Rapid Immunoassay Research Articles

Advancements in electrochemical immunosensors towards point-of-care detection of cardiac biomarkers.

Cardiovascular disease remains the leading cause of death worldwide, with mortality rates increasing annually. This underscores the urgent need for accurate diagnostic and monitoring tools. Electrochemical detection has emerged as a promising method for swiftly and precisely measuring specific biomarkers in bodily fluids. This approach is not only cost-effective and efficient compared to traditional clinical methods, but it can also be tailored to detect individual biomarkers, which makes it particularly well-suited for point-of-care (POC) applications. The ability to conduct testing at the point of care is crucial for timely interventions and personalized disease management, empowering healthcare providers to tailor treatment plans based on real-time biomarker data. Thanks to recent advancements in nanomaterials, we've seen significant progress in electrochemical detection, leading to the development of specialized rapid immunoassay systems. These systems utilize specific antibodies to target molecules, expanding the range of detectable biomarkers. This innovation has the potential to revolutionize the diagnosis and treatment of cardiovascular diseases by enhancing detection sensitivity and specificity. Ultimately, these advancements aim to improve patient outcomes by enabling earlier diagnosis, more precise monitoring, and personalized therapeutic interventions, which will contribute to more effective management of cardiovascular health globally.

Long afterglow nanoprobes labeled image enhancement using deep learning in rapid and sensitive lateral flow immunoassay

Point-of-care testing (POCT) is currently the predominant method of in vitro diagnostics, with lateral flow immunoassay(LFIA) being commonly utilized in POCT. The performance of the probe in LFIA will directly affect the results of the detection. Therefore, the selection of probes is particularly important. However, the current commercially produced probes generally suffer from short luminescence lifetime, high detection cost, and poor anti-interference capability. Meanwhile, the traditional detection system has difficulties in detecting weakly positive samples. These ultimately lead to the problem of poor detection sensitivity. In order to address the above issues, we have synthesized an ultralong organic phosphorescence nanoprobe (UOPN). The UOPNs exhibit a long afterglow phosphorescence signal of around 300 ms after being excited, which can be used to eliminate endogenous interference. Meanwhile, focusing on the problem of poor detection of weakly positive samples in conventional detection systems, and the weak inherent signal of UOPNs, resulting in a low signal-to-noise ratio, we have developed a detection system that combines image processing and neural network-driven analysis for long afterglow image analysis. Through the analysis of the long afterglow images of serum amyloid A (SAA) in human blood, a detection limit of 0.01 μg/mL and a linear range of 0.1–251 μg/mL were achieved. The whole analysis was completed within 5 minutes. After pre-processing with the denoising algorithm, the system demonstrated an impressive classification accuracy of 99.24 %, validating the reliability of the detection system. This approach has further enhanced the sensitivity of immunoassay, contributing to the advancement of point-of-care testing.

The development of rapid immunoassays for the urinary analysis of 1-hydroxypyrene glucuronide facilitate both laboratory and on-site polycyclic aromatic hydrocarbon biomonitoring

Polycylic Aromatic Hydrocarbons (PAHs) are produced during the incomplete burning of organic materials. PAH sources include vehicle exhaust, tobacco smoke and waste incineration. Environmental and occupational exposures to PAHs are known to occur. Cancer is a significant endpoint of PAH exposure and several occupations associated with high PAH exposure have been classified by IARC as carcinogenic to humans (Group 1).Pyrene is a common component of PAH mixtures and metabolism of pyrene leads to the excretion of 1-hydroxypyrene glucuronide (1-OHPyrG) in urine. Laboratory measurement of urinary 1-OHPyrG is employed in occupational and environmental biomonitoring programmes. The production of an anti-1-OHPyrG monoclonal antibody would allow the development of a PAH biomonitoring ELISA facilitating large scale laboratory screening and routine testing.The development of a lateral flow immunoassay and the production of a field test (point of use test) would greatly increase the value of biomonitoring. A novel Lateral Flow has been developed which employs an anti-1-OHPyrG sheep monoclonal antibody (Mab) to capture the PAH metabolite. The captured metabolite is visualised through a second Mab raised against the Mab-1-OHPyrG immune complex. This sandwich assay provides a positive correlation between the assay signal and biomarker concentration.A Smartphone camera allows signal measurement and a carefully considered ‘app’ provides result interpretation and data analysis. Results are provided in an exposed/not-exposed format. Performance of the lateral flow was confirmed through a comparative study and field trial. The development of a lateral flow test provides "real-time" analysis to occupational health professionals. On-site screening allows the immediate confirmation of safe working practice, provides immediate reassurance to those involved in potentially hazardous activities and greatly increases the efficacy of biomonitoring.

B-258 Rapid clozapine blood levels on the Beckman Coulter DxC 500 AU

Abstract Background Clozapine is the most effective drug for treatment-resistant schizophrenia. Multiple guidelines strongly recommend clozapine therapeutic drug monitoring (TDM). Measuring clozapine blood levels provides essential information when there are questions of medication adherence, efficacy, toxicity, medication interactions, or concern over other factors that may influence clozapine levels. Turnaround time is essential for effective clinical decisions. However, with liquid-chromatography/mass spectrometry, clozapine levels can take days to report results. To address the medical need for a faster turnaround time, a rapid clozapine immunoassay was developed for automated clinical chemistry analyzers. The immunoassay measuring range is based on established APA Practice Guidelines and ASCP/AGNP Consensus Statement. We developed an analyzer application for the DxC 500 AU (time to first result < 9 minutes) to expand the availability of rapid clozapine blood level testing. Methods The performance of the FDA-cleared clozapine assay was evaluated on the DxC 500 AU according to CLSI guidelines. Precision testing was performed with three clozapine spiked pools over five days with five replicates per sample. Precision sample recovery was assessed to their theoretical spike concentration. Linearity was verified with five spiked serum samples covering the measuring range. Method comparison was performed with 40 remnant patient samples vs. the Beckman Coulter AU480. Results Repeatability CV was ≤5.5% and within-laboratory precision CV was ≤6.1%. Individual deviation was ±20% and mean deviation was ±2% for all samples. Deviation from linearity was <3%. Deming regression statistics for the method comparison were a slope of 0.987, an intercept of 5.7, and an R of 0.9948. The methods are statistically equivalent: the 99% confidence limits contained 1 and 0 for the slope and intercept respectively. Conclusions The precision, recovery, linearity, and method comparison results for clozapine on the DxC 500 AU demonstrate that the analyzer can be used to generate robust results for clozapine.

B-295 Sensitive and Rapid Homogeneous Immunoassay for the Detection of Zolpidem and its Major Metabolite in Urine

Abstract Background Zolpidem, a schedule IV controlled substance under the Controlled Substances Act, is commonly known as Ambien and Ambien CR, a prescription only treatment for insomnia. Zolpidem is one of the most common prescribed sleep aids in the U.S. due to its rapid sedative effects. The recommended duration of treatment is between two days and four weeks, as long-term use of zolpidem increases the risk of habit formation. Addiction to zolpidem can cause severe side effects, including memory loss, delusion, and in extreme cases, fatal overdose. From 2005-2010, Zolpidem-related emergency room visits doubled, underscoring the need for accurate testing as zolpidem testing plays an important role in cases of misuse, diversion, and forensics. The sole commercially available homogeneous immunoassay for the detection of zolpidem in urine has a relatively high cutoff at 20 ng/mL and poor cross-reactivity (0.02%) to major metabolite zolpidem 4-carboxylic acid. ARK Diagnostics has developed the ARK™ Zolpidem Assay to detect zolpidem at a cutoff concentration of 10 ng/mL with high cross-reactivity to its metabolite, Zolpidem phenyl 4-carboxylic acid and no cross-reactivity to zaleplon and zopiclone at concentrations below 100,000 ng/mL. Methods The ARK™ Zolpidem Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff concentration of 10 ng/mL and semi-quantitative range up to 40 ng/mL. Preliminary performance characterization for this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, analytical recovery, specificity, Histogram Overlap Analysis of ±50% controls and the cutoff, and method comparison with LC-MS/MS were evaluated. Results In semi-quantitative mode, total precision ranged from 2.6 to 3.1% CV. Spiked recovery of zolpidem ranged from 88.6% (2.5 ng/mL) to 96.8% (10 ng/mL). The major metabolite, zolpidem phenyl-4-carboxylic acid, showed an approximate equivalence to the 10 ng/mL zolpidem cutoff at 30 ng/mL (33.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 100% (15 positives and 100 negatives). Conclusions The ARK™ Zolpidem Assay measures zolpidem and its major metabolite zolpidem phenyl-4-carboxylic acid in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.

Multivalent bifunctional nanobody to enhance the sensitivity of direct competitive chemiluminescence immunoassay for the detection of microcystin LR in lake water

Microcystin-LR (MC-LR), a toxic cyanobacterial toxin in freshwater, poses significant health and ecological risks due to its ability to induce cell apoptosis and liver damage. Sensitive detection of MC-LR is crucial for public health and water safety. In this work, we engineered a multivalent bifunctional nanobody (A2.3-C4-SBP) by fusing the anti-MC-LR nanobody gene (A2.3) with self-assembling peptides (C4) and a streptavidin-binding peptide (SBP). A2.3-C4-SBP was directionally immobilized on the ELISA microplate via streptavidin-mediated to develop a multivalent bifunctional nanobody-based chemiluminescent immunoassay (MBN-CLIA) for MC-LR detection in lake water. The IC50 of the A2.3-C4-SBP heptamer based CLIA was 5.80 ng/mL, and the LOD (IC10) was 0.33 ng/mL, which were 9.51-fold and 1.82-fold lower, respectively, than those of the A2.3-SBP monomer based CLIA. Additionally, the IC50 and LOD were 1.26-fold and 1.82-fold lower, respectively, than those of the A2.3-C4-SBP heptamer without streptavidin-mediated directional immobilization. In summary, this work developed a sensitive, rapid and simple immunoassay for the detection of MC-LR in lake water based on multivalent bifunctional nanobodies. Furthermore, the proposed combined strategy of nanobody multimerization and directed immobilization is simple to operate and has great potential to improve the sensitivity and signal amplification of various immunoassays.

Dual improved enzyme-linked immunosorbent assay based on multivalent bifunctional nanobody and polymerized horseradish peroxide for the ultrasensitive detection of Ochratoxin A in coffee samples

Enzyme-linked immunosorbent assay (ELISA) is favored for its high throughput, low cost, and simplicity, but the limited sensitivity of the traditional colorimetric ELISA restricts its wider application. Therefore, developing improvement strategies to enhance its sensitivity is a current research hotspot. In this work, we developed a multivalent bifunctional nanobody (Nb28-C4-SBP) by fusing a nanobody gene (Nb28) against ochratoxin A (OTA) with a self-assembling peptide (C4) and a streptavidin-conjugated peptide (SBP), which can introduce streptavidin-conjugated polymerized horseradish peroxidase (SA-PolyHRP) as a probe into the detection system. Subsequently, a dual improved ELISA (DI-ELISA) was developed using the higher affinity multivalent bifunctional nanobody (Nb28-C4-SBP) and the more enzyme-loaded probe (SA-PolyHRP) for the ultrasensitive detection of OTA in coffee samples. The DI-ELISA demonstrated a detection limit of 1 pg/mL, which was about 20-fold lower than that of the Nb28-C4-SBP and SA-HRP based ELISA and 266-fold lower than that of the Nb28-SBP and SA-PolyHRP based ELISA, respectively. In summary, we developed an ultrasensitive, rapid, and simple immunoassay for detecting OTA in coffee samples. Additionally, the proposed improvement strategy based on multivalent nanobody and polymerized horseradish peroxide is straightforward to operate and holds great potential for enhancing the sensitivity in various immunoassays.

Enhancing Differential Diagnosis Related to Oxidative Stress, Nitrous Oxide, and Nutrition by Rapid Plasma Homocysteine Measurement.

Historically used as a marker for inherited disorders, the current interest in plasma homocysteine measurement lies in its ability to provide valuable information about the metabolic and nutritional status of patients. Specifically, nitrous oxide (N2O) abuse can lead to functional vitamin B12 deficiency by oxidation and increase oxidative stress, resulting in elevated plasma homocysteine levels, which mimic neurological conditions such as Guillain-Barré syndrome. Rapid identification of hyperhomocysteinemia is crucial for timely intervention and avoiding costly, unnecessary treatments. This study evaluates the performance of a rapid immunoassay technique (Snibe) compared to mass spectrometry (LC-MS/MS) for measuring plasma homocysteine levels in patients with nitrous oxide abuse and non-inherited caused of elevated homocysteine, aiming to enhance differential diagnosis related to oxidative stress. 235 patients from Lille University Hospital were included. EDTA blood samples were collected and analyzed using both rapid immunoassay (Snibe) and LC-MS/MS. Neurological assessment was performed using the peripheral neuropathy disability (PND) score. Firstly, significant elevations in plasma homocysteine levels were observed in patients abusing nitrous oxide measured by LC-MS/MS. Secondly, the immunoassay provided rapid results, essential for early clinical decision-making, but tended to underestimate high values compared to LC-MS/MS. A good correlation was found between the methods for low and moderate values. The immunoassay tended to underestimate high-value samples compared to LC-MS/MS, which is a common problem with the competitive methodology. The rapid immunoassay technique is effective for initial screening and early intervention, aiding in the differential diagnosis of conditions related to oxidative stress. Therefore, it is recommended to use the CLIA method for initial screening and confirm with mass spectrometry if there are abnormal samples. Integrating both techniques can enhance diagnostic accuracy and improve patient outcomes.

Customizable OpenGUS immunoassay: A homogeneous detection system using β-glucuronidase switch and label-free antibody

We developed a customizable OpenGUS immunoassay that enables rapid and sensitive detection of analytes without requiring antibody modification. This immunoassay employs label-free whole antibodies, an antibody-binding Z domain (ZD) derived from Staphylococcal protein A, and a β-glucuronidase (GUS) switch mutant, allowing for easy replacement of antibodies to tailor the immunoassays for various targeted antigens. The working principle is that the OpenGUS probe, the fusion protein of ZD and a GUS switch, converts the antibody-antigen interaction into GUS activation in a one-pot reaction. To enhance the signal-to-background ratio of the immunoassay, a GUS switch mutant that exhibits reduced background activation was developed by screening several additional mutations at the diagonal interface residue H514. Moreover, we optimized the composition of the reaction buffer, including organic solvents, salt, and surfactant. Under optimal conditions, we customized OpenGUS immunoassays for Cry j 1, human C-reactive protein, and human lactoferrin, achieving around 10–20-fold maximum fluorescence (15 min) or colorimetric (2 h) responses with picomolar to low nanomolar level detection limit, simply by using commercially available IgGs. Additionally, the three analytes were successfully detected in complex matrices similar to those used in practical applications. We believe that this customizable OpenGUS immunoassay will pave the way for the prompt development of rapid and sensitive homogeneous immunoassays for point-of-care diagnostics, high-throughput testing, and onsite environmental assessments.

Lessons learned: drive-through COVID-19 clinic testing during an adaptive epidemic response and a point-of-care test assessment of a computer-read rapid lateral flow immunoassay with fluorescence-based detection.

Background. The COVID-19 pandemic demonstrated a need for robust SARS-CoV-2 test evaluation infrastructure to underpin biosecurity and protect the population during a pandemic health emergency.Gap statement. The first generation of rapid antigen tests was less accurate than molecular methods due to their inherent sensitivity and specificity shortfalls, compounded by the consequences of self-testing. This created a need for more accurate point-of-care SARS-CoV-2 detection methods.Aim. Here we present the lessons-learned during the COVID-19 emergency response in Western Australia including the detailed set-up, evaluation and operation of rapid antigen test in a state-run drive-through sample collection service during the COVID-19 pandemic after the strict border shutdown ended.Methods. We report a conformity assessment of a novel, second-generation rapid antigen test (Virulizer) comprising a technician-operated rapid lateral flow immunoassay with fluorescence-based detection.Results. The Virulizer rapid antigen test demonstrated up to 100% sensitivity (95% CI: 61.0-100%), 91.94% specificity (95% CI: 82.5-96.5%) and 92.65% accuracy when compared to a commercial PCR assay method. Wide confidence intervals in our series reflect the limits of small sample size. Nevertheless, the Virulizer assay performance was well-suited to point-of-care screening for SARS-CoV-2 in a drive-through clinic setting.Conclusion. The adaptive evaluation process necessary under changing pandemic conditions enabled assessment of a simple sample collection and point-of-care testing process, and showed how this system could be rapidly deployed for SARS-CoV-2 testing, including to regional and remote settings.

Intestinal parasitic co-infections associated with Helicobacter pylori among paediatric patients with gastrointestinal illness attending a general hospital in southern Ethiopia

BackgroundConcurrent infections or co-infections caused by intestinal parasites and Helicobacter pylori are quite rampant in paediatrics living in endemic areas of sub-Saharan Africa, including Ethiopia, and if left untreated, can result in severe complications and hence must be addressed to ensure their health and well-being.ObjectivesTo determine the prevalence of intestinal parasitic and H. pylori co-infections and associated factors among paediatric patients with gastrointestinal symptoms who attended the Arba Minch General Hospital (AMGH), Arba Minch, southern Ethiopia, from September to November 2020.MethodsA cross-sectional study was conducted among a study population of 299 paediatric patients with gastrointestinal symptoms who visited AMGH. Stool samples were collected and analysed to detect H. pylori and intestinal parasites. A rapid lateral flow chromatographic immunoassay was employed to identify the H. pylori copra antigen, whereas the latter was detected using wet mount saline preparation and formol-ether concentration method. Socio-demographic, clinical, behavioural and other factors were obtained by means of a pre-tested structured questionnaire. Descriptive statistics and logistic regression analysis were done by Statistical Package for Social Service (SPSS) version 25; P values < 0.05 were considered statistically significant.ResultsThe prevalence of Helicobacter pylori and intestinal parasites was 14% (n = 42) and 37.1% (n = 111), respectively, whereas that of the co-infections with these pathogens was 6.4% (n = 19). Giardia lamblia was the most prevailing parasite, 21.4% (n = 64). Informal maternal education [AOR = 5.14; 95% CI: 1.98–15.70] and lack of hand washing practice were significantly associated with the extent of co-infections [AOR = 4.18; 95% CI: 1.36–12.80].ConclusionNearly one in twenty pediatric patients with gastrointestinal symptoms had intestinal parasitic infections and H. pylori co-infections, representing a silent health problem that is to be addressed through effective control strategies. Health administrators should consider the importance of co-infections in clinical diagnosis and planning aimed at its prevention.

Rapid pre-analysis derivatization-enabled high-throughput immunoassay for nitrite detection in foods

Nitrite is a well-known food additive and water pollutant that has garnered extensive attention due to both of its adverse and beneficial effects on human health, calling for rapid and reliable analytical methods. However, the simple and diminutive structure of nitrite has impeded the development of screening immunoassay for this substance. In this work, based on the rapid and selective derivatization of nitrite into benzotriazole (BTA) by o-phenylenediamine, two haptens targeting the structure of BTA were designed and synthesized, and a specific monoclonal antibody against BTA was generated. Using this antibody, an indirect competitive enzyme-linked immunosorbent assay (ELISA), involving a rapid pre-analysis derivatization, was developed for the detection of nitrite in foods. The limits of detection in drinking water, pickle, and ham sausage were 2.0 μg/L, 0.05 mg/kg, and 0.05 mg/kg, respectively. The recoveries ranged from 82.7% to 114.6%, and the results of this ELISA correlated well with those from the standard spectrophotometry method. This pioneering immunoassay for nitrite holds great promise for routine monitoring, ensuring food safety and consumer health.

HIV Diagnostic Algorithms in A Resource-Limited Setting: A Comparative Study

Background: Adoption of appropriate HIV testing algorithm is essential for quality HIV diagnostic results. Whether rapid enzyme immunoassay (rEIA) or combination of rapid enzyme immunoassay and enzyme-linked immunosorbent assay (rEIA-ELISA) alternative confirmatory algorithm is enough to make accurate HIV diagnosis has been a subject of controversies. Recent evaluation of national algorithm for HIV testing presented some discrepancies in results. Again, current guideline prescribed discontinuity of Western blot supplemental test and its replacement with Geenius HIV-1/ HIV-2 differentiation test followed by nucleic acid testing. The objective of this study is to compare HIV-1 testing algorithms using ELISA-NAT algorithm as the gold standard for HIV-1 diagnosis, and rEIA-Western blot and ELISA-Western blot as alternative confirmatory algorithms for HIV-2 diagnosis. Methods: This is a comparative study involving a cohort of 173 (98.9%) HIV-positive subjects and two (1.1%) HIV-negative subjects initially diagnosed on the basis of rEIA algorithm. Plasma samples separated from ethylene diamine tetra-acetic acid anticoagulated blood were used for further analyses. Fourth generation HIV antigen-antibody kit was used for further testing to make up rEIA-ELISA algorithm. Analyses with Western blot 1 and 2 assays were done as supplemental tests to make up alternative rEIA-Western blot and ELISA-Western blot confirmatory algorithms. Research subjects were further tested for HIV-RNA under PEPFAR plan. The sensitivity, specificity, positive predictive value and negative predictive value of algorithms were compared using the descriptive statistics of the SPSS version 17. Results: The HIV testing analyses showed 100% sensitivity, specificity, positive predictive value and negative predictive value from algorithm to algorithm especially for HIV-1 diagnosis. Use of combined ELISA-NAT confirmatory algorithm validated outcomes of rEIA-Western blot and ELISA-Western blot alternative confirmatory algorithms for HIV-1 diagnosis. Among HIV-1 positive samples tested, 50.0-60.7% HIV-2 Western blot indeterminate result was obtained and 7.1-10.7% HIV-1/HIV-2 co-infections were observed. Conclusion: The rEIA or national algorithm and its combination with enzyme-linked immunosorbent assay are reliable alternative confirmatory algorithms for the diagnosis of established HIV-1infection in resource-limited settings but not for HIV-2. Diagnosing acute HIV infection with current algorithm is limited and still require further review.

Presepsin in Human Milk Is Delivery Mode and Gender Dependent.

Breast milk (BM) is a unique food due to its nutritional composition and anti-inflammatory characteristics. Evidence has emerged on the role of Presepsin (PSEP) as a reliable marker of early sepsis diagnosis. In the present study, we aimed to investigate the measurability of PSEP in BM according to different maturation stages (colostrum, C; transition, Tr; and mature milks, Mt) and corrected for delivery mode and gender. We conducted a multicenter prospective case-control study in women who had delivered 22 term (T) and 22 preterm (PT) infants. A total of 44 human milk samples were collected and stored at -80 °C. BM PSEP (pg/mL) levels were measured by using a rapid chemiluminescent enzyme immunoassay. PSEP was detected in all samples analyzed. Higher (p < 0.05) BM PSEP concentrations were observed in the PT compared to the T infants. According to the grade of maturation, higher (p < 0.05) levels of PSEP in C compared to Tr and Mt milks were observed in the whole study population. The BM subtypes' degrees of maturation were delivery mode and gender dependent. We found that PSEP at high concentrations supports its antimicrobial action both in PT and T infants. These results open the door to further studies investigating the role of PSEP.

A rapid and facile immunoassay for C-reactive protein using PDMS-based digital magnetofluidics

BackgroundC-reactive protein has been reported as a biomarker of inflammation caused by acute injury, infection or tissue damage and also a prediction marker of cardiovascular diseases. Commonly, the gold standard for the detection of CRP is enzyme-linked immunosorbent assays (ELISAs). Normally, traditional immunoassays in multiwell plates typically suffer from prolonged assay time due to slow mass transport controlled by diffusion. Herein, a PDMS based magnetofluidic approach has been applied for a rapid and facile immunoassay using a sandwich enzyme-linked immunosorbent assay (ELISA) for the analysis of CRP. ResultsDue to the superhydrophobic PDMS, droplets of reagent and sample solutions were obtained when pipetting all solutions onto the PDMS substrate. These droplets were individually controlled by an external magnet to perform the assays. Magnetic beads immobilized with a capture antibody were not only used for immunomagnetic separation (IMS) of the captured CRP from the sample matrix, but also used as a carrier for droplet movement on the magnetofluidic device, expediting the immunoassay procedure, especially washing steps. The immunoassay of CRP was successfully performed within 1 h with a limit of detection of 0.015 mg L−1 in the concentration range of 0.1–10 mg L−1. The recovery percentages of CRP spiked in human serum were found in the range of 90–114 % with %RSD of less than 5 %, indicating acceptable accuracy and precision. SignificanceBy individually controlling the droplet movement using an external magnet, all steps of immunoassays were simply and rapidly performed. In addition, the microfluidic format allows for small volumes of reagents and samples and rapid assay kinetics. Therefore, the proposed magnetofluidic approach has shown its potential of becoming a rapid, facile and cost-effective method to perform traditional immunoassays in a variety of applications. In addition, the proposed approach is also particularly well-suited for analyses/reactions with multiple steps.

Detection of serum CC16 by a rapid and ultrasensitive magnetic chemiluminescence immunoassay for lung disease diagnosis.

It has been reported that serum Clara cell secreted protein 16 (CC16) is a potential biomarker for lung injury diseases, but currently, there is no other method that is faster, more accurate, or more sensitive being applied in clinical practice apart from ELISA. The current study was designed to established a magnetic nanoparticles chemiluminescence immunoassay (MNPs-CLIA) for highly sensitive automated detection of serum Clara cell secretory protein 16 (CC16), and validated its diagnostic performance for lung disease. The study included the expression of CC16 recombinant protein, the preparation and screening of its monoclonal antibody (MAb), as well as the construction, optimization and analytical evaluation of the MNPs-CLIA method. The clinical application value of this method was investigated by detecting CC16 level in 296 serum samples. The linear range of the MNPs-CLIA assay system was 0.2-50 ng/mL, and the limit of detection was 0.037 ng/mL. Performance parameters such as specificity, recovery rate, and precision can meet the industry standards of invitro diagnostic reagents. The established method reveals consistent results with ELISA (R2=0.9962) currently used clinically, and it also exhibits satisfactory diagnostic efficacy of silicosis, chronic obstructive pulmonary disease (COPD), and pulmonary sarcoidosis, with areas under the curve (AUC) of 0.9748, 0.8428 and 0.9128, respectively. Our established MNPs-CLIA method has the advantages of automation, high throughput, rapidity, and simplicity, and can be promoted for widely popularized in clinical applications. MNPs-CLIA detection of serum CC16 has efficient diagnostic potentiality for predicting and diagnosing lung diseases.

Rapid lateral flow immunoassay for fluorescence detection of canine distemper virus (CDV).

Canine distemper virus (CDV) is a highly contagious and potentially lethal virus that affects dogs and other members of the Canidae family, including wolves, foxes, and coyotes. Here, we present a fluorescent lateral flow immunoassay (FLFA) platform for the detection of CDV, which utilizes fluorescent microspheres - fusion protein monoclonal antibody (mAb)-labeled monoclonal antibody. The assay detected CDV within 5 min, with a detection limit threshold of 3 × 102 TCID50/mL. Notably, the assay demonstrated no cross-reactivity with canine parvovirus, canine coronavirus, canine adenovirus, feline calicivirus, feline herpesvirus, or feline parvovirus. Field and clinical applicability of the assay was evaluated using 63 field samples, including 30 canine fecal samples, 18 swab samples, and 15 blood samples. The coincidence rate between the detection results of clinical samples obtained through FLFA and reverse transcription polymerase chain reaction (RT-PCR) was 96.83%. Thus, this assay offers a significant advancement for the rapid diagnosis of CDV at the point of care.

A sandwich chemiluminescent magnetic microparticle immunoassay for cryptococcal antigen detection

ABSTRACT Background Cryptococcosis is a global invasive mycosis associated with significant morbidity and mortality. Cryptococcal antigen (CrAg) testing from serum and cerebrospinal fluid (CSF) has been regarded as a gold standard for early diagnosis. This study aimed to develop and validate a rapid and sensitive sandwich chemiluminescent magnetic microparticle immunoassay (CMIA) for quantitative detection of CrAg in sera. Research design and methods CMIA is based on magnetic beads modified with capture antibodies and biotinylated antibodies and Streptavidin-polyHRP, where biotinylated antibodies functioned as the recognition element and Streptavidin-polyHRP as the signal component. Assay parameters were first optimized, and then assay performances were evaluated. Results Under optimized conditions, the total runtime of the CMIA was 22 min. The assay had a wide linear range (2 –10,000 ng/mL) and high analytical sensitivity (0.24 ng/mL), together with acceptable reproducibility, accuracy, and stability. Besides, it exhibited no cross-reactivity with other pathogens. Importantly, the assay showed 92.91% (95% CI, 80.97–93.02%) overall qualitative agreement with a commercial ELISA kit in a retrospective cohort of 55 cases with confirmed cryptococcal infection, and 72 controls without evidence of invasive fungal disease (IFD). Conclusion These results demonstrated that the present study paved a novel strategy for reliable quantitative detection of CrAg in sera.

Endoparasite Infections in Captive Inland Bearded Dragons (Pogona vitticeps) in Italy.

The inland bearded dragon (Pogona vitticeps) is a lizard species commonly kept as a pet worldwide. Endoparasites are among the most important pathogens affecting bearded dragons. The aim of this study was to evaluate the endoparasites in captive P. vitticeps in Italy. Faecal samples from 30 P. vitticeps were analysed by fresh faecal smears, flotation tests, the Mini-FLOTAC technique, and a rapid immunoassay to detect Cryptosporidium spp. To search for microsporidia, PCR and sequencing were performed on the faecal samples. Data were statistically analysed. The overall positivity rate for endoparasites was 83.3% (25/30). The identified endoparasites were oxyurids (17/30, 56.7%), Isosospora amphiboluri (13/30, 43.3%), Encephalitozoon pogonae (4/18, 22.22%), and Cryptosporidium sp. (1/30, 3.33%). The positivity for protozoa was significantly higher in juveniles compared to adults. Moreover, the frequency of clinical signs was significantly higher in the positive animals. The results obtained here emphasize the importance of regular veterinary examinations of captive P. vitticeps, aimed at the diagnosis, treatment, and control of endoparasites. This study is one of the largest surveys on microsporidia infections in living bearded dragons, suggesting that E. pogonae may be widespread in this lizard.

Accuracy of rapid lateral flow immunoassays for human leptospirosis diagnosis: A systematic review and meta-analysis.

In the last two decades, several rapid lateral flow immunoassays (LFIs) for the diagnosis of human leptospirosis were developed and commercialized. However, the accuracy and reliability of these LFIs are not well understood. In this study, we aimed to evaluate the accuracy of leptospirosis LFIs as well as the factors affecting the test efficiency using systematic review and meta-analysis. Original articles reporting the accuracy of human leptospirosis LFIs against microagglutination tests (MAT) or immunofluorescent assays (IFA) were searched from PubMed, Embase, and Scopus, and selected as per pre-set inclusion and exclusion criteria. A total of 49 data entries extracted from 24 eligible records published between 2003 and 2023 were included for meta-analysis. A meta-analysis was performed using STATA. The quality of the included studies was assessed according to the revised QUADAS-2. Only nine studies (32.1%) were considered to have a low risk of bias and no concern for applicability. Pooled sensitivity and specificity were calculated to be 68% (95% confidence interval, CI: 57-78) and 93% (95% CI: 90-95), respectively. However, the ranges of sensitivity (3.6 - 100%) and specificity (53.5 - 100%) of individual entries are dramatically broad, possibly due to the heterogeneity found in both study designs and LFIs themselves. Subgroup analysis demonstrated that IgM detection has better sensitivity than detection of IgG alone. Moreover, the test performance seems to be unaffected by samples from different phases of infection. The pooled specificity of LFIs observed is somewhat acceptable, but the pooled sensitivity is low. These results, however, must be interpreted with caution because of substantial heterogeneity. Further evaluations of the LFIs with well-standardized design and reference test will be needed for a greater understanding of the test performance. Additionally, IgM detection type should be employed when leptospirosis LFIs are developed in the future.