixing test–specific cutoff (MTC), as a reference assay (Cohen’s kappa coefficient, >0.90 [range, 0.94-1.00]). A combination of the index of circulating anticoagulant and NSCR was optimal for interpreting the activated partial thromboplastin time–based test, whereas a combination of the MTC and NSCR was suitable for the diluted Russell’s viper venom time–based test. ConclusionAll laboratory algorithms showed equivalent diagnostic performance. Establishing the best method of interpretation for each assay is recommended to improve LA detection performance.

Evaluation of Mitochondrial Respiratory Function in Murine Splenocytes.

Accumulated evidence has demonstrated a key role of mitochondria in the onset and progression of autoimmune disease. Understanding and modulation of mitochondrial dysfunction could provide new molecular targets for both preventive and therapeutic intervention in disease management. The ability to assess mitochondrial function has enabled rheumatologists to advance the understanding of the contribution of cellular metabolism in cellular physiology and disease pathology and etiology. Direct measurement of oxygen consumption rate using an Agilent Seahorse XF measurement system has been widely used as the gold-standard assay for evaluating mitochondrial function in cells. Using this assay system, measurement of parameters of basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and nonmitochondrial respiration can be achieved. An optimized method which works well in mouse splenocytes and a Jurkat cell line is presented in this chapter.

(Invited) Lab-in-a-Tube Design Enables a Rapid Diagnosis of Infectious Disease

Disease diagnosis increasingly relies on molecular tests that are often limited by infrastructure constraints. For example, tuberculosis (TB) remains a leading cause of death from infectious disease, but an estimated 4.2 million (39.6%) new TB cases were not diagnosed in 2021 and only 57% were confirmed by gold-standard sputum culture or PCR-based tests, partially due to limited accessibility. New point-of-care tests are thus urgently needed to address coverage disparities for chronic, malignant, and infectious diseases.We have developed two smartphone-based assays that can address two diagnostic shortfalls: an on-chip interferon-gamma release assay (IGRA) that detects a T-cell activation response induced by pathogen antigens in fingerstick blood samples, and a lab-in-tube assay that employs CRISPR to sensitively detect pathogen DNA in sputum. Both approaches translate current diagnostic approaches to point-of-care devices that require minimal equipment, user expertise, and time.Results from our on-chip IGRA also correlated with clinical IGRA results when we employed two proteins associated with TNF-alpha receptor signaling (4-1BB and OX-40) to analyze T-cell activation, indicating this approach could detect infections in individuals with compromised interferon-gamma responses; as observed in HIV patients, who are at increased risk for TB. On-chip IGRA results from a SARS-CoV-2-specific assay also corresponded with both infection and vaccination history, suggesting this data could be employed as a high-throughput, high-capacity test to evaluate the effect of vaccination or previous infection to confer a protective immune response, evaluate its change over time, and predict the response to new variant strains to guide healthcare decisions. Mitogen-induced results could also guide immunotherapy decisions, as reduced IGRA values in patients receiving immune checkpoint inhibitors predict poor treatment response, reduced progression-free survival, and increased risk for interstitial pneumonia, while their change after adoptive T-cell therapy can be an independent predictor for overall survival. Results from our lab-in-tube TB DNA assay also had robust diagnostic performance when compared to gold-standard clinical assays but lacked their more extensive personnel, workflow, and infrastructure requirements and were performed in a closed-tube system that protected user from exposure to infectious material. This lab-in-tube approach also permits parallel multiplex analyses to be performed in a single tube, and thus can be employed to detect mutations associated with drug-resistance to guide treatment decisions. For example, we found that an assay with an optimized guide RNA accurately detected a single-nucleotide polymorphism (rpoB S450L) responsible for the majority of rifampin-resistant TB cases.Both these self-contained handheld devices can employ lyophilized reagents to limit cold-chain concerns, can be readily adapted to diagnose other disease conditions by substituting other disease-specific antibodies or guide RNAs, use rechargeable batteries to avoid infrastructure limitations, and can directly transmit results to central sites to improve disease control efforts.