Antigen Immunoassay Research Articles

Development and Evaluation of an Immunochromatographic Strip and a Magnetic Chemiluminescence Immunoassay for Detection of Porcine Circovirus Type 2 Antigen

Porcine circovirus type 2 (PCV2) is the main and primary causative agent of Postweaning Multisystemic Wasting Syndrome (PMWS). To date, immunoperoxidase monolayer assay (IPMA), indirect immunofluorescent assay (IFA), and enzyme linked immunosorbent assay (ELISA) are the most commonly diagnostic methods for detecting PCV2 antigens. However, these methods require specialized equipment and technical expertise and are suitable for laboratory use only. This study aims to develop an immunochromatographic strip and a magnetic chemiluminescence immunoassay for the detection of PCV2 antigens. The recombinant protein was constructed using a prokaryotic expression system, and the polyclonal antibody was obtained by animal experiments. Polystyrene microspheres are used as solid phase carriers to covalently bind to the amino groups of proteins to form immunoprobes. Monodisperse beads are covalently bound to antigens or antibodies as solid phases to bind antibodies or antigens in the liquid phase in a superior manner, thereby capturing and separating antigens and antibodies in the liquid phase. The immunochromatographic strip is qualitative detection method, this method can detect PCV2a strain, PCV2b strain, and PCV2d strain. The immunochromatographic strip had minimum detection limits of 102.89TCID50/0.1 mL, 103.19TCID50/0.1 mL, and 103.49TCID50/0.1 mL for PCV2a/LG, PCV2b/SH, and PCV2d/JH. The results of testing PEDV (CV777 strain), PRV (HB2000 strain), CSFV (WH-09 strain), PRRS (JXA1-R strain), PPV (WH-1 strain), and ASFV (SD strain) were negative. The agreement between the immunochromatographic strip and the ELISA kit was 93.33% (140/150) and the Kappa was 0.866 (Kappa > 0.81). On the premise of ensuring sensitivity, the most important feature of the immunochromatographic strip is that this method can save time when testing; results can be obtained within 5 to 10 min. Magnetic chemiluminescence immunoassay is quantitative detection method; this method can detect PCV2 Cap proteins in swine serum, the linear range of this method was 0.25 ng/mL to 32 ng/mL and R2 of the standard curve was 0.9993. The limit of detection (LOD) is 0.051 ng/mL and the limit of quantitation (LOQ) is 0.068 ng/mL. The agreement between the magnetic chemiluminescence immunoassay and the ELISA kit (test PCV2 Cap proteins) was 97.14% (68/70). This method took less than 30 min to achieve results, which is less than the ELISA kit. The results of this study show that immunochromatographic strip and magnetic chemiluminescence immunoassay for PCV2 antigens had great sensitivity and specificity, which lays the foundation for PCV2 clinical detection.

Facing Clostridioides difficile infection in a resource - Limiting setting.

An HIV- positive patient with Kaposi's sarcoma and undergoing chemotherapy, had been on antibiotic therapy for around 14 days with ceftriaxone and imipenem. He was admitted with a 5-day evolution of watery diarrhea followed by rectorrhagia. His CD4 level was 226 cells/ mm3. Colonoscopy was done with visualization of scattered pseudomembranes separated by areas of healthy mucosa, covering the entire intestine. Due to low resources the diagnosis of Clostridioides difficile (C.D) infection was proposed and treatment with oral metronidazole were made. Diarrhea improved after 5 days of treatment. Endoscopic findings were suggestive of CD infection. Other less common causes of pseudomembranous colitis, were unlikely according to clinical history. The probability of CD infection increased due the combination of risk factors such as HIV immunosuppression, chemotherapy, antibiotic therapy and endoscopic findings. The lack of Polymerase chain reaction, enzyme immunoassay for CD glutamate dehydrogenase antigen and toxins A and B was a diagnosis limitation. All these diagnostic tools were not available in the hospital. Oral vancomycin is the treatment of choice, however it was not available in the hospital, so treatment with oral metronidazole was made and the previous antibiotics used were discontinued. The patient progressed with clinical improvement.

Positive impact of a diagnostic stewardship intervention on syndromic panel ordering practices and inappropriate C. difficile treatment.

Multiplex polymerase chain reaction (PCR) panels for stool testing may be used to diagnose Clostridioides difficile, which can circumvent more appropriate targeted C. difficile testing, resulting in treatment of incidentally detected colonization. We sought to reduce C. difficile diagnosis via a gastrointestinal pathogen panel (GIPP). Quasi-experimental, pre/post, retrospective cohort study from January 1, 2022, to January 31, 2024. Mayo Clinic Arizona-a single academic medical center and associated clinics. Adult patients receiving C. difficile testing and/or treatment. Preferred C. difficile testing consisted of glutamate dehydrogenase and toxin antigen immunoassay, followed by toxin gene testing for discrepant results. The GIPP contained 22 targets during the baseline period with C. difficile removed during the postintervention period. Surveys were provided to provider and nursing groups, separately, to identify C. difficile ordering practices and knowledge gaps. At baseline, from January 1, 2022, to January 31, 2023, 2,772 GIPPs were completed for 2,307 unique patients (∼7 per day), primarily for outpatients (1,805 of 2,772, 65%). The most common positive target was C. difficile (517 of 1,018, 51%), which resulted in treatment for C. difficile infection in 94.9% (337 of 355) of cases. Following GIPP C. difficile target removal, GIPP orders decreased from 3.23 to 2.7 per 1,000 patient visits (P < .001). Prescribing of C. difficile treatments decreased in the postintervention period in inpatient and outpatient settings. There were no cases of delayed C. difficile diagnosis during the postintervention period. Removing C. difficile from the GIPP resulted in effective diagnostic and antimicrobial stewardship without resulting in delayed diagnoses.

Screening and Characterization of Sialic Acid-Binding Variable Lymphocyte Receptors from Hagfish.

Sialic acid is a diverse group of monosaccharides often found on the termini of N- and O-linked glycans as well as being components of glycoconjugates. Hypersialylation has been associated with the progression of chronic inflammation-mediated diseases such as cardiovascular disease and cancer. Given its role in infection and disease-related processes, sialic acid is a promising target for therapeutic approaches that utilize carbohydrate-binding molecules. In this study, we screened for sialic acid-recognizing variable lymphocyte receptors (VLRBs) or ccombodies from inshore hagfish (Eptatretus burgeri) using a synthetic Neu5Ac-glycoconjugate as an antigen in immunoassay. Resulting ccombodies, 2D8, 5G11, 4A1, and 5F8 were further characterized in terms of their binding activity and specificity. A competitive ELISA using free haptens showed strong inhibition using either N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). The half-maximal inhibitory concentrations (IC50) for Neu5Ac ranged from 7.02 to 17.06 mM, with candidates 4A1 and 5G11 requiring the least and highest amounts, respectively. IC50 values for Neu5Gc ranged from 8.12 to 13.91 mM, for 4A1 and 5G11, respectively. Candidate ccombodies also detected naturally occurring sialic acid from known sialoglycoproteins using a dot blot assay. Neu5Gc-5G11 and Neu5Ac-2D8 yielded the strongest and weakest docking interactions with affinity values of -5.9 kcal/mol and -4.9 kcal/mol, respectively. Hydrogen bonding and hydrophobic interactions were predicted to be the predominant noncovalent forces observed between the ccombodies and sialic acid. This study demonstrates that glycan-binding VLRBs from hagfish hold promise in augmenting the glycobiologists' toolkit in investigating the roles of glycans in human and animal health and disease.

B-267 Validation of the Newly Developed Fully Automated Chemiluminescent Enzyme Immunoassay (CLEIA) Reagent for Measurement of Cyclosporine in the Blood

Abstract Background Cyclosporine (CsA) is an immunosuppressive drug requiring therapeutic drug monitoring (TDM) for its narrow-ranged medication level. Approximately 60% of CsA in the blood exist as molecular complexes with proteins like cyclophilin in lymphocytes and erythrocytes. Measurement of CsA in the blood by immunoassay or liquid chromatography-tandem mass spectrometry (LC-MS/MS) requires an extraction process for CsA from the whole blood specimen, which is often manual complicated and time-consuming process causing a risk of variation in the measurement values among different operators. In addition, the small (cyclic polypeptide composed of 11 amino acids) and hydrophobic property of CsA, make it difficult to establish antibodies for a sandwich immunoassay. In this study, we have validated a novel fully automated sandwich immunoassay reagent for measurement of blood CsA that used a combination of newly-established CsA antibodies and iTACT® (immunoassay for total antigen including complex via pretreatment) technology. Methods The reagent was developed and validated on a fully automated immunoassay system, LUMIPULSE® L2400 (FUJIREBIO INC). Twenty uL whole blood specimens are sampled after mixed with suction/discharge stirring to prevent that blood cells settle to the bottom of a container on the analyzer, and then treated with a pretreatment solution to obtain free CsA. The treated samples are assayed by a two-step sandwich immunoassay. Limit of Detection (LoD), Limit of Quantitation (LoQ), within-laboratory precision, linearity, no interference, and the correlation of this assay and LC-MS/MS were validated, following the current CLSI guidelines protocols. Data were analyzed using Microsoft Excel statistical tool Analyse-it. Results The LoD and the LoQ were 2.7 ng/mL and 10.8 ng/mL respectively. The within-laboratory precision showed CV 1.6-2.9 %. Linearity was demonstrated over the range 13.1-2381.5 ng/mL. No interference was observed with unconjugated (≤19.7 mg/dL) or conjugated bilirubin (≤20.1 mg/dL), chyle (≤1480 FTU), RF (rheumatoid factor, ≤1000 IU/mL), and HAMA (human anti-mouse antibody, ≤1010 ng/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.0 and 1.0, respectively (N=120). Conclusions The performance of our novel CsA assay, which use LUMIPULSE L2400, was satisfactory for routine analysis. A unique fully-automated process, including the pretreatment process of whole blood specimens, for measurement would contribute the low LoQ and the low Within-laboratory CV. In addition, the results are obtained in 35-minute. We expect this novel immunoassay could reduce the burden on operators and turnaround time.

Comparison of a dual antibody and antigen HCV immunoassay to standard of care algorithmic testing.

The Centers for Disease Control and Prevention (CDC) guidelines for hepatitis C virus (HCV) testing, although effective, may miss crucial diagnostic opportunities. The goal of this study was to assess the utility of an antibody (Ab) and antigen (Ag) combination immunoassay as an alternative to traditional HCV screening. Remnant specimens from 1,341 patients with concurrent third-generation serologic (Roche anti-HCV-II) and nucleic acid amplification testing (NAAT) were assessed using the HCV Duo Ab/Ag immunoassay (Roche). Patient demographics, risk factors, and standard of care (SOC) laboratory results from the medical records were recorded. Overall, 99.0% (197/199) of the HCV Duo Ab+/Ag+specimens accurately identified active infections as confirmed by NAAT, and 99.9% (670/671) Ab-/Ag- samples corresponded to those without HCV infections. Individually, the HCV Duo Ab component demonstrated a 95.6% positive percent agreement (PPA) (95% CI = 93.8-96.9) and 99.1% negative percent agreement (NPA) (98.8-99.6) compared with SOC anti-HCV II Ab assay. The HCV Duo Ag had a 73.5% PPA (67.9-78.4) and 99.8% NPA (99.3-100) with NAAT. Among RNA+ specimens, 73.4% (197/267) were HCV Duo Ag+, and 265/267 (99.3%) were successfully detected on the HCV Duo Ab component. Notably, 5/7 (71.4%) Ab-/RNA +specimens were detected by HCV Duo, which would have been missed by traditional algorithmic testing. Fourth generation HCV Duo Ab/Ag assay demonstrated comparable performance to SOC testing and shortens the diagnostic window but does not eliminate the need for NAAT in all patients. Ab/Ag testing identified several Ab-/RNA+ cases, a subgroup often undiagnosed by current algorithmic testing, demonstrating promise for improved diagnostic efficiency and accuracy in HCV detection.IMPORTANCEThis study highlights the potential of a combined hepatitis C virus (HCV) Duo antibody (Ab) and antigen (Ag) immunoassay to improve early detection of HCV infections. Traditional Ab-only screening methods recommended by the Centers for Disease Control and Prevention may miss early-stage infections. The HCV Duo assay showed high accuracy, detecting nearly all active infections confirmed by nucleic acid amplification testing. Dual detection of HCV Ab and Ag shortens the diagnostic window, enabling intervention and treatment in a single visit, which is crucial for improving patient outcomes and reducing HCV transmission, especially in areas with limited access to confirmatory molecular testing.

Nanozyme-mediated signal amplification on g-C3N4/NaBiO3 Z-scheme heterojunction photoelectrode toward ultrasensitive photoelectrochemical immunoassay for prostate-specific antigen

A sandwich-type photoelectrochemical (PEC) immunosensor for prostate-specific antigen (PSA) detection was developed using the g-C3N4/NaBiO3 (CN/NBO) Z-scheme heterojunction as the photoelectrode and glutathione-Cu/Cu2O nanozyme (GSH-Cu/Cu2O NPs) as the signal amplifier. The unique Z-scheme charge-carrier migration pathway of the CN/NBO heterojunction significantly promotes the separation and migration of photogenerated electron-hole pairs, producing a high photocurrent response of the photoelectrode. GSH-Cu/Cu2O NPs with ascorbic acid oxidase (AAO) activity were introduced into the PEC immunosensing system through immunoreactions for signal amplification. Signal amplification was achieved through double photocurrent quenching of the GSH-Cu/Cu2O NPs on the photoelectrode. On the one hand, GSH-Cu/Cu2O NPs as AAO catalyzed ascorbic acid (AA), an electron donor in the PEC system, to produce dehydroascorbic acid (DHA), leading to a decrease in the photocurrent response. On the other hand, the GSH-Cu/Cu2O NPs prevented AA transfer from the detection solution to the interface of the photoelectrode, further aggravating the PEC signal quenching effect. Benefiting from the excellent performance of the Z-scheme heterojunction-based photoelectrode, the dual signal quenching effect of GSH-Cu/Cu2O NPs, the PEC immunosensor showed an ultralow detection limit of 5.1 × 10−15 g mL−1. By coupling a Z-scheme heterojunction-based photoelectrode with a nanozyme-mediated signal amplification strategy, this study provides a novel avenue for the construction of ultrasensitive and convenient biosensing systems.

Clinical performance of Circulating HBV RNA and iTACT-HBcrAg Assays in HBeAg-negative and HBsAg-cleared Chronic Hepatitis B Patients.

Hepatitis B virus (HBV) RNA and hepatitis B core-related antigen (HBcrAg) have been reported to reflect the transcriptional activity of covalently closed circular HBV DNA. We retrospectively investigated the proportions of quantifiable serum HBV RNA and immunoassay for total antigen including complex via pretreatment-hepatitis B core-related antigen (iTACT-HBcrAg) in chronic hepatitis B patients negative for hepatitis B e antigen (HBeAg) and/or with hepatitis B surface antigen (HBsAg) seroclearance. This study included 246 HBeAg-negative HBV-infected patients, who comprised 13 with liver cirrhosis (LC, the LC group), 118 chronic hepatitis (CH, the CH group), and 115 inactive carriers (IC, the IC group), and 44 patients with HBsAg seroclearance. iTACT-HBcrAg and HBV RNA levels were determined using stored serum samples. Higher proportions of the patients had quantifiable iTACT-HBcrAg than HBV RNA in all groups of HBeAg-negative patients (iTACT-HBcrAg: 84.6%, 90.7%, 35.7%, HBV RNA: 23.1%, 26.3%, 14.8%, for the LC, CH, IC groups). With HBsAg seroclearance (HBsAg <0.05 IU/mL), the proportions of quantifiable samples for HBV RNA were also lower than iTACT-HBcrAg (0% for HBV RNA). Thus, iTACT-HBcrAg was more often detectable than circulating HBV RNA in this study population. Further long-term prospective evaluation of iTACT-HBcrAg is desirable for its utilization in clinical practice.

Comparative performance of the Platelia Aspergillus Antigen and Aspergillus Galactomannan antigen Virclia Monotest immunoassays in serum and lower respiratory tract specimens: a "real-life" experience.

The Platelia Aspergillus Antigen immunoassay is the "gold standard" for Aspergillus galactomannan (GLM) measurement in sera and bronchoalveolar lavage (BAL) for the diagnosis of invasive pulmonary aspergillosis (IPA). We evaluated the performance of the Aspergillus GLM antigen Virclia Monotest compared to the Platelia assay. A total of 535 specimens [320 sera, 86 bronchial aspirates (BAs), 70 BAL, and 59 tracheal aspirates (TAs)] from 177 adult patients (72 hematological, 32 Intensive Care Unit, and 73 hospitalized in other wards) were processed for GLM testing upon clinical request. One patient had proven IPA, and 11 had probable disease. After excluding indeterminate Virclia results (n = 38), 396 specimens yielded concordant results (56 positive and 340 negative) and 101 discordant results (Virclia positive/Platelia negative, n = 95). The overall agreement between immunoassays was higher for sera (κ 0.56) than for BAL (κ ≤ 0.24) or BAS and TA (κ ≤ 0.22). When considering all specimen types in combination, the overall sensitivity and specificity of the Virclia assay for the diagnosis of proven/probable IPA were 100% and 65%, respectively, and for the Platelia immunoassay, sensitivity and specificity were 91.7% and 89.4%, respectively. The correlation between index values by both immunoassays was strong for serum/BAL (ρ = 0.73; P < 0.001) and moderate for BAS/TA (Rho = 0.52; P = 0.001). The conversion of Virclia index values into the Platelia index could be derived by the formula y = (11.97 * X)/3.62 + X). Data from GLM-positive serum/BAL clinical specimens fitted the regression model optimally (R2 = 0.94), whereas that of BAS and TA data did not (R2 = 0.11). Further studies are needed to determine whether the Virclia assay may be an alternative to the Platelia assay for GLM measurement in sera and lower respiratory tract specimens.IMPORTANCEGalactomannan detection in serum or bronchoalveolar fluid specimens is pivotal for the diagnosis of invasive pulmonary aspergillosis (IPA). The Platelia Aspergillus Antigen immunoassay has become the "gold standard" for Aspergillus GLM measurement. Here, we provide data suggesting that the Virclia Monotest assay, which displays several operational advantages compared with the Platelia assay, may become an alternative to the Platelia assay, although further studies are needed to validate this assumption. We also provide a formula allowing the conversion of Virclia index values into Platelia values. The study may contribute toward positioning the Virclia assay within the diagnostic algorithm of IPA.

Engineering nickel-supported osmium bimetallic nanozymes with specifically improved peroxidase-like activity for immunoassay

Researchers have shown significant interest in modulating the peroxidase-like activity of nanozymes. Among these, bimetallic nanozymes have shown superior peroxidase-like activity over monometallic counterparts, offering enhanced performance and cost-efficiency in nanozyme designs. Herein, bimetallic nanozymes comprising nickel (Ni) and osmium (Os) incorporated into hyaluronate (HA) have been developed, resulting in HA-Nin/Os nanoclusters. Subsequently, comprehensive characterizations have been conducted. Further investigation has revealed that HA-Nin/Os efficiently catalyzed 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation with hydrogen peroxide (H2O2), confirming its peroxidase-like behavior and role as a nanozyme. Impressively, HA-Ni2/Os (Ni/Os = 2:1) displays heightened substrate affinity, accelerated reaction rates, enhanced hydroxyl radical production in acidic conditions, and exhibits activity unit of 1224 U/mg, representing more than two-fold increase compared to non-Ni-supported Os nanozyme. Theoretical calculations indicate that Ni support enhances the peroxidase-like process of Os nanozyme by improving H2O2 adsorption and TMB oxidation. Crucially, the support of Ni does not significantly alter the other enzyme-like activities of Os nanozymes, thereby enabling Ni to selectively enhance their peroxidase-like activity. In terms of application, the peroxidase-like ability of HA-Ni2/Os, facilitated by HA's carboxyl groups enabling crosslinking, proves effective in a squamous carcinoma antigen immunoassay. Moreover, HA-Ni2/Os exhibit reliable stability, promising as a peroxidase substitute. This work underscores the advantages of incorporating Ni into Os, specifically enhancing peroxidase-like activity, highlighting the potential of Os bimetallic nanozymes for peroxidase-based applications.

Hematuria Due to Possible Histoplasma-Associated Urinary Bladder Pseudotumor With Negative Serologic and Urine Antigen Testing

Abstract Histoplasma capsulatum is a ubiquitous dimorphic fungus causing multiple infectious syndromes, ranging from subclinical to severe disseminated disease. We present an unusual case of hematuria due to pedunculated urinary bladder mass in an immunocompetent host. Although the gold standard for diagnosis of histoplasmosis is through demonstration of characteristic yeast forms on histopathologic examination of infected tissue, or observation of typical mycelial growth in culture of clinical specimens, investigational multiplex polymerase chain reaction of formalinized tissue was helpful in this case due to conflicting serologic testing, equivocal morphologic findings on histopathologic examination and, surprisingly, a negative urine Histoplasma antigen despite anatomically proximal location within the urinary bladder. Although antigen immunoassay and serology are commonly used proxy diagnostics in Histoplasma-associated disease, varying performance characteristics in certain disease states, such as cases of locally proliferative infection mimicking neoplastic growth similar to this report, may lead to elusive diagnosis.

Surface plasmon resonance-enhanced photoelectrochemical immunoassay with Cu-doped porous Bi2WO6 nanosheets

The development of rapid screening sensing platforms to improve pre-screening mechanisms in community healthcare is necessary to meet the significant need for portable testing in biomarker diagnostics. Here, we designed a portable smartphone-based photoelectrochemical (PEC) immunoassay for carcinoembryonic antigen (CEA) detection using Cu-doped ultrathin porous Bi2WO6 (CuBWO) nanosheets as the photoactive material. The CuBWO nanosheets exhibit a fast photocurrent response and excellent electrical transmission rate under UV light due to their surface plasmon resonance effect (SPR). The method uses glucose oxidase-labeled secondary antibody as a signal indicator for sandwich-type immune conjugation. In the presence of the target CEA, the electrons and holes generated at the surface of the photo-excited ultrathin porous CuBWO were rapidly consumed by the production of H2O2 from glucose oxidase oxidizing glucose, resulting in a weakened photocurrent signal. The photocurrent intensity increased logarithmically and linearly with increasing CEA concentration (0.02–50 ng mL−1), with a detection limit of 15.0 pg mL−1 (S/N = 3). The system provides a broader idea for inferring the electron-hole transport mechanism in ultrathin porous nanosheet layer materials and developing efficient PEC sensors.

Highly efficient ratiometric electrochemiluminescence biosensor with amyloid aggregation as antifouling coating and g-C3N4 as internal standard for carcinoembryonic antigen immunoassay

The electrochemical antifouling sensing interface can effectively resist the adsorption of non-specific biomolecules, which provides a powerful means for the sensitive determination of various biomarkers in complex media. Therefore, the development of simple and efficient antifouling coatings is particularly important. Herein, we utilized tris (2-carboxyethyl) phosphine to rapidly reduce disulfide bonds in bovine serum albumin (BSA) to generate amyloid aggregation (PTB), thus forming a macroscopic two-dimensional film through one-step self-assembly. Compared to other antifouling interfaces, PTB has the advantages of simple preparation means, high stability and reproducibility, as well as good antifouling properties. At the same time, a layer of polyaniline (PANI) was electrodeposited on the modified electrode to improve the electron transmission rate, which improves the detection efficiency of the sensor. Furthermore, g-C3N4 was introduced as an internal standard substance to eliminate instrument errors. According to the ratio of electrochemiluminescence (ECL) signals of [Ru(bpy)3]2+ and g-C3N4, a ratiometric sensing strategy was designed to improve detection accuracy and sensitivity. Based on this, the proposed biosensor can achieve sensitive detection of CEA with a detection limit as low as 0.43 fg/mL (S/N = 3), which has important significance for early clinical diagnosis.

Low-Cost Biosensor Technologies for Rapid Detection of COVID-19 and Future Pandemics.

Many systems have been designed for the detection of SARS-CoV-2, which is the virus that causes COVID-19. SARS-CoV-2 is readily transmitted, resulting in the rapid spread of disease in human populations. Frequent testing at the point of care (POC) is a key aspect for controlling outbreaks caused by SARS-CoV-2 and other emerging pathogens, as the early identification of infected individuals can then be followed by appropriate measures of isolation or treatment, maximizing the chances of recovery and preventing infectious spread. Diagnostic tools used for high-frequency testing should be inexpensive, provide a rapid diagnostic response without sophisticated equipment, and be amenable to manufacturing on a large scale. The application of these devices should enable large-scale data collection, help control viral transmission, and prevent disease propagation. Here we review functional nanomaterial-based optical and electrochemical biosensors for accessible POC testing for COVID-19. These biosensors incorporate nanomaterials coupled with paper-based analytical devices and other inexpensive substrates, traditional lateral flow technology (antigen and antibody immunoassays), and innovative biosensing methods. We critically discuss the advantages and disadvantages of nanobiosensor-based approaches compared to widely used technologies such as PCR, ELISA, and LAMP. Moreover, we delineate the main technological, (bio)chemical, translational, and regulatory challenges associated with developing functional and reliable biosensors, which have prevented their translation into the clinic. Finally, we highlight how nanobiosensors, given their unique advantages over existing diagnostic tests, may help in future pandemics.

Rapid multiplex assay of SARS-CoV-2 antigens based on magnetic Janus photonic barcodes

SARS-CoV-2 has been causing severe impacts on all aspects of society for more than three years. Antigen immunoassay is the main detection method of SARS-CoV-2 at present. Here, a magnetic photonic crystal microsphere with a Janus structure is designed for synchronous detection of SARS-CoV-2-associated antigens. One hemisphere of the Janus microsphere is a photonic crystal assembled by silica nanoparticles, where the structural color serves as the encoding basis for multiplex detection and the surface is functionalized with the capture antibodies. The other hemisphere of the Janus microsphere is assembled by magnetic nanoparticles, allowing the controlled movement of the microspheres in a magnetic field, thereby enhancing antigen capture efficiency. It is demonstrated that this ingeniously designed detection platform can rapidly and simultaneously detect spike protein and nucleocapsid protein of SARS-CoV-2 based on a double-antibody sandwich immunoassay strategy. The limit of detection (LOD) is 0.478 ng/mL for spike protein and 0.255 ng/mL for nucleocapsid protein. Hence, the Janus photonic barcodes provide a new avenue for multiplex detection of SARS-CoV-2-associated antigens and have the potential to be utilized in the assay of other disease-related biomarkers.

Natural history of adenoviral conjunctivitis in a US-based population: Viral load, signs, and symptoms

PurposeTo report the clinical signs, symptoms, and viral clearance in individuals in the United States with adenoviral conjunctivitis (Ad-Cs). MethodsIndividuals ≥ 18 years presenting within 4 days of symptoms of Ad-Cs who met eligibility criteria and tested positive with both point-of-care immunoassay antigen and quantitative polymerase chain reaction (qPCR) testing were enrolled. Patient-reported symptoms, clinician-graded signs, and qPCR viral titers were collected at baseline, days 1–2, 4 (days 3–5), 7 (days 6–10), 14 (days 11–17) and 21 (days 18–21). ResultsThere was no detectable viral titers by the day 14 visit in 6/8 patients. By day 21, there was no detectable viral titers in the 7 participants who completed the visit; however, signs and symptoms persisted including: blurry vision (5/7), discomfort (2/7) or redness (1/7). Masked clinicians also noted conjunctival redness (4/7), follicular conjunctivitis (4/7) and bulbar edema (3/7). ConclusionMany patient-reported symptoms and clinical signs persist after viral titers are no longer detectable by qPCR. Using clinical signs and symptoms to determine quarantine duration may result in patients being furloughed longer than the time that the patient is infectious.

Oxygen-bridged W-Pd atomic pairs enable H2O2 activation for sensitive immunoassays.

Regulating the performance of peroxidase (POD)-like nanozymes is a prerequisite for achieving highly sensitive and accurate immunoassays. Inspired by natural enzyme catalysis, we design a highly active and selective nanozyme by loading atomically dispersed tungsten (W) sites on Pd metallene (W-O-Pdene) to construct an artificial three-dimensional (3D) catalytic center. The 3D asymmetric W-O-Pd atomic pairs can effectively stretch the O-O bonds in H2O2 and further promote the desorption of H2O to enhance POD-like activity. Moreover, the W-O-Pd sites with unique spatial structures demonstrate satisfactory specificity for H2O2 activation, effectively preventing the interference of dissolved oxygen. Accordingly, the highly active and specific W-O-Pdene nanozymes are utilized for sensitive and accurate prostate-specific antigen (PSA) immunoassay with a low detection limit of 1.92 pg mL-1, superior to commercial enzyme-linked immunosorbent assay.

False-positive Aspergillus galactomannan immunoassay in the glucose component of total parenteral nutrition products.

In October 2022, we experienced a significant increase in samples showing high galactomannan (GM) indexes ranging from 6.22 to 10.58, as determined by the Platelia Aspergillus antigen immunoassay, also known as the GM test. After reviewing the medical records of nine GM antigenemia cases that did not show evidence of invasive aspergillosis, we found that these patients had received total parenteral nutrition (TPN) products from the same manufacturer, whose supplier of the glucose component had recently changed. The TPN products supplied by the specific manufacturer in October were subjected to a GM assay. The glucose component of the products from three different lot numbers exhibited strong positive results in the GM assay. Microbiological investigations through fungal culture and PCR on the TPN products were negative. The present study demonstrated that the glucose component of the TPN products contained a high level of GM antigen, which caused false-positive GM test results. The source of GM in the glucose component was glucoamylase, which was produced from Aspergillus niger to obtain glucose monohydrate from starch. Investigation of three commercially available glucoamylase products exhibited positive GM and 1,3-β-D-glucan tests with various titers positive up to 1:1,000 dilutions, while fungal cultures were all negative. Quality assurance measures of TPN products to prevent GM contamination should be emphasized during the manufacturing process to avoid unnecessary additional diagnostic procedures and overtreatment of invasive aspergillosis due to false-positive GM tests.IMPORTANCEThis manuscript describes an occurrence of false-positive GM tests in patients receiving TPN products from a manufacturer who had recently changed the supplier of the glucose component. We describe the clinical presentation of nine false-positive cases and the results of serologic and microbiological investigations of the TPN products suspected of contamination with GM. Attempts to detect GM in parenteral nutrition products were made since the detection of GM in sodium gluconate-containing solutions in 2007, but none of them identified the source of elevated GM indexes in TPN products. However, the present study demonstrated that the glucose component of the TPN products contained a high level of GM antigen, which caused false-positive GM assay results. The source of GM was glucoamylase, which was derived from A. niger in the manufacturing process. Physicians and clinical microbiology laboratories should be aware of this issue to improve interpretation and patient care.

Distinguishing SARS-CoV-2 infection and vaccine responses up to 18 months post-infection using nucleocapsid protein and receptor-binding domain antibodies.

The prediction of the durability of immunity against COVID-19 is relevant, and longitudinal studies are essential for unraveling the details regarding protective SARS-CoV-2 antibody responses. It has become challenging to discriminate between COVID-19 vaccine- and infection-induced immune responses since all approved vaccines in Europe and the USA are based on the viral spike (S) protein, which is also the most commonly used antigen in immunoassays measuring immunoglobulins (Igs) against SARS-CoV-2. We have developed a nucleocapsid (N) protein-based sandwich ELISA for detecting pan anti-SARS-CoV-2 Ig with a sensitivity and specificity of 97%. Generalized mixed models were used to determine the degree of long-term humoral immunity against the N protein and the receptor-binding domain (RBD) of the S protein in a cohort of infected individuals to distinguish between COVID-19 vaccine- and infection-induced immunity. N-specific waning could be observed in individuals who did not experience reinfection, while individuals who experienced reinfection had a new significant increase in N-specific Ig levels. In individuals that seroconverted without a reinfection, 70.1% remained anti-N seropositive after 550 days. The anti-RBD Ig dynamics were unaffected by reinfection but exhibited a clear increase in RBD-specific Ig when vaccination was initiated. In conclusion, a clear difference in the dynamics of the antibody response against N protein and RBD was observed over time. Anti-N protein-specific Igs can be detected up to 18 months after SARS-CoV-2 infection allowing long-term discrimination of infectious and vaccine antibody responses.IMPORTANCELongitudinal studies are essential to unravel details regarding the protective antibody responses after COVID-19 infection and vaccination. It has become challenging to distinguish long-term immune responses to SARS-CoV-2 infection and vaccination since most approved vaccines are based on the viral spike (S) protein, which is also mostly used in immunoassays measuring immunoglobulins (Igs) against SARS-CoV-2. We have developed a novel nucleocapsid (N) protein-based sandwich ELISA for detecting pan-anti-SARS-CoV-2 Ig, exhibiting high sensitivity and specificity. Generalized mixed models were used to determine long-term humoral immunity in a cohort of infected individuals from the Faroe Islands, distinguishing between COVID-19 vaccine- and infection-induced immunity. A clear difference in the dynamics of the antibody response against N protein and S protein was observed over time, and the anti-N protein-specific Igs could be detected up to 18 months after SARS-CoV-2 infection. This enables long-term discrimination between natural infection and vaccine-dependent antibody responses.

Smartphone-based photoelectrochemical immunoassay for carcinoembryonic antigen based on BiOCl/CuBi2O4 heterojunction

Photoelectrochemical (PEC) immunoassay has been widely developed for biomarker detection, but most include heavy and expensive instruments that are not suited for portable and on-site detection. In this work, the PEC immunoassay platform for mobile phones was reported for flexible, rapid, low-cost detection of carcinoembryonic antigen (CEA). The PEC detection platform was successfully composed of disposable screen-printed carbon electrodes, a micro-electrochemical workstation, a flashlight (the excitation light source), and a smartphone with a companion software with a micro-electrochemical workstation for rapid and on-site detection of target biomarkers. In this portable smartphone-based PEC system, the S-scheme heterojunction BiOCl/CuBi2O4 was effectively excited due to the efficient electron transfer rate and excellent photocurrent response under visible light. Specifically, the sandwich-type immunoreaction for capturing target biomarkers introduced alkaline phosphatase (ALP) labeled gold nanoparticles (Au NPs). The addition of CEA increased the ascorbic acid (AA) content and enhanced the photocurrent. The proposed immunoassay presented a good linear with the logarithm of CEA concentrations range within 0.01–40 ng mL−1, and the detection limit of 3.5 pg mL−1 (S/N = 3). Therefore, the portable detection platform offered an implementable approach to the development of miniaturized and portable photoelectrochemical detectors and on-site detection technology.