Conventional ELISA Research Articles

Multifunctional self-assembled nanoparticles serving as the signal labels of fluorescent immunoassays

Nanolabels composed of multiple small molecules have shown great potential as the signal tags of immunoassays. However, their practical applications are limited by the complex preparation and modification procedures. This study proposed the design of multifunctional nanoparticles as the signal labels of fluorescent immunoassays, which were prepared by the self-assembly of biotinylated pyrene-phenylalanine (bio-PyF) monomers. The recognition elements of biotin moieties on the nanoparticles enabled their attachment on the sensor surface through streptavidin–biotin interactions. The tetrameric streptavidin proteins serving as the crosslinkers induced the in-situ assembly of bio-PyF nanoparticles (bio-PyFNPs) into network architectures on the sensor interface, thus achieving the second signal amplification. The captured nanoparticles were disrupted into a large number of signal reporters with pyrene tags under external surfactant stimulus. The inherent fluorescent property of pyrene allowed for the detection of targets with high simplicity and sensitivity. The analytical performances of the immunoassays were evaluated by determining alpha-fetoprotein (AFP) in buffer and serum samples, achieving a detection limit down to 0.5 pg/mL. This method offers a simple, sensitive, and selective alternative to conventional enzyme-linked immunosorbent assays for clinical diagnosis. The proposed strategy holds great promise for the development of novel signal labels and self-assembly-based biosensors.

B-292 Detection of natural toxins derived from potatoes by biotin-streptavidin direct ELISA

Abstract Background α-solanine (SO) and α-chaconine (CHA) are natural toxins of potato and often cause food poisoning worldwide. We have previously developed a polyclonal antibody (anti-Sold antibody) that bind to SO and CHA and used it in an enzyme-linked immunosorbent assay (ELISA). Our previously constructed ELISA (conventional ELISA) has a weak point of low sensitivity in detecting SO and CHA, we have tried to improve it in this study. Methods Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Anti-Sold antibody was then purified from rabbit serum using affinity column chromatography. In our ELISA method, named “B-S direct ELISA”, biotin-labeled anti-Sold antibody is bound to SO and CHA in the sample, followed by the binding of peroxidase-labeled streptavidin. To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers. Results The absorbance of the samples containing SO and CHA prepared in 10 mM phosphate buffer saline (PBS) was about 5-fold higher than that of the conventional ELISA. Furthermore, the absorbance of the samples containing SO and CHA prepared in human serum and urine was enhanced about 2.5- and 1.6-fold, respectively, compared to that of the conventional ELISA. The limit of detection in B-S direct ELISA was 0.32 ng/mL in 10 mM PBS, 2.08 ng/mL in serum, and 4.88 ng/mL in urine, respectively. Using this ELISA, it was also possible to measure the content of SO and CHA in extracts of potato tubers, peels, and sprouts. Both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.4- and 2.2-fold higher, respectively, in each part than those in the extracts from fresh potatoes. Furthermore, the potato sprout extract contained approximately 50-fold more SO and CHA than the tuber and 9-fold more than the peel. On the other hand, B-S direct ELISA cross-reacted with solanidine and solasodine, which have similar chemical structures to SO and CHA, and also with cholesterol and vitamin D3 in serum. Conclusions Based on our results, B-S direct ELISA presented greater performance in detecting SO and CHA than our conventional ELISA. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although this ELISA has a detection performance applicable to human biological samples, cross-reactivity should be fully considered when judging the results.

Development of novel FRET aptasensor based on the quenching ability of iron oxide-gold nanostars for the detection of aflatoxin M1

Detecting Aflatoxin M1 (AFM1) in food products is crucial due to its high toxicity and health risks. This study introduced a fluorescence donor material using Rhodamine-B-Isothiocyanate (RBITC)-doped silica nanoparticles (RDSN) combined with iron oxide‑gold nanostars (IOGNS) as a quencher. The composite aptasensor (RDSN/IOGNS) served as a Förster Resonance Energy Transfer (FRET) nanoprobe for sensitive and selective AFM1 detection. The fluorescence of aptamer-conjugated RDSN (apt-RDSN) was effectively quenched by complementary DNA-conjugated IOGNS (cDNA-IOGNS). Upon AFM1 introduction, apt-RDSN dissociated from the IOGNS surface, restoring the fluorescence signal. The fluorescence intensity correlated linearly with AFM1 concentration, achieving a detection limit of 0.15 ng/mL. Compared to conventional enzyme-linked immunosorbent assay (ELISA), this FRET aptasensor showed excellent recovery rate and relative standard deviation (RSD) in milk samples, highlighting its potential for practical AFM1 detection applications.

OptoAssay-Light-controlled dynamic bioassay using optogenetic switches.

Circumventing the limitations of current bioassays, we introduce a light-controlled assay, OptoAssay, toward wash- and pump-free point-of-care diagnostics. Extending the capabilities of standard bioassays with light-dependent and reversible interaction of optogenetic switches, OptoAssays enable a bidirectional movement of assay components, only by changing the wavelength of light. Demonstrating exceptional versatility, the OptoAssay showcases its efficacy on various substrates, delivering a dynamic bioassay format. The applicability of the OptoAssay is successfully demonstrated by the calibration of a competitive model assay, resulting in a superior limit of detection of 8 pg ml-1, which is beyond those of conventional ELISA tests. In the future, combined with smartphones, OptoAssays could obviate the need for external flow control systems such as pumps or valves and signal readout devices, enabling on-site analysis in resource-limited settings.

Circulating Interleukins as Biomarkers in Non-Small Cell Lung Cancer Patients: A Pilot Study Compared to Normal Individuals.

Identifying biomarkers in non-small cell lung cancer (NSCLC) can improve diagnosis and patient stratification. We evaluated plasmas and sera for interleukins (IL)-11, IL-6, IL-8, IL-17A, and IL-33 as biomarkers in primary NSCLC patients undergoing surgical treatment against normal volunteers. Exhaled-breath condensates (EBCs), a potential source without invasive procedures, were explored in normal individuals. Due to separate recruitment criteria and intrinsic cohort differences, the NSCLC and control cohorts were not well matched for age (median age: 65 vs. 40 years; p < 0.0001) and smoking status (p = 0.0058). Interleukins were first assessed through conventional ELISA. IL-11 was elevated in NSCLC plasma compared to controls (49.71 ± 16.90 vs. 27.67 ± 14.06 pg/mL, respectively, p < 0.0001) but undetectable in sera and EBCs by conventional ELISA. Therefore, high-sensitivity PCR-based IL-11 ELISA was repeated, albeit with concentration discrepancies. IL11 gene and protein upregulation by RT-qPCR and immunohistochemistry, respectively, were validated in NSCLC tumors. The lack of detection sensitivity across IL-6, IL-8, IL-17A, and IL-33 suggests the need for further, precise assays. Surprisingly, biomarker concentrations can be dissimilar across paired plasmas and sera. Our results identified a need to optimize detection limits for biomarker detection and caution against over-reliance on just one form of blood sample for biomarker assessment.

Integrating lateral flow device with controllable gold in situ growth for sensitive detection of staphylococcal enterotoxin A in milk

Gold nanoparticle-based lateral flow immunoassays (AuNP-LFIA) are widely used for pathogen monitoring to prevent foodborne illness outbreaks. However, conventional AuNP-LFIA exhibits poor sensitivity and limited quantitative capacity due to the low colorimetric signal intensity of AuNPs. Herein, we introduced a low-background gold in situ growth (GISG) strategy by lowering the pH of the growth solution to weaken the reducibility of hydroxylamine, thereby enhancing the sensitivity of AuNP-LFIA. Additionally, we developed a universal and manufacturable lateral flow device to streamline the GISG process. We applied this device to detect staphylococcal enterotoxin A (SEA), an exotoxin produced by Staphylococcus aureus. Under optimal conditions, the proposed device demonstrated superior practicality and excellent sensitivity for SEA detection, achieving a detection limit of 0.061 ng/mL with the total detection time of 37 min, showing 311 times more sensitive than the unamplified AuNP-LFIA. Furthermore, SEA detection in milk samples showed a strong correlation (R2 = 0.8845) with results obtained from a conventional ELISA kit. Therefore, this promising LFIA device offers a novel strategy with high sensitivity and practicality for in-field detection of Staphylococcus aureus and can be easily adapted for screening other foodborne pathogens.

A silver auto-catalyzed plasmonic enzyme-linked immunosorbent assay for colorimetric and fluorescent detection of neutrophil gelatinase associated lipocalin (NGAL)

The conventional ELISA lacked sufficient sensitivity and accuracy for analyzing neutrophil gelatinase associated lipocalin (NGAL), a potential biomarker of ulcerative colitis. Here, we developed a dual-readout plasmonic ELISA with cascade amplification of alkaline phosphatase hydrolysis and O-phenylenediamine oxidation. In this novel plasmonic ELISA, alkaline phosphatase-conjugated antibodies capture NGAL from samples, alkaline phosphatase hydrolyzes 2-phospho-L-ascorbic acid into ascorbic acid, and then ascorbic acid blocks silver-catalyzed O-phenylenediamine oxidation by turning silver ions into silver microwires. Therefore, NGAL concentration is proportional to the decrease in chromogenic and fluorescent response. This novel plasmonic ELISA exhibited a linear range of 0.5–80 ng/mL, and the LOD was as low as 0.5 ng/mL. The sensitivity was superior to that of conventional ELISA and previous studies. The plasmonic ELISA exhibited favorable selectivity because of the specific antibody-antigen interaction and the robust cascade amplification. Besides, the dual-readout feature endowed the plasmonic ELISA with advantages in terms of convenience and compatibility. In summary, our study provided a novel and sensitive plasmonic ELISA for the diagnosis of ulcerative colitis.

Straightforward high-temperature antibody immobilization for rapid and simple microfluidic paper-based ELISA for detection of β’-component (Onc k 5) protein, a major IgE-binding protein in salmon roe

Detection of allergenic proteins in food is required for maintaining the health of persons with food allergies. Enzyme-linked immunosorbent assay (ELISA) is a common technique for the detection of proteins. However, conventional ELISA is expensive, time-consuming, and needs technical expertise. Here, a microfluidic paper-based analytical device (µPAD) combined with a sandwich ELISA technique is developed for rapid, simple, and sensitive colorimetric detection of β’-component protein, a major allergen in salmon roe. The developed µPAD-ELISA employs a capture antibody and enzyme-labeled detection antibody. In addition to the µPAD, a technique for immobilization of the capture antibody has been developed, which completes the immobilization within 5 min by drying the antibody solution on a paper substrate in an oven at 50 °C without any chemical modification. The immobilized antibody exhibited higher binding capability to an antigen and stability against washing processes than those immobilized at room temperature. Since this technique allows simple and rapid preparation of the ELISA in the µPAD, the present µPAD-ELISA completed the analysis within only 15 min, including the immobilization step, compared to a conventional ELISA, which takes about 19 h. This µPAD-ELISA offered a good limit of detection of 1.59 ng mL−1 and reduced the amounts of reagents by a factor of about 17–67, which decreases the analysis cost. This µPAD-ELISA approach employing the present antibody immobilization is expected to be an effective strategy for the detection of allergenic proteins in food.

Quantitative Detection of the CP4-EPSPS Protein in Genetically Modified Soybeans Using a Novel Fluorescence Immunoassay Assay.

Protein-based detection methods, enzyme-linked immunosorbent assays (ELISAs) and lateral flow strips, have been widely used for rapid, specific, and sensitive detection of genetically modified organisms (GMOs). However, the traditional ELISA method for the quantitative detection of GMOs has certain limitations. Herein, a quantum dot (QD)-based fluorescence-linked immunosorbent assay was developed using QDs as fluorescent markers for the detection of glyphosate-resistant protein (CP4-EPSPS) in the MON89788 soybean. The end-point fluorescent detection system was carried out using QDs conjugated with a goat anti-rabbit secondary antibody. Compared with the conventional sandwich ELISA method, the newly developed fluorescence-linked immunosorbent assay was highly sensitive and accurate for detecting the CP4-EPSPS protein. The quantified linearity was achieved in the range of 0.05-5% (w/w) for the MON89788 soybean sample. The recovery of protein extracted from mixed MON89788 soybean samples ranged from 87.67% to 116.83%. The limits of detection and limits of quantification were 0.7101 and 2.152 pg/mL, respectively. All of the results indicated that the QD-based fluorescence-linked immunosorbent assay was a highly specific and sensitive method for monitoring the CP4-EPSPS protein in GMOs.

Performance evaluation of microfluidic microplate-based fluorescent ELISA for qualitative detection of SARS-CoV-2–specific IgG and IgM

We evaluated the diagnostic performance of newly developed microfluidic microplate-based fluorescent ELISA for anti-SARS-CoV-2 antibody detection: the Veri-Q opti COVID-19 IgG and IgM ELISAs (hereafter, “Opti IgG/M”; MiCo BioMed, Gyeonggi-do, Republic of Korea), in comparison with conventional ELISAs. A total of 270 serum samples were analyzed, among which 90 samples were serially obtained from 25 COVID-19 patients. Another 180 samples were collected from 180 SARS-CoV-2–negative individuals. As comparative assays, we used SCoV-2 Detect IgG/M ELISA (hereafter, “InBios IgG/M”; InBios, Seattle, WA, USA) and Veri-Q COVID-19 IgG/IgM ELISA (hereafter, “Veri-Q IgG/M”; MiCo BioMed). Compared with conventional ELISAs, the Opti IgG yielded 97.1–100.0% positive percent agreement, 95.2–98.0% negative percent agreement, 96.3–97.8% total percent agreement, and kappa values of 0.90–0.94. Between the Opti IgM and the InBios IgM, the values were 93.7%, 96.6%, 95.9%, and 0.89, respectively. For the Opti IgG, sensitivities for the samples collected from 0–7, 8–14, 15–21, and ≥ 22 days after symptom onset were 40.0, 58.3, 94.1, and 100.0%, respectively. The values for the Opti IgM were 30.0, 54.2, 88.2, and 80%, respectively. The diagnostic specificities of the Opti IgG and IgM were 99.4 and 97.2%, respectively. The microfluidic microplate-based fluorescent ELISAs showed comparable diagnostic performance to conventional ELISAs for detecting anti-SARS-CoV-2 antibodies. With the combination of high throughput, a simplified workflow, and the ability to analyze reduced volumes, this new technology has great potential for improving SARS-CoV-2 serologic testing.

Practical Considerations for the Use of the Rapid AcuStar® ADAMTS13 Activity Assay in the Diagnosis of Acute Thrombotic Thrombocytopenic Purpura (TTP).

Introduction: Conventional practice in the management of acute TTP entails empirical treatment of suspected cases whilst awaiting confirmatory ADAMTS13 deficiency testing. Rapid ADAMTS13 assays offer increased accessibility and rapid diagnostics. The new automated HemosIL AcuStar® ADAMTS13 assay has seen increasing use among UK TTP Specialist Centres alongside the traditional ELISA method to confirm severe ADAMTS13 deficiency. Methods: A multi-centre retrospective case-control study was performed to review patients demonstrating discrepant ADAMTS13 activity results measured using rapid (AcuStar®) and ELISA assays in parallel from September 2019 to December 2021. Cases were compared with a cohort of suspected TTP patients exhibiting no difference in assay results and in relation to their presenting characteristics and pre-test probability of a diagnosis of TTP. Results: Where the clinical index of suspicion for TTP was high at presentation, acute TTP was confirmed using the AcuStar® assay < 0.2 IU/dL and subsequently < 10 IU/dL by ELISA with zero incidence of discrepancy. For patients with low clinical suspicion of acute TTP, a discrepancy between the AcuStar® and ELISA assay results was observed in 2% of cases; 5-10 IU/dL in AcuStar®, confirmed as >20 IU/dL by ELISA. A concurrent cancer diagnosis or sepsis was observed in 40% of discrepant cases. Conclusions: Where acute TTP is strongly suspected, there is a good correlation between the rapid AcuStar® ADAMTS13 assay and the conventional ELISA assay. Where the clinical suspicion of acute TTP is low, caution should be exercised in the interpretation of the ADAMTS13 activity using the AcuStar® assay. Accurate interpretation requires robust ADAMTS13 testing algorithms to be incorporated into diagnostic pathways.

Evaluation of Methodologies in Anti-nephrin Autoantibody Detection.

Recent studies discovered the prominent presence of anti-nephrin autoantibodies in minimal change disease, steroid-sensitive nephrotic syndrome and/or post-transplant recurrent focal segmental glomerulosclerosis (FSGS). However, widely different, and often unconventional autoantibody detection methods were used among these studies, making it challenging to assess the pathogenic role for the antibodies. Here we examined methods of conventional ELISA, magnetic on-beads ELISA, immunoprecipitation-immunoblotting (IP-IB), and cell- and tissue-based antibody assays with 127 plasma samples of kidney and non-kidney diseases. On the antigen side, we compared commercially available recombinant human nephrin extracelluar domain (ECD) produced from human or mouse cell lines, as well as lab-made full length, ECD, and series of ECD truncates for measuring autoantibody reactivity and specificity. Surprisingly, different assay methods and different antigen preparations led to observation of assay-specific false-positive and false-negative results. In general, a set of tests that combines magnetic beads-enhanced ELISA, followed by IP-IB, and epitope mapping showed the most robust results for anti-nephrin autoantibodies, detected in two primary FSGS patients among all cases tested. It is interesting to note that cell/tissue-based results, also supported by antigen truncation studies, clearly suggest steric hindrance of reactive epitopes, as in full length nephrin that forms compact self-associated complexes. In conclusion, anti-nephrin positivity is rare among the tested patients (2/127), including those with FSGS (2/42), and autoantibody results can be affected by the choice of detection methods.

Diagnosis of Human Cystic Echinococcosis by detecting Antigen B in Serum and Urine using Nanomagnetic Beads-Enzyme Linked Immunosorbent Assay

Abstract Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the larval stage; hydatid cyst, of E. granulosus. The disease is usually asymptomatic, and mostly presents by pressure symptoms according to its location. The diagnosis of CE is currently based on imaging techniques, which may not be available in some areas and antibody detection in serum, with several known drawbacks. Conversely, antigen detection has proven its efficacy in serodiagnosis of CE. The study aimed to test the diagnostic efficacy of Nanomagnetic beads-enzyme linked immunosorbent assay (NMB-sandwich ELISA) for diagnosis of CE by detection of E. granulosus AgB in serum and urine samples in comparison to conventional sandwich ELISA. The study included 42 individuals classified into 3 groups; CE infection group (Gp I), other parasitic infections group (Gp II) and healthy control group (Gp III). E. granulosus AgB was prepared from human hydatid cysts and used to produce anti- E. granulosus AgB-IgG-pAbs in rabbit which was used to detect AgB level in serum and urine samples by sandwich ELISA and NMB-sandwich ELISA. Using sandwich ELISA, AgB was detected in serum samples of 88.9% of CE patients with sensitivity and specificity of 88.9% and 91.7%, respectively. In urine samples, sandwich ELISA detected AgB in 83.3% of CE patients and the sensitivity and specificity were 83.3% and 87.5%, respectively. NMB-sandwich ELISA detected AgB in serum samples of 94.4% of CE patients with a sensitivity and specificity of 94.4% and 95.8%, respectively, whereas in urine samples it was detected in 88.9% of CE cases and the sensitivity and specificity were 88.9% and 95.8%, respectively. There was no statistically significant difference (p &amp;gt; 0.05) between the use of serum and urine samples for detection of AgB when examined by either sandwich ELISA or NMB- sandwich ELISA. The best test accuracy was obtained with NMB-sandwich ELISA detecting AgB in serum samples (95.2%), whereas, it showed a slightly lower accuracy of 92.9% when using urine samples. It was concluded that NMB-sandwich ELISA is efficient for detection of AgB in serum samples. Moreover, urine samples might be a good alternative to serum samples for the detection of AgB showing reasonable sensitivity, specificity and accuracy.

Diagnosis of Clinically Suspected Amoebic Liver Abscess Using Conventional PCR and Antigen ELISA in Liver Aspirate and Serum

Diagnosis of Clinically Suspected Amoebic Liver Abscess Using Conventional PCR and Antigen ELISA in Liver Aspirate and Serum

Point-of-care personalized rapid diagnosis of allergies using peptide epitopes and SPR multiplexed detection

Allergy is the most common chronic disease in Europe. Up to 20 % of patients with allergies live with a severe debilitating form of their condition, struggling with the fear of a possible asthma attack, anaphylactic shock, or even death from an allergic reaction. Allergic reactions vary among different patients, due to the presence of different antibodies, which may also define the severity of the allergic reaction. Diagnostic tools are still suboptimal, as they require an oral food challenge for diagnostic, which is personnel, time, and cost consuming and bear the risk of severe allergic reactions for the patient. Also, there is a major gap in food allergy diagnosis to easily distinguish between sensitization and allergy. Therefore, a fast point-of-care multiplexed analytical platform for allergy assessment, supporting low-risk minimally invasive personalized diagnostics and treatment monitoring is advanced. The novel portable Surface Plasmon Resonance system integrates microfluidics and a low-fouling, multiple-ligand biosensor based on recently identified peptide epitopes for appraising allergen-specific serum antibodies. Aiming at personalized diagnosis of allergy, we successfully tested and validated the platform on hazelnuts and peanuts, two of the most threatening and frequent food allergens. Moreover, in contrast to the conventional SPR analysis and ELISA, by using an innovative analysis of the entire sensing area where the peptides are immobilized, it was even possible to discern different sera related to the same allergen.

Fully Integrated and High-Throughput Microfluidic System for Multiplexed Point-Of-Care Testing.

For every epidemic outbreak, the prevention and treatments in resource-limited areas are always out of reach. Critical to this is that high accuracy, stability, and more comprehensive analytical techniques always rely on expensive and bulky instruments and large laboratories. Here, a fully integrated and high-throughput microfluidic system is proposed for ultra-multiple point-of-care immunoassay, termed Dac system. Specifically, the Dac system only requires a handheld portable device to automatically recycle repetitive multi-step reactions including on-demand liquid releasing, dispensing, metering, collecting, oscillatory mixing, and discharging. The Dac system performs high-precision enzyme-linked immunosorbentassays for up to 17 samples or targets simultaneously on a single chip. Furthermore, reagent consumption is only 2% compared to conventional ELISA, and microbubble-accelerated reactions shorten the assay time by more than half. As a proof of concept, the multiplexed detections are achieved by detecting at least four infection targets for two samples simultaneously on a singular chip. Furthermore, the barcode-based multi-target results can rapidly distinguish between five similar cases, allowing for accurate therapeutic interventions. Compared to bulky clinical instruments, the accuracy of clinical inflammation classification is 92.38% (n = 105), with a quantitative correlation coefficient of R2 = 0.9838, while the clinical specificity is 100% and the sensitivity is 98.93%.

Identification of novel biomarkers for anti-Toxoplasma gondii IgM detection and the potential application in rapid diagnostic fluorescent tests.

Toxoplasmosis, while often asymptomatic and prevalent as a foodborne disease, poses a considerable mortality risk for immunocompromised individuals during pregnancy. Point-of-care serological tests that detect specific IgG and IgM in patient sera are critical for disease management under limited resources. Despite many efforts to replace the T. gondii total lysate antigens (TLAs) by recombinant antigens (rAgs) in commercial kits, while IgG detection provides significant specificity and sensitivity, IgM detection remains comparatively low in sensitivity. In this study, we attempted to identify novel antigens targeting IgM in early infection, thereby establishing an IgM on-site detection kit. Using two-dimensional gel electrophoresis (2DE) and mouse serum immunoblotting, three novel antigens, including EF1γ, PGKI, and GAP50, were indicated to target T. gondii IgM. However, rAg EF1γ was undetectable by IgM of mice sera in Western blotting verification experiments, and ELISA coated with PGKI did not eliminate cross-reactivity, in contrast to GAP50. Subsequently, the lateral flow reaction employing a strip coated with 0.3 mg/mL purified rAg GAP50 and exhibited remarkable sensitivity compared with the conventional ELISA based on tachyzoite TLA, which successfully identified IgM in mouse sera infected with tachyzoites, ranging from 103 to 104 at 5 dpi and 104 at 7 dpi, respectively. Furthermore, by using standard T. gondii-infected human sera from WHO, the limit of detection (LOD) for the rapid fluorescence immunochromatographic test (FICT) using GAP50 was observed at 0.65 IU (international unit). These findings underline the particular immunoreactivity of GAP50, suggesting its potential as a specific biomarker for increasing the sensitivity of the FICT in IgM detection.

Automated enzyme-linked immunosorbent assay for point-of-care COVID-19 testing

The COVID-19 pandemic pushed the demand for readily available monitoring tools, leading to the development of electrochemical point-of-care devices. This work presents an electrochemical capillary-driven immunoassay (eCaDI) device based on polyethylene terephthalate (PET) and adhesive films featuring a screen-printed carbon electrode (SPCE) for the detection of SARS-CoV-2 nucleocapsid (N) protein in clinical samples. The SPCE was modified with protein A and capture antibodies (Ab1) to ensure biomolecule immobilization and enhance sensitivity to detect N protein. The eCaDI automates an enzyme-linked immunosorbent assay (ELISA) through sequential reagent delivery, allowing for one-step sample addition. This process involves capturing the N protein by Ab1, followed by the automated delivery of HRP-labelled antibody and 3,3’,5,5’-tetramethylbenzidine (TMB) followed by electrochemical detection through TMB reduction. The SPCEs are sensitive and selective for SARS-CoV-2 N protein and stable for four weeks. Inactivated virus was used to determine a limit of detection (LOD) of 627 PFU/mL. The results obtained through immunoassay on eCADI were consistent with those of the RT-PCR method, showing a coefficient of determination (R2) of 0.816 for 27 nasal swab samples from SARS-CoV-2 infected individuals. The developed eCADI is an alternative to conventional ELISA, given the device's low cost (less than $1), low consumption of samples and reagents, low waste generation, and short analysis time (5 min). Moreover, the immunoassay has shown potential for diagnosing COVID-19 in decentralized areas.

Relationship Between Cerebrospinal Fluid Alzheimer's Disease Biomarker Values Measured via Lumipulse Assays and Conventional ELISA: Single-Center Experience and Systematic Review.

Although Lumipulse assays and conventional ELISA are strongly correlated, the precise relationship between their measured values remains undetermined. To determine the relationship between Lumipulse and ELISA measurement values. Patients who underwent cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarker measurements and consented to biobanking between December 2021 and June 2023 were included. The relationship between values measured via Lumipulse assays and conventional ELISA were evaluated by Passing-Bablok analyses for amyloid-β 1-42 (Aβ42), total tau (t-tau), and phospho-tau 181 (p-tau 181). Studies using both assays were systematically searched for in PubMed and summarized after quality assessment. Regression line slopes and intercepts were 1.41 (1.23 to 1.60) and -77.8 (-198.4 to 44.5) for Aβ42, 0.94 (0.88 to 1.01) and 98.2 (76.9 to 114.4) for t-tau, and 1.60 (1.43 to 1.75) and -21.1 (-26.9 to -15.6) for p-tau181. Spearman's correlation coefficients were 0.90, 0.95, and 0.95 for Aβ42, t-tau, and p-tau181, respectively. We identified 13 other studies that included 2,117 patients in total. Aβ42 slope varied among studies, suggesting inter-lab difference of ELISA. The slope and intercept of t-tau were approximately 1 and 0, respectively, suggesting small proportional and systematic differences. Conversely, the p-tau181 slope was significantly higher than 1, distributed between 1.5-2 in most studies, with intercepts significantly lower than 0, suggesting proportional and systematic differences. We characterized different relationship between measurement values for each biomarker, which may be useful for understanding the differences in CSF biomarker measurement values on different platforms and for future global harmonization.

Novel Immunosensor Based on Metal Single-Atom Nanozymes with Enhanced Oxidase-Like Activity for Capsaicin Analysis in Spicy Food.

Capsaicin (CAP) is a primary indicator for assessing the level of pungency. Herein, iron-based single-atom nanozymes (SAzymes) (Fe/NC) with exceptional oxidase-like activity were used to construct an immunosensor for CAP analysis. Fe/NC could imitate oxidase actions by transforming O2 to •O2- radicals in the absence of hydrogen peroxide (H2O2), which could avoid complex operations and unstable results. By regulating the Fe atom loads, an optimal Fe0.7/NC atom usage rate could improve the catalytic activity (Michaelis-Menten constant (Km) = 0.09 mM). Fe0.7/NC was integrated with goat antimouse IgG by facile mix incubation to develop a competitive enzyme-linked immunosorbent assay (ELISA). Our Fe0.7/NC immunosensing platform is anticipated to outperform the conventional ELISA in terms of stability and shelf life. The proposed immunosensor provided color responses across 0.01-1000 ng/mL CAP concentrations, with a detection limit of 0.046 ng/mL. Fe/NC may have potential as nanozymes for CAP detection in spicy foods, with promising applications in food biosensing.