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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. • A novel lateral flow allows in the field/on-site PAH biomonitoring. • LF testing and integration with mobile technology provides new biomonitoring methods. • On-site testing greatly increases the value of occupational biomonitoring. • A new PAH biomonitoring ELISA facilitates large scale screening programmes. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Our objectives were to develop and evaluate an integrated system consisting of a lateral flow immunoassay (LFIA) and an electronic portable imaging device for determination of pregnancy status of cows based on plasma concentrations of pregnancy-specific protein B (PSPB). Experiment 1 was conducted to test the performance of the LFIA for PSPB (PSPB-LFIA), whereas experiment 2 was conducted to evaluate the performance of the integrated system, including both the LFIA and imaging device. The PSPB-LFIA strips were made of nitrocellulose membrane with polystreptavidin, anti-mouse antibody, europium anti-PSPB conjugates, and biotin PSPB. After adding buffer and plasma in a 96-well plate, strips were dipped to initiate flow and were read in a fluorescence microscope to estimate PSPB concentrations based on the test-to-control line signal (T/C ratio). The T/C ratio of standards was linearly associated with PSPB (R2 = 0.99 in both experiments) concentrations. To test the ability to identify pregnant cows of the PSPB-LFIA only or the integrated system, plasma samples were collected and transrectal ultrasonography (TUS) was conducted 29 to 35 d after AI in lactating Holstein cows (experiment 1: n = 83; experiment 2: n = 205). A cow was considered pregnant (Preg) if concentrations of PSPB in plasma obtained by ELISA were ≥2 ng/mL or if an embryo was visible by TUS. In experiment 1, the accuracy of the PSPB-LFIA compared with ELISA was 92.7% (91.2% sensitivity [Se]; 96.1% specificity [Sp]; 98.1% positive predictive value [PPV]; 83.3% negative predictive value [NPV]) and compared with TUS was 90.4% (100% Se; 78.9% Sp; 84.9% PPV; 100% NPV). The agreement between LFIA and ELISA (kappa = 0.84; 95% CI 0.71–0.96) or LFIA and TUS (kappa = 0.80; 95% CI 0.67–0.93) as methods to classify cows as Preg or nonpregnant (Non-Preg) was high. In experiment 2, the accuracy of the PSPB-LFIA compared with ELISA was 96.1% (93.8% Se; 100% Sp; 100% PPV; 90.5% NPV) and compared with TUS was 92.2% (99.0% Se; 84.7% Sp; 87.6% PPV; 98.8% NPV). The agreement between LFIA and ELISA (kappa = 0.92; 95% CI 0.86–0.97) or LFIA and TUS (kappa = 0.84; 95% CI 0.77–0.92) as methods to classify cows as Preg or Non-Preg was high. We conclude that a system integrating a fluorescence-based LFIA and an optical reader was effective for classifying cows as pregnant or not pregnant based on estimations of plasma concentrations of PSPB. This novel system serves as a platform for further development of on-farm pregnancy testing tools based on measurement of biomarkers of pregnancy in bodily fluids of cattle. [ABSTRACT FROM AUTHOR]

HLA-DQ molecules drive unwanted alloimmune responses after solid-organ transplants and several autoimmune diseases, including type 1 diabetes and celiac disease. Biologics with HLA molecules as part of the design are emerging therapeutic options for these allo- and autoimmune conditions. However, the soluble a and b chains of class II HLA molecules do not dimerize efficiently without their transmembrane domains, which hinders their production. In this study, we examined the feasibility of interchain disulfide engineering by introducing paired cysteines to juxtaposed positions in the a and b chains of HLA-DQ7, encoded by HLA-DQA1*05:01 and HLADQB1*03:01 respectively. We identified three variant peptideHLA-DQ7-Fc fusion proteins (DQ7Fc) with increased expression and production yield, namely Y19C-D6C (YCDC), A83CE5C (ACEC), and A84C-N33C (ACNC). The mutated residues were conserved across all HLA-DQ proteins and had limited solvent exposure. Further characterizations of the YCDC variant showed that the expression of the fusion protein is peptide-dependent; inclusion of a higher-affinity peptide correlated with increased protein expression. However, highaffinity peptide alone was insufficient for stabilizing the DQ7 complex without the engineered disulfide bond. Multiple DQ7Fc variants demonstrated expected binding characteristics with commercial anti-DQ antibodies in two immunoassays and by a cell-based assay. Lastly, DQ7Fc variants demonstrated dose-dependent killing of DQ7-specific B cell hybridomas in a flow cytometric, complement-dependent cytotoxicity assay. These data support inter-chain disulfide engineering as a novel approach to efficiently producing functional HLA-DQ molecules and potentially other class II HLA molecules as candidate therapeutic agents. [ABSTRACT FROM AUTHOR]

Serological tests of non-treponemal and treponemal types are the most frequently used for syphilis diagnosis. Nontreponemal tests are used to monitor disease activity. Toluidine red unheated serum test (TRUST), as one of nontreponemal tests, is generally applicable to hospitals at different levels. However, accurate judgment of TRUST results is inseparable from an experienced and accurate operator. To reduce current shortcomings of manual TRUST method, we attempted to convert the manual TRUST test into automatic TRUST test, that is, to determine the degree of aggregation of toluidine red particles by detecting the absorbance value of serum after reaction with toluidine red particles. 50 μL of serum sample and 80 μL toluidine red particles were added to 96-well plate. Then, the 96-well plate was placed on a microplate reader at medium grade for 8 min to mix. Then, plasma reagin reacted with toluidine red particles and promoted the aggregation of toluidine red particles to form a large clot, which eventually caused a decrease in the absorbance at 540 nm. The results showed that the specificity of the automatic TRUST test was 100%, the sensitivity was 87%. And this method showed 93.5% correlation with manual TRUST test. The developed method is simple and involves less subjectivity in reading results, opening new avenues for syphilis diagnostic testing. Turbidimetric immunoassay can avoid the shortcomings of subjective interpretation, time-consuming and manual operation of manual TRUST method, and is more suitable for large-scale screening in health examination. [ABSTRACT FROM AUTHOR]

Findings from previous studies on maternal 25(OH)D levels during pregnancy and offspring schizophrenia are limited and inconsistent. We used nationwide population-based register data with a nested case-control design to examine the association between maternal 25(OH)D levels during pregnancy and offspring schizophrenia. The cases of schizophrenia (n = 1145) were born from 1987 to 1997, and received a diagnosis of schizophrenia by 2017, and were matched with equal number of controls. A quantitative immunoassay was used to measure maternal 25(OH)D in archived maternal serum in the national biobank of the Finnish Maternity Cohort, collected during the first and early second trimesters. Conditional logistic regression models examined the association between maternal 25(OH)D levels and offspring schizophrenia. No significant association was found between log-transformed maternal 25(OH)D levels and schizophrenia in unadjusted (OR 0.96, 95 % CI 0.78–1.17, p = 0.69) or adjusted analyses (aOR 0.98, 95 % CI 0.79–1.22, p = 0.89). Analyses by quintiles also revealed no association between the lowest quintile of maternal 25(OH)D levels and schizophrenia (OR 1.09, 95 % CI 0.81–1.45, p = 0.55; aOR 1.06, 95 % CI 0.78–1.45, p = 0.71). Maternal 25(OH)D levels, measured in categories, either in deficient category (OR 1.07 (0.85–1.35), p = 0.52; aOR 1.05 (0.81–1.34), p = 0.88) or insufficient category (OR 1.13, 95 % CI 0.92–1.40, p = 0.23; aOR 1.13, 95 % CI 0.90–1.41, p = 0.27) were also not associated with offspring schizophrenia. Maternal vitamin D levels in early pregnancy were not associated with offspring schizophrenia. Future studies measuring vitamin D during different stages of gestation are needed to draw firm conclusions. [ABSTRACT FROM AUTHOR]

This study evaluated whether IgG avidity measured by chemiluminescent microparticle immunoassay (CMIA) compared with enzyme-linked immunosorbent assay (ELISA) was useful to detect primary T. gondii infection during pregnancy and to estimate the risk for congenital T. gondii infection. One hundred six women with positive tests for T. gondii IgG and T. gondii IgM, comprising 21 women (19.8%) with low (<30%), 6 (5.7%) with borderline (30%–35%), and 79 (74.5%) with high (>35%) IgG avidity measured by ELISA were selected. Their stored sera were used for T. gondii IgG avidity measurements by CMIA. In CMIA, 72 (67.9%) women had low (<50%), 12 (11.3%) had borderline (50%–59.9%), and 22 (20.8%) had high (≥60%) IgG avidity. The ratio of low T. gondii IgG avidity index in CMIA was more than three-fold than that in ELISA. Eighteen (85.7%) of 21 women with ELISA low avidity also had CMIA low avidity, and 26 (96.3%) of 27 women with ELISA low or borderline avidity corresponded to CMIA low or borderline avidity, whereas 21 (26.6%) of 79 women with ELISA high avidity were diagnosed with CMIA low avidity. All three cases with congenital T. gondii infection showed coincidentally low IgG avidity in both methods. A positive correlation in IgG avidity indices was found between of ELISA and CMIA. CMIA for T. gondii avidity measurements compared with ELISA was clinically useful to detect pregnant women at a high risk of developing congenital T. gondii infection. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is common in older rabbits. The renal biomarkers blood urea and creatinine can be insensitive as indicators of renal disease in rabbits. Symmetric dimethylarginine (SDMA) has been shown to be an earlier and more sensitive biomarker for the assessment of glomerular filtration rate and the evaluation of CKD in several animal species. An immunoassay for SDMA has been validated in a number of species. The purpose of this study was to validate the SDMA immunoassay for rabbit serum and plasma and to determine the normal rabbit reference interval (RI) for SDMA. Clinically healthy rabbits (n = 167) between 5 months and 8 years of age, of any breed or sex, participated in the study. Heparinized plasma or serum was collected for clinical chemistry and used for assay validation. Analytical validation of the SDMA immunoassay for rabbit serum and plasma was performed and included assessment of assay accuracy, precision, analytical sensitivity, linearity, interference testing, and plasma/serum bias. The SDMA reference interval (RI) was determined. The SDMA immunoassay met all set quality requirements assessed in analytical validation. The RI for SDMA was determined to be between 4–18 µg/dL. The SDMA immunoassay demonstrated acceptable performance in both rabbit serum and heparinized plasma as determined by the validation criteria. The utility of SDMA as a biomarker of renal disease in rabbits has not been determined yet. [ABSTRACT FROM AUTHOR]

• Tested the effect of ozone disinfection on blood, saliva, and semen stains. • Ozone decreased the sensitivity of luminol and immunoassay tests of diluted blood. • It lowered the sensitivity of STK Lab and PSA immunoassay of diluted seminal fluid. • Ozone treatment did not affect microscopic identification of spermatozoa. • Ozone disinfection is not recommended with saliva stains. Good laboratory practice minimizes the biological hazard posed by potentially infectious casework samples. In certain scenarios, when the casework sample is contaminated with highly contagious pathogens, additional safety procedures such as disinfection might be advised. It was previously proven that ozone gas treatment does not hamper STR analysis, but there is no data on how the disinfection affects other steps of the forensic analysis. In this study, we aimed to assess the interference of ozone disinfection with forensic tests used to identify biological stains. A dilution series of blood, saliva, and semen samples were pipetted onto cotton fabric and let completely dry. Half of the samples were subjected to ozone treatment, while the rest served as controls. All the samples were tested with specific lateral flow immunochromatographic assays and for specific RNA markers with quantitative real-time PCR. Additionally, luminol test was carried out on blood spots, Phadebas® Amylase Test on saliva stains, and semen stains were examined with STK Lab kit and light microscope following Christmas Tree or Hematoxylin-Eosin staining. Ozone treatment had no detrimental effect on the microscopic identification of sperm cells. Undiluted blood samples were detected with luminol and immunoassay, but at higher dilution, the sensitivity of the test decreased after disinfection. The same decrease in sensitivity was observed in the detection of semen stains using STK Lab kit from STK® Sperm Tracker, and in the case of the immunoassay specific for prostate-specific antigen (PSA). Ozone treatment almost completely inhibited the enzymatic activity of amylase. The sensitivity of antibody-based detection of amylase was also greatly reduced. RNA markers showed degradation but remained detectable in blood and semen samples after incubation in the presence of ozone. In saliva, the higher Ct values of the mRNA markers were close to the detection limit, even before ozone treatment. [ABSTRACT FROM AUTHOR]

The application of pesticides (mostly insecticides and fungicides) during the tea-planting process will undoubtedly increase the dietary risk associated with drinking tea. Thus, it is necessary to ascertain whether pesticide residues in tea products exceed the maximum residue limits. However, the complex matrices present in tea samples comprise a major challenge in the analytical detection of pesticide residues. In this study, nine types of lateral flow immunochromatographic strips (LFICSs) were developed to detect the pesticides of interest (fenpropathrin, chlorpyrifos, imidacloprid, thiamethoxam, acetamiprid, carbendazim, chlorothalonil, pyraclostrobin, and iprodione). To reduce the interference of tea substrates on the assay sensitivity, the pretreatment conditions for tea samples, including the extraction solvent, extraction time, and purification agent, were optimized for the simultaneous detection of these pesticides. The entire testing procedure (including pretreatment and detection) could be completed within 30 min. The detected results of authentic tea samples were confirmed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which suggest that the LFICS coupled with sample rapid pretreatment can be used for on-site rapid screening of the target pesticide in tea products prior to their market release. [Display omitted] • Rapid test for multi-residue detection of nine pesticides in tea leaves was developed. • An extract-purify-dilute method was optimized for sample rapid pretreatment. • The entire test is easy-to-use, cost-effective, and can be finished within 30 min. [ABSTRACT FROM AUTHOR]

Current national severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination policy covers children aged >12 years. Unvaccinated, uninfected children remain susceptible to SARS-CoV-2 and play a role in community transmission, as paediatric infection is mostly mild or asymptomatic. To estimate the proportion of susceptible children in a community for public health measures, there is a need to assess the extent of natural infection. We performed a cross-sectional household serosurvey of SARS-CoV-2 antibodies in unvaccinated children aged between 6 and 18 years after the second COVID-19 wave. Anti-SARS-CoV-2 immunoglobin G (IgG) testing in serum was done using chemiluminescence immunoassay. We used a logistic regression model to investigate predicted factors of seropositivity. We observed a high prevalence (weighted average: 68.3%) of anti-SARS-CoV-2 IgG in 2700 enrolled children. Logistic regression for predictors of IgG seropositivity showed lower odds in households with completely vaccinated adults (adjusted odds ratio [OR]: 0.43, 95% confidence interval [CI]: 0.26–0.71, P = 0.0011) compared with households with unvaccinated adults. Other factors for low seropositivity included frontline workers as family members (adjusted OR: 0.69, 95% CI: 0.52–0.91, P = 0.0091) and non-crowded households (adjusted OR: 0.74, 95% CI: 0.61–0.89, P = 0.0019). A high SARS-CoV-2 IgG prevalence in unvaccinated children was indicative of previous exposure to potentially infected contacts. This implies in-person academic activities for children can be continued during future community transmission. Comparatively lower seropositivity in children of completely vaccinated households or frontline workers suggests decreased transmission due to vaccination-induced immunity of family members. Vaccination will still be required in these children to maintain protective IgG levels, particularly in low seroprevalence groups. [Display omitted] [ABSTRACT FROM AUTHOR]

• Higher prevalence of vitamin D deficiency was demonstrated in PD patients compared to control subjects. • Severity and stage of Parkinson's disease were significantly associated with lower 25OHD concentration. • A higher frequency of G allele of CYP2R1 polymorphism was observed in all subgroups with vitamin D deficiency. Vitamin D is a steroid hormone, known to be involved in the pathogenesis of various neurodegenerative disorders, including Parkinson's disease (PD). We aimed to clarify the relationship between hypovitaminosis D and the predisposition for PD and its clinical presentation. An additional aim was to examine the specific gene polymorphisms associated with vitamin D level. Total level of 25(OH)-vitamin D (25(OH)D) was measured in the serum of parkinsonian patients (n = 113) and controls (n = 82) using a commercial immunoassay. Genetic analyses were performed using Taqman assays on Real Time PCR amplification system. Higher frequency of vitamin D deficiency (<50 nmol/L) was observed in PD patients, compared to controls (40.7% and 23.2%, respectively, P = 0.010). It was also a positive predictive marker of PD (OR, 2.27; 95% CI, 1.206–4.298; P < 0.011). Significantly higher UPDRS (35.85 ± 1.35 and 32.09 ± 0.99, respectively, P = 0.023) and HY scores (2(1.5–2.5) and 1.5(1.0–2.0), respectively, P = 0.005) were present in patients with 25(OH)D level < 50 nmol/L compared to patients with 25(OH)D level ≥ 50 nmol/L. Despite some trends observed, differences in allelic and genotypic distribution between controls and patients, as well as between subgroups, did not reach the level of significance (P > 0.05). Findings of this study confirm the hypothesis of a significant relationship between hypovitaminosis D and PD. We demonstrated higher prevalence of vitamin D deficiency in PD patients, as well as its predictive potential for the onset and progression of PD. [ABSTRACT FROM AUTHOR]

To ensure proper dosage of a drug, analytical quantification of it in biofluid is necessary. Liquid chromatography mass spectrometry (LC-MS) is the conventional method of choice as it permits accurate identification and quantification. However, it requires expensive instrumentation and is not appropriate for bedside use. Using soluble epoxide hydrolase (sEH) inhibitors (EC5026 and TPPU) as examples, we report development of a nanobody-based enzyme-linked immunosorbent assay (ELISA) for such small molecules and its use to accurately quantify the drug chemicals in human samples. Under optimized conditions, two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL, respectively, and two order of magnitude linear ranges with high precision and accuracy. The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026. In addition, the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency. The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds. These methods could be easily implemented by the bedside, in the field in remote areas or in veterinary practice. This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy. Attributes of nanobody based pharmaceutical assays are discussed. [Display omitted] • High-affinity nanobodies against soluble epoxide hydrolase (sEH) inhibitors were reported. • Rapid and sensitive nanobody-based ELISA to quantify sEH inhibitors was developed. • The new nanobody–based ELISA correlated well with LC-MS analysis for the detection of sEH inhibitors. • The new ELISA was used to analyze urine samples of human dosed with EC5026. • Future role of nanobodies in pharmaceutical discovery and application was discussed. [ABSTRACT FROM AUTHOR]

Enzyme-linked i2mmunosorbent assays (ELISAs) that measure circulating phylloquinone have become commercially available, but their validity is uncertain. The objective of this study was to compare plasma phylloquinone concentrations measured using two commercially available ELISAs with concentrations measured using a validated high-performance liquid chromatography (HPLC) assay in 108 samples obtained from participants in a depletion (~10 mcg phylloquinone/d)-supplementation (~500 mcg phylloquinone/d) study. The geometric mean of plasma phylloquinone measured with ELISA A was 0.70 nmol/L, 37% lower than that measured with HPLC. The mean of the ELISA B measures was 12.4 nmol/L, >700% higher than the HPLC measures. Plasma phylloquinone measured using HPLC was significantly lower during phylloquinone depletion than supplementation (0.4 ± 0.1 compared with 1.2 ± 0.2 nmol/L; P < 0.001). Neither of the two ELISAs detected any significant difference in plasma phylloquinone concentrations between depletion and supplementation (ELISA A, P = 0.76; ELISA B, P = 0.29). These findings reinforce the need to validate plasma phylloquinone assays as they become available. [ABSTRACT FROM AUTHOR]

• Reference interval of IGF-1 immunoassay is not transferable to the IGF-1 LC-MS-MS assay due to lack of agreement. • Only IGF-1 assay methodology and not traceability led to significant bias in IGF-1 measurement. • Strong agreement between LC-MS assays enabled the transference of IGF-1 reference interval. • Verification of the transferred RIs showed that 90% of the reference samples fell within the transferred RIs (inclusive of the 95% CIs), meeting CLSI EP28-A3c guidelines. In this study, we aimed to determine the feasibility of transferring IGF-1 reference intervals between two liquid chromatography-mass spectrometry assays with distinct assay formats and calibration traceability. To adopt a reference interval (RI) for our new assay we have conducted RI transference and verification studies according to the CLSI EP28-A3c and EP9c guidelines. Specifically, the analytical agreement between the assays was evaluated using the linear model and the appropriateness of the linear model for RI transference was assessed using Deming regression, correlation coefficients, Q-Q plot, difference plot and studentized residues for the LC-MS/MS against DiaSorin LiaisonXL IGF-1 immunoassay and the liquid chromatography-high resolution mass spectrometry (LC-MS/HRMS) IGF-1 assay. Both Diasorin immunoassay and LC-MS/HRMS assays are traceable to WHO, 02/254. Our study showed a strong correlation (R2 > 0.93) and agreement (slope = 1.006, negligible intercept) between LC-MS/MS and LC-MS/HRMS regardless of their traceability and all statistical criteria were met per CLSI guidelines. Conversely, while the LC-MS/MS and Diasorin immunoassay results showed a strong correlation (R2 > 0.97, slope = 1.055), they failed to meet all statistical criteria for RI transference due to the bias (−44.91) and non-normal distribution of the residues. The RI verification study showed that 90% of the local LC-MS results fell within the RIs transferred from the reference LC-MS method, thus meeting CLSI EP28-A3c guidelines and permitting the transference of the reference LC-MS RIs. Taken together, this study provides data to suggest excellent agreement between assays traceable to distinct reference standards for IGF-1. [ABSTRACT FROM AUTHOR]

Anti-spike receptor binding domain (S-RBD) antibody against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which best correlates with virus-neutralizing antibody is useful for estimating the period of protection and identifying the timing of additional booster doses. Long-term transition of the S-RBD antibody titer and the antibody responses among healthy individuals remain unclear. In the present study, therefore, we monitored the S-RBD antibody titers of 16 healthcare workers every 4 weeks for 76 weeks after vaccination with a fourth dose of mRNA-1273 (Moderna) following three doses of BNT162b2 (Pfizer/BioNTech) using two commercial automated immunoassays (Roche and Abbott). Two antibody responses to the vaccine were similar with an up-down change before and after the second (weeks 3), third (weeks 40) and fourth (week 72) vaccinations, but the titer did not fall below the assay's positivity threshold in any individual. The peak level of the geometric mean titer (GMT) in the Roche assay was highest after the third vaccination, and that in Abbott assay was highest after the fourth vaccination but almost equal to that after the third vaccination. Both the geometric mean fold rise (GMFR) demonstrated by the Roche and Abbott assays were highest after the third vaccination. Antibody titers determined by the Roche and Abbott assays showed a positive strong correlation (correlation coefficient: 0.70 to 0.99), but the ratio (Roche/Abbott) of antibodies demonstrated by both assays increased 0.46- to 8.26-fold between weeks 3 and 76. These findings will be helpful for clinicians when interpreting results for SARS-CoV-2 antibody levels and considering future vaccination strategies. [ABSTRACT FROM AUTHOR]

Hay un interés creciente en el estudio de los anticuerpos antifosfolípidos (aPL) no criterio, incluyendo anticuerpos contra el dominio 1 de la B2 glicoproteína 1 (anti-D1 B2GP1) y anticuerpos antifosfatidilserina/protrombina (PS/PT). Nuestro objetivo fue analizar un panel de aPL convencionales y no criterio en una cohorte de pacientes con lupus eritematoso sistémico (LES) y síndrome antifosfolípido primario (SAF), para describir si hay diferencias en los títulos de aPL entre los grupos, y evaluar asociaciones clínicas incluyendo el riesgo de eventos recurrentes con aPL novedosos. Estudio observacional que evaluó los anticuerpos anti-D1 B2GP1 y anti-PS/PT de manera basal. Los anticuerpos anti-D1 B2GP1 se evaluaron a través de inmunoanálisis por quimioluminiscencia. Los anticuerpos anti-PS/PT, anticardiolipinas (aCL) y anti-B2GP1 fueron evaluados por técnicas de ELISA. Finalmente, los pacientes fueron seguidos en el tiempo para identificar nuevos eventos trombóticos. Se incluyeron 133 pacientes con LES y 23 pacientes con SAF primario. Las principales manifestaciones de SAF fueron TVP (27%), morbilidad obstétrica (22%) y trombosis arterial (10,1%). Los títulos de anticuerpos anti-PS/PT IgM fueron 46,5 (20,6-127) vs. 21,9 (11,2-39,2) U/ml, p < 0,001, en pacientes con SAF primario vs. LES con SAF secundario, respectivamente. Los anti-D1 B2GP1, anti-PS/PT IgG e IgM se asociaron con manifestaciones trombóticas y no trombóticas. Durante el seguimiento, los anticuerpos IgG B2GP1 se relacionaron con un riesgo acumulativo significativo de trombosis. Se encontraron diferencias estadísticamente significativas en títulos séricos de aPL no criterio en pacientes con SAF primario vs. pacientes con LES y SAF secundario. Si los títulos de aPL no criterio son útiles para diferenciar entre SAF primario y SAF secundario, se requieren más análisis en otras poblaciones para poder confirmar si los títulos de aPL no criterio. There is an increasing interest in the study of non-criteria antiphospholipid antibodies (aPL) including antibodies targeting domain 1 of the B2 glycoprotein 1 (anti-D1 B2GP1) and antibodies anti phosphatidylserine/ prothrombin (PS/PT). Our aim was to analyze a panel of conventional and non-criteria aPL in a cohort of patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (APS), to describe if there are differences in aPL titers among groups, to evaluate clinical associations including risk of recurrent events of novel aPL. Observational study that evaluated at baseline antibodies against anti-D1 B2GP1 and anti PS/PT. Anti-D1 B2GP1 antibodies were tested using a chemiluminescent immunoassay. IgG and IgM anti PS/PT, aCL and anti B2GP1 by ELISA techniques. Therefore, patients were followed in order to identify new thrombotic events. 133 patients with SLE and 23 with primary APS patients were included. Main APS manifestations were DVT (27%), obstetric morbidity (22%) and arterial thrombosis (10.1%). IgM anti PS/PT antibodies levels were (20.6 - 127) vs 21.9 (11.2 - 39.2) U/ml, p< 0.001 in primary APS vs SLE with APS, respectively. Anti-D1 B2GP1, IgG and IgM anti PS/PT were associated with thrombotic and non-thrombotic manifestations. During follow-up, IgG B2GP1 were related with a significant cumulative risk of thrombosis. We found significant differences in serum titers of non-criteria aPL among patients with primary APS vs SLE with APS. Whether non-criteria aPL antibodies titers are useful to differentiate patients with primary and secondary APS requires further analysis in other populations. [ABSTRACT FROM AUTHOR]

• Analysis of 58 fentanyl analogs as detected by the ARK II and Immunalysis fentanyl immunoassays. • Prediction of each immunoassay epitope allows for hypothesis of future analog detection. • Comparison of assay performance between the two vendors. • Comparison to native patient samples indicates both assays are sensitive and specific. • Both assays detect the majority of analogs indicating "false positive" screens that do not confirm for fentanyl may be detecting analogs instead. The ability to detect fentanyl analogs in urine aids in patient management. Little is published about the new ARK™ Fentanyl II Assay formulation's ability to detect fentanyl analogs. Norfentanyl (fentanyl metabolite) cross-reactivity with the ARK II assays is 7%, while the Immunalysis SEFRIA assay norfentanyl cross-reactivity is approximately 0.005%. The purpose of this study was to determine the new ARK II and SEFRIA fentanyl assays' detection of 58 fentanyl analogs. Drug-free urine was fortified with 0–100 ng/mL (0–0.297 µmol/L) of the fentanyl analog and analyzed using the previously evaluated immunoassays. Results were compared to molecular structure. Of the 58 analogs tested at ≤ 100 ng/mL (0–0.297 µmol/L), the ARK II and SEFRIA assays produced 51 and 57 positive results respectively. The cross-reactivity of the assay was predominantly determined by the location of the modification. Most modifications to the aniline ring and/or amide group did not affect the ARK II or SEFRIA assay. Modifications to the piperidine ring decreased detection by ARK II assay. Of the 7 compounds which were undetected by the ARK II assay, all had modifications to the N -alkyl chain. Norsufentanil was not detected by either assay and was the only analog not detected by the SEFRIA assay. The ARK II and Immunalysis fentanyl immunoassays can detect a range of fentanyl analogs with acryl, butyryl, or furanyl modifications to the amide group or aniline ring of the molecule. N -alkyl chain and piperidine ring modifications significantly affect the ARK II assay's ability to detect the analogs, while the SEFRIA assay appeared less affected and detected all analogs tested except for norsufentanil, which was also not detected by the ARK II assay. [ABSTRACT FROM AUTHOR]

Solriamfetol is a schedule IV-controlled substance used to treat excessive daytime sleepiness resulting from narcolepsy or obstructive sleep apnea. We present a patient prescribed solriamfetol who tested positive for amphetamines on a routine urinary toxicology screen despite patient denial of illicit drug use, raising the possibility of a false positive amphetamine screen. Spiking studies were performed on negative urine, and different concentrations of solriamfetol drug on 2 different amphetamine assays: the commonly used Beckman Emit® II Plus Amphetamines Assay, and the Citrine™ Triple Quad™ MS/MS Systems. The Beckman yielded positive results for amphetamines at solriamfetol concentrations of 200 μg/mL and 2000 μg/mL and negative results at 0.2 μg/mL and 2 μg/mL. However, the Citrine™ Triple Quad™ MS/MS Systems was negative at all concentrations. The Beckman Emit® II Plus Amphetamine Assay gave false positive results for amphetamines due to solriamfetol drug usage, a finding of relevance to prescribers of solriamfetol. [ABSTRACT FROM AUTHOR]

• Serum AMH levels can predict ovarian response following a GnRH antagonist cycle • A strong positive correlation was observed between AMH level and oocyte yield • AMH cut-offs for excluding a low or high response were 6.4 and 14.2 pmol/l • 47% of the variation in ovarian response could be attributed to AMH alone What is the capability of serum anti-Müllerian hormone (AMH) measured using the automated Elecsys® AMH immunoassay to (Roche Diagnostics International Ltd) determine ovarian response after fertility treatment? Single-centre, retrospective, observational, cohort study including women undergoing ovarian stimulation. Serum AMH concentrations were determined using the Elecsys AMH immunoassay based on one blood sample drawn 6 months or less before treatment. Stimulation was conducted in accordance with a gonadotrophin-releasing hormone (GnRH) antagonist protocol. Patients were divided into four ovarian response categories based on their oocyte yield: low (0–3), suboptimal (4–9), optimal (10–15) and high (>15). Areas under the curve were calculated for each ovarian response group. Overall, 1248 patients were enrolled. The AMH concentration had a strong positive correlation with oocyte yield (Spearman's rho = 0.74, P < 0.001). Areas under the curve (95% CI) for AMH predicting ovarian response were 0.85 (0.83 to 0.88) for low and 0.89 (0.87 to 0.91) for high response. Optimal serum AMH cut-offs for predicting a low and high response using the Elecsys AMH immunoassay were 6.4 pmol/l (0.89 ng/ml) and 14.2 pmol/l (1.99 ng/ml), respectively. Multivariable regression analysis showed that 47% (R2 = 0.470) of variation in ovarian response could be attributed to AMH alone, increasing to 50.9% (R2 = 0.509) with the addition of age, body weight, and total dose of gonadotrophin. Ovarian response and oocyte yield after stimulation in a GnRH antagonist cycle can be predicted with high accuracy using a single determination of serum AMH before ovarian stimulation. [ABSTRACT FROM AUTHOR]

• Discrepancy in IgE concentrations was greater in patients with the IgG4-IgE complex. • The ratio of IgG4-bound to free IgE correlated with the inter-method discrepancy. • The IgG4-IgE complex interferes with IgE measurements, depending on the method. Patients with immunoglobulin G4 (IgG4)-related disease (IgG4-RD) have elevated immunoglobulin E (IgE) concentration compared to that in healthy individuals, which suggests the occurrence of IgE-mediated allergic reactions. We have previously shown that IgG4 and IgE form a complex in some patients with IgG4-RD. However, it is currently unknown whether and how the presence of the IgG4-IgE complex affects IgE concentration measurements by different assays. Twenty patients with confirmed presence or absence of IgG4-IgE complex were evaluated. We compared IgE concentrations measured by ST AIA-PACK IgE II (AIA-PACK), Elecsys IgE II Immunoassay (Elecsys), and Iatroace IgE (Iatroace) and evaluated to what extent the IgG4-IgE complex interfered with these measurements. In patients with the IgG4-IgE complex, IgE concentrations measured using Iatroace were significantly lower than those measured using Elecsys and tended to be lower than those measured using AIA-PACK. IgE concentrations determined by Iatroace were significantly different in patients with and without the IgG4-IgE complex, whereas no significant differences between these groups were detected when IgE concentrations were measured by AIA-PACK or Elecsys. The formation of the IgG4-IgE complex underestimates measured IgE concentrations depending on the method used. Therefore, caution should be exercised when selecting a specific IgE assay for patients with IgG4-RD. [ABSTRACT FROM AUTHOR]

• Parathyroid hormone plays an important role in the assessment of parathyroid function. • HA, biotin, PTH fragments, oxPTH, phosphorylated PTH, and some preanalytical factors are the main interference factors. • Some interferences can be mitigated with the development of mass spectrometry. Automated immunoassays used to evaluate parathyroid function are vulnerable to different types of interference, which can affect clinical practices. This review provides a detailed overview of the six main types of interference known to affect the measurement of parathyroid hormone (PTH): heterophilic antibodies, biotin, PTH fragments, oxidized PTH (oxPTH), phosphorylated PTH, and some preanalytical factors. Because the prevalence of some of these conditions has been reported to approach 11.7%, and the frequency of testing for parathyroid function is important, the scale of the problem might be tremendous. Potential interference in parathyroid function testing should always be suspected whenever clinical or biochemical discrepancies arise. Their identification typically relies on additional laboratory tests, including method comparison, serial dilution, blocking reagent studies, affinity adsorption, and polyethylene glycol precipitation. Moreover, some of these issues can be mitigated with the development of mass spectrometry. This review also evaluated the clinical impact of parathyroid interference on immunoassays, including misdiagnosis, inappropriate parathyroidectomy; and delay in receiving appropriate therapy. Hence, strong communication should be maintained between the clinician and laboratory to avoid such scenarios. [ABSTRACT FROM AUTHOR]

• Investigated test-line response curve for a qualitative serum hCG lateral flow assay. • Image analysis was performed to obtain the Test line integral (S) vs [hCG]. • S vs [hCG] was well-characterized by a hyperbola: S/Smax = [hCG]/([hCG] + K). • K was 202 mIU/mL; interval between 10% and 90% Smax was a factor of 81 in [hCG]. • At claimed sensitivity of [hCG] = 10 mIU/mL, S/N was 2.7. As an adjunct to verification of performance characteristics of a qualitative serum hCG lateral flow immunoassay (LFI), we performed image analysis to characterize the dose vs response curve (visibility of the test line), as a means of understanding the transition from negative to positive as a function of increasing [hCG]. Using serum samples of known [hCG], device images were obtained using a scanner at the prescribed reading time (5 min). Image analysis (using Python and R) was used to obtain the integral (S) of the test-line color as a function of [hCG]. Data for S as a function of [hCG] were well characterized by a simple hyperbola: S = Smax [hCG]/([hCG] + K), where K = 202 mIU/ml (r = 0.997). Replicates of S at K had CV of 7.3 %. By eye, uncertainty of test results among users occurred only below the assay's stated sensitivity of 10 mIU/ml, in region of S < 3 % of Smax, and signal:noise ratio < 3. By image analysis, the dose vs response (Test line integral) for this qualitative serum hCG LFI was a simple hyperbola. Characterization of the dose vs response curve was useful in verification of the assay's performance characteristics. [ABSTRACT FROM AUTHOR]

• An HPLC-DMS/MS/MS method was developed to assay steroid hormones in human serum. • A novel DMSPE method was developed to purify and to enrich the analytes in serum. • In situ derivatization was conducted on the adsorbent after DMSPE procedures. Androgens play important roles in polycystic ovarian syndrome (PCOS). However, measures of androgens based on mass spectrometry (MS) remain complex due to endogenous inferences of isomers or compounds with similar structures. Lack of sensitivity can also affect the accurate quantification of androgens, especially for very low level of 11-oxygenated androgens. We developed a fast and sensitive high-performance liquid chromatography-differential mobility spectrometry tandem mass spectrometry (HPLC-DMS/MS/MS) method for the simultaneous determination of seven androgens and 17-hydroxyprogesterone. Dispersive magnetic solid phase extraction (DMSPE) was conducted with core–shell structured nanoparticles of magnetic graphene oxide (Fe 3 O 4 @GO). In situ derivatization was performed using Girard's Reagent P. Linear ranges of the eight analytes were set in terms of clinical use. Intra- and inter-run precisions were < 16.7 % and 12.9 % for all the analytes and relative error was − 14.7–13.3 % and − 9.3–11.0 %, respectively. Extraction recoveries were 54.0–92.7 % for different analytes. The method was validated and was applied to assay 432 clinical samples. The developed method is green, fast, sensitive and accurate for the determination of endogenous androgens. It can be readily implemented in medical laboratories to provide superior analytical performance over the traditional electrochemiluminescence immunoassay method. [ABSTRACT FROM AUTHOR]

Hyaluronic acid is a commercially important polysaccharide with wide applications. Along with the rapid development of hyaluronic acid-based products, their authenticity has aroused considerable attention from consumers. In the present study, a carbohydrate-binding module (CBM) SrCBM70 was cloned and expressed. The protein could specifically bind to hyaluronic acid with a strong affinity. A novel method for the identification of hyaluronic acid was subsequently established by integrating SrCBM70 into the lateral flow immunoassay (LFIA). Its detection limit for hyaluronic acid was approximately 0.1 μg/mL, and the assay could be completed in 5 min. The feasibility of this method in the authenticity identification of commercialized products containing hyaluronic acid was confirmed. The establishment of the SrCBM70-based LFIA method provided a solution for the on-site authenticity identification and would facilitate the market supervision of hyaluronic acid-based products. • A hyaluronic acid-specific CBM SrCBM70 was discovered and characterized. • A novel method was established by integrating SrCBM70 into LFIA. • This method was competent in the on-site identification of hyaluronic acid. • Its detection limit was about 0.1 μg/mL, and detection could be completed in 5 min. • The CBM-based LFIA strategy was proposed for identifying polysaccharides. [ABSTRACT FROM AUTHOR]

• Hepatitis B surface antigen was measured by two tests from representative samples. • Rapid diagnosic test and dried blood spot-based immunoassay had a high agreement. • Rapid diagnosic test is less costly, but dried blood spot test allows testing for multiple diseases. • The cost and advantages of each testing method should be considered. This study aimed to determine the agreement between a rapid diagnostic test (RDT) and a dried blood spot (DBS)-based electrochemiluminescence immunoassay (ECLIA) of hepatitis B surface antigen and to compare the costs of conducting serosurveys using RDTs and DBS in a field setting. A serosurvey was conducted in the South Central Coast region of Vietnam in May 2019. Participants aged 1-39 years were recruited using a four-stage random sampling method and tested for hepatitis B surface antigen using an RDT kit (Alere Determine) and a DBS-based ECLIA. The agreement between the RDT and the DBS-based ECLIA was assessed using cross-tabulation and Cohen kappa. Cost data were categorized by input (personnel, transportation, field consumables, laboratory consumables, and capital item/overhead) and survey phase (survey preparation, data/biospecimen collection, laboratory testing, and coordination). A total of 2072 participants were analyzed. There was a 99% agreement between the RDT and the DBS-based ECLIA results, with a Cohen kappa of 0.9. The estimated cost of conducting a serosurvey by DBS was UD $75,291, whereas RDT was $53,182. RDTs and DBS-based ECLIA provide test results with high agreements. RDTs are a better option in terms of cost, whereas the DBS-based ECLIA may be useful when evaluating multiple infectious diseases. [ABSTRACT FROM AUTHOR]

Circulation of influenza A virus (IAV), especially within poultry and pigs, continues to threaten public health. A simple and universal detecting method is important for monitoring IAV infection in different species. Recently, nanobodies, which show advantages of easy gene editing and low cost of production, are a promising novel diagnostic tool for the monitoring and control of global IAVs. In the present study, five nano-bodies against the nucleoprotein of H9N2 IAV were screened from the immunized Bactrian camel by phage display and modified with horseradish peroxidase (HRP) tags. Out of which, we determined that H9N2-NP-Nb5-HRP can crossreact with different subtypes of IAVs, and this reaction is also blocked by positive sera for antibodies against different IAV subtypes. Epitope mapping showed that the nanobody-HRP fusion recognized a conserved conformational epitope in all subtypes of IAVs. Subsequently, we developed a nanobody-based competitive ELISA (cELISA) for detecting anti-IAV antibodies in different species. The optimized amount of coating antigen and dilutions of the fusion and testing sera were 100 ng/well, 1:4000, and 1:10, respectively. The time for operating the cELISA was approximately 35 min. The cELISA showed high sensitivity, specificity, reproducibility, and stability. In addition, we found that the cELISA and hemagglutination inhibition test showed a consistency of 100% and 87.91% for clinical and challenged chicken sera, respectively. Furthermore, the agreement rates were 90.4% and 85.7% between the cELISA and commercial IEDXX ELISA kit. Collectively, our developed nanobody-HRP fusion-based cELISA is an ideal method for monitoring IAV infection in different species. [ABSTRACT FROM AUTHOR]

Fluorescent nanoprobes are widely applied in innovate enzyme-linked immunosorbent assays (ELISA) for detection of fluoroquinolones (FQs) residue in foodstuffs. Nevertheless, the complicated synthesis of nanoprobes hampers their practical applications. Herein, a nanomaterial-independent and fluorescent ELISA for sensitive detection of FQs is developed using the Eu-micelles as signal probe. Non-nanostructured Eu-micelles with high quantum yield and stability are facilely synthesized through the assembly of Eu3+ and ligands. Alkaline phosphatase catalyzes hydrolysis of 4-nitrophenyl phosphate to 4-nitrophenol. The fluorescent Eu-micelles can be readily quenched by 4-nitrophenol via static quenching. The signal generation mechanism integrates well with conventional ELISA systems. The established fluorescent ELISA achieves sensitive detection of FQs with a limit of detection of 0.03 μg/kg. The validation results from LC-MS show that the fluorescent ELISA exhibits good accuracy and recoveries. Our study presents a nanomaterial-independent strategy for developing the rapid immunoassay for FQs, which holds good promise for practical applications. • Highly fluorescent Eu-micelles are facilely prepared through self-assembly strategy. • Fluorescent and nanomaterial-independent immunoassay is developed using Eu-micelles. • The immunoassay has improved sensitivity for the detection of FQs in chicken. [ABSTRACT FROM AUTHOR]

Schematic representation of sensing of BNP antigen using the upconversion probe AuNS@Ab@PAA@Mn-NaYF 4 :Yb/Er UCNPs. [Display omitted] • Developed a photon upconversion-based immunoprobe for detecting Brain Natriuretic Peptide (BNP), a heart failure biomarker. • Probe utilizes resonance energy transfer (RET) between Mn2+ ions doped NaYF 4 :Yb/Er upconversion nanoparticles (UCNPs) and gold nanostars (AuNS). • Specific antigen–antibody interactions induce turn-on luminescence response upon BNP detection. • Exhibits high sensitivity with detection range of 30 pg/mL–303 pg/mL and low detection limit of 1.76 pg/mL. • Capable of detecting BNP in human serum within a 10-min incubation with a recovery percentage of 83.76 %–112.94 %. Brain Natriuretic Peptide (BNP) holds significant clinical importance as a biomarker for heart-related conditions aiding in diagnosis, prognosis and monitoring of heart failure, providing valuable insights for timely intercession and patient management. Photon upconversion nanomaterials exhibit captivating ability to convert low energy photons to high energy ones, enabling enhanced sensitivity in sensing application. In this perspective, we develop a new photon upconversion based immunosensor for the detection of a heart failure biomarker, Brain Natriuretic Peptide (BNP) by Resonance Energy Transfer (RET) between Polyacrylic acid capped Mn2+ ions doped NaYF 4 :Yb/Er Upconversion Nanoparticles (PAA@Mn-NaYF 4 :Yb/Er UCNPs) as the donor and gold nanostar (AuNS) as the acceptor. BNP antibody was conjugated on the surface of PAA@Mn-NaYF 4 :Yb/Er UCNPs. The immunosensor can produce strong turn on luminescence intensity in proportional to increasing BNP antigen concentration through specific antigen–antibody interactions. The proposed photon upconversion based immunosensor can achieve turn on behaviour in the linear range from 30 pg/mL to 303 pg/mL with a Limit of Detection (LOD) of 1.76 pg/mL. The immunosensor exhibited appreciable selective and sensitive response towards BNP. [ABSTRACT FROM AUTHOR]

[Display omitted] • The ratiometric fluorescence immunoassay for the detection of AFB1 was established based on Ce4+ oxidated OPD and PVP-CuNCs. • The ratiometric fluorescence immunoassay reduced the interference of the background signal. • The ratiometric fluorescence immunoassay had a low LOD. • The ratiometric fluorescence immunoassay was simple in operation and had high specificity. • PVP-CuNCs was stable and easier to obtain. As the most toxic mycotoxin, aflatoxin B1 (AFB1) can cause a variety of health problems in humans and animals. Therefore, it is very important and urgent to establish a sensitive and convenient method for the detection of AFB1. In this study, a ratiometric fluorescence method is established to detect AFB1 based on Ce4+ oxidized o-phthalylenediamine (OPD) and polyvinylpyrrolidone protected copper nanoclusters (PVP-CuNCs). The free Ce4+ with strong oxidative activity can oxidize OPD to 2,3-diaminophenazine (DAP) and emit intense fluorescence. Meanwhile, the fluorescence of PVP-CuNCs can be quenched by DAP. In the catalysis of alkaline phosphatase (ALP), adenosine triphosphate (ATP) is hydrolyzed and releases phosphate ions (PO 4 3-). PO 4 3- has a strong affinity for Ce4+ which reduces the free Ce4+ in solution. Thus, the OPD cannot be oxidized to DAP, and the fluorescence of PVP-CuNCs at 430 nm cannot be quenched. The limit of detection (LOD) of the new immunoassay is 26.79 pg/mL, and the linear detection range is 50–250 pg/mL. Besides, the recovery of AFB1 ranges from 84.66 % to 105.21 % and coefficient of variation (CV) is in the range of 1.18 %–4.88 %. Meanwhile, the new immunoassay exhibits satisfactory selectivity. These results indicate the ratiometric fluorescence immunoassay is sensitive and reliable for detection of AFB1 in actual samples. [ABSTRACT FROM AUTHOR]

Peptidyl arginine deiminase 4 (PAD4) plays a critical role in many autoimmune diseases including rheumatoid arthritis. Herein, a trypsin assisted highly immunoassay method was established to determine PAD4 activity and screen potent inhibitors from herbal plants extracts and purified natural products. The method was applied to determine endogenous PAD4 activity in both cell and tissue lysates, as well as the inhibitory effects of 20 herbal plants and 50 purified natural products. The Cinnamomi ramulus extract showed strongest inhibitory potency with IC 50 value lower than 5 μg/mL. Meanwhile, pyrroloquinoline quinone (PQQ), widely used as a dietary supplement, was discovered as a promising PAD4 inhibitor with an IC 50 value lower than 4 μM. The inhibition kinetic analysis, drug affinity response target stability (DARTS) and molecular docking were performed to confirm the interaction between PQQ and PAD4. This method has great potential for researchers to monitor activities and discover potential inhibitors of PAD4. [Display omitted] • A trypsin assisted highly sensitive immunoassay method was developed for PAD4 activity assay. • The method was used to screen PAD4 inhibitors from herbal plant extracts and natural products. • Cinnamomi ramulus extracts were found to hold high inhibitory activity against PAD4. • PQQ was discovered as a promising PAD4 inhibitor with an IC 50 value lower than 4 μM. • PQQ had good interaction with PAD4 by both DARTS and molecular docking verification analysis. [ABSTRACT FROM AUTHOR]

ELISA has become the gold standard for detecting harmful substances due to its specific antibody recognition and sensitive enzyme-catalyzed reactions. In this study, multifunctional magnetic Prussian blue nanolabels (MPBNs) were synthesized using a simple gentle two-step method to achieve a dual-readout mode. The MPBNs provide a sensitive colorimetric signal by efficiently catalyzing the oxidation of TMB and exhibit prominent photocatalytic degradation activity towards Rhodamine B (RhB). Supplemented by the quenching effect of oxTMB, the fluorescence was enabled to serve as a sensitive second signal. The magnetic property of the labels facilitates the separation and enrichment of the target, thereby improving sensitivity. Utilizing the versatile MPBNs, the visual limit of detection (vLOD) for Staphylococcus aureus is as low as 100 CFU/mL, with a quantitative analysis range of 102–108 CFU/mL. The introduction of photocatalytic reactions into immunoassay has opened up a new signal response system with strong momentum for development and application. • A multifunctional magnetic probe was constructed with peroxidase activity and photocatalytic ability. • Colorimetric and fluorescent dual-mode ELISA was developed for sensitive pathogen detection. • This work is poised to advance the utilization of photocatalytic active materials in immunosensors. [ABSTRACT FROM AUTHOR]

• Florfenicol half-antigen transformed; 8 specific monoclonal antibodies identified. • Monoclonal antibodies against florfenicol were coupled with quantum dots. • New method detects florfenicol in foods and pig feed with heightened sensitivity. • Method aligns with HPLC-MS/MS, quantitatively detecting florfenicol. Florfenicol (F), an antimicrobial agent exclusive to veterinary use within the chloramphenicol class, is extensively applied as a broad-spectrum remedy for animal diseases. Despite its efficacy, concerns arise over potential deleterious residues in animal-derived edibles, posing threats to human health. This study pioneers an innovative approach, introducing a quantum dot fluorescence-based immunoassay (FLISA) for the meticulous detection of F residues in animal-derived foods and feeds. This method demonstrates heightened sensitivity, with a detection limit of 0.3 ng/mL and a quantitative detection range of 0.6–30.4 ng/mL. Method validation, applied to diverse food sources, yields recoveries from 90.4 % to 109.7 %, featuring RSDs within 1.3 % to 8.7 %, the results showed high consistency with the national standard HPLC-MS/MS detection method. These findings underscore the method's accuracy and precision, positioning it as a promising tool for swift and reliable F residue detection, with substantial implications for fortifying food safety monitoring. [ABSTRACT FROM AUTHOR]

Immunoassays are considered important methods for detecting disease biomarkers for biomedical diagnosis. Particularly, enzyme-linked immunosorbent assay (ELISA) is a commonly-used immunoassay method due to its high reliability and low cost. Nowadays, automatic analyzers integrating all steps of ELISA can achieve detection without manual operation to save labor, increase detection efficiency and avoid manual error. However, most ELISA analyzers require more than 30 min for one detection, which are not fast enough for on-site point-of-care testing (POCT). Herein, based on the reciprocating-flowing immunobinding strategy, we developed a miniaturized reciprocating-flowing POCT platform (RF-POCT platform) for rapid immunodetection. RF-POCT platform, which included an electronic control module, a fluid control module, a mechanical control module and a detection module, enabled fast immunobinding of antigen and antibody. Taking ELISA as an example of immunoassay methods for RF-POCT platform, we developed a miniaturized reciprocating-flowing ELISA analyzer (RF-ELISA analyzer) to automatically detect SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 N protein) and dust mite-specific immunoglobulin E (DM-IgE), with the LOD of 5.2–6.6 pg/mL and the CV less than 3 %. The detection required only 3.5 min. Afterwards, we detected the SARS-CoV-2 N protein in nasal swab samples and the DM-IgE in serum samples; the specificities were 85.7–100 %, the sensitivities were 96.3–100 %. The quantitative results from RF-ELISA analyzer were generally-consistent with those from large ELISA analyzers in hospitals. Therefore, RF-ELISA analyzer demonstrated excellent performance on ultrafast, highly-sensitive, and on-site immunoassay, with potential to support on-site POCT for primary hospitals and community clinics. • A miniaturized point-of-care testing (POCT) platform enables fast immunobinding of antigen and antibody. • A miniaturized ELISA analyzer based on this platform achieves automatic detection of multiple disease biomarkers. • This ELISA analyzer requires only 3.5 min for a detection, may be the fastest ELISA analyzer so far. • Compared to existing miniaturized ELISA analyzers, the LODs of this ELISA analyzer are much lower. • This ELISA analyzer could strongly support the on-site POCT for primary hospitals and community clinics. [ABSTRACT FROM AUTHOR]

Staphylococcal enterotoxin B (SEB), the major virulence factor of Staphylococcus aureus (S. aureus), brings about grievous food poisoning by infiltrating the food supply chain at diverse phases. ELISA based on traditional antibody and single signal analysis is slightly less specific and stable in SEB detection. Here, we innovatively reported an immunoassay based on a bifunctional protein reconstructed by nanobody, greatly enhancing the specificity of ELISA. Meanwhile, this is complemented by the second signal response, quenching effect of oxidative chromogenic substances on fluorochrome, to heighten the accuracy with dual mode. Under optimal conditions, the dual-mode immunoassay showed a wide linear range from 0.31 to 2500 ng/mL and a lower limit of detection (LOD) with 0.12 ng/mL for colorimetric mode and 0.24 ng/mL for fluorescence mode. There was no nonspecific binding occurred under the interference of S. aureus and other enterotoxins, demonstrating the splendid specificity. Moreover, the proposed assay was successfully employed in detecting SEB in food samples with satisfying practicability. This work integrated the nanobody modification technology with a dual signal sensing strategy, providing a reference for the development of a more specific and feasible platform for food safety. [Display omitted] • It reported a stable and sensitive dual-mode immunoassay based on a bifunctional protein RANbody. • RANbody (SEB27-vHRP) with both recognition and catalysis facilitated the immunoassay with convenience and efficiency. • The assay using nanobodies as recognition elements avoided the interference of Staphylococcus aureus and other enterotoxins. • The dual-mode immunoassay was used successfully to evaluate SEB in food samples. [ABSTRACT FROM AUTHOR]

Antibody pairs-based immunoassay platforms served as essential and effective tools in the field of pathogen detection. However, the cumbersome preparation and limited detection sensitivity of antibody pairs challenge in establishment of a highly sensitive detection platform. In this study, using COVID-19 testing as a case, we utilized readily accessible nanobodies as detection antibodies and further proposed an accurate design concept with a more scientific and efficient screening strategy to obtain ultrasensitive antibody pairs. We employed nanobodies capable of binding different antigenic epitopes of the nucleocapsid (NP) or receptor-binding domain (RBD) antigens sandwich as substitutes for monoclonal antibodies (mAbs) sandwich in fast detection formats and utilized time-resolved fluorescence (TRF) microspheres as the signal probe. Consequently, we developed a multi-epitope nanobody sandwich-based fluorescence lateral flow immunoassay (FLFA) strip. Our results suggest that the NP antigen had a detection limit of 12.01pg/mL, while the RBD antigen had a limit of 6.51 pg/mL using our FLFA strip. Based on double mAb sandwiches, the values presented herein demonstrated 4 to 32-fold enhancements in sensitivity, and 32 to 256-fold enhancements compared to commercially available antigen lateral flow assay kits. Furthermore, we demonstrated the excellent characteristics of the proposed test strip, including its specificity, stability, accuracy, and repeatability, which underscores its the prospective utility. Indeed, these findings indicate that our established screening strategy along with the multi-epitope nanobody sandwich mode provides an optimized strategy in the field of pathogen detection. [ABSTRACT FROM AUTHOR]

1-Aminohydantoin (AHD), the residual marker of nitrofurantoin, is usually detected after derivatisation using the derivatisation reagent 2-nitrobenzaldehyde. Avoiding the antibody recognition of the derivatisation reagent is essential for the accurate detection of AHD residues. In this paper, a novel hapten called hapten D was designed, and then, a monoclonal antibody that did not recognise 2-nitrobenzaldehyde was prepared based on this novel hapten. An ultra-sensitive indirect competitive enzyme linked–immunosorbent assay (icELISA) was established under optimal conditions. The 50% inhibition concentration and limit of detection of AHD were 0.056 and 0.0060 ng/mL, respectively, which improved the sensitivity by 9–37-fold compared with the previously reported icELISA methods. The average recovery rates were 88.1%–97.3%, and the coefficient of variation was <8.6%. The accuracy and reliability of the icELISA were verified using liquid chromatography–tandem mass spectrometry. These results demonstrated that the developed icELISA is a useful and reliable tool. [Display omitted] • Novel haptens of nitrofurantoin metabolite were designed and synthesised. • A high-affinity monoclonal antibody to the nitrofurantoin metabolite was prepared. • An ultrasensitive immunoassay method was developed for nitrofurantoin metabolite. [ABSTRACT FROM AUTHOR]

Adulterating hazardous bisoxatin (BSO) and bisoxatin acetate (BSOA) in slimming foods poses a threat to public health. A rapid synchronous detection method is urgently needed. Herein, the precise design of four novel haptens based on the general skeleton of BSO and BSOA was driven by computer-chemical visualization strategy, which was used to raise monoclonal antibody (mAb) toward both target compounds. The generated mAb 1F1 recognized BSO and BSOA with maximal half-inhibitory concentration (IC 50) of 0.26 and 16.85 ng/mL, respectively. The molecular mechanism governing the duplex-recognition of mAb was elucidated by homology modeling and molecular docking. Finally, an immunochromatography (ICA) was developed for identifying BSO and BSOA, demonstrating a detection capability for screening (CCβ) estimated to be 10–500 ng/g in candy tablets, jellies, and oral liquids. This study provides a robust approach for determining adulteration in food and offers insights into hapten design to improve antibody recognition spectrum. [Display omitted] • Computer-chemical visualization strategy was proposed for precise hapten design. • Duplex-recognition antibody toward bisoxatin adulterants was first reported. • Molecular mechanism governing the antibody recognition spectrum was elucidated. • Visual immunoassay was developed for authenticating bisoxatin adulterants in food. [ABSTRACT FROM AUTHOR]

[Display omitted] • The CuO@Ag composite was synthesized by metal self-reducing triggered green etching. • Flower-like morphology of the CuO@Ag improved its binding ability with antibodies. • The CuO@Ag has excellent photothermal conversion efficiency. • The dual-mode immunosensor can achieve high-sensitive detection of clenbuterol. Photothermal materials has aroused extensive attention for building multi-signal immunosensors. Herein, a metal self-reducing triggered green etching strategy was employed for the synthesis of CuO@Ag nanoparticles (NPs) with high photothermal conversion efficiency and better antibody coupling efficiency. The flower-like morphology of the CuO@Ag NPs has improved its binding ability with antibodies, which enables them to serve as the multifunctional signal label of the lateral flow immunoassay (LFIA). Then, a colorimetric-photothermal dual-mode LFIA (CuO@Ag-based LFIA) was designed for the highly sensitive detection of clenbuterol hydrochloride (CLE). After direct, the CuO@Ag NPs endowed T-line to be irradiated by an 808 nm laser to obtain photothermal signal owing to their strong photothermal conversion efficiency of CuO@Ag. Under optimal conditions, the detection limits of the CuO@Ag-based LFIA are 0.205 ng mL−1 and 0.131 ng mL−1 under colorimetric and photothermal modes, respectively, which are 13 and 20 times sensitive than that of the conventional AuNP-based LFIA. Additionally, the LFIA was assessed by measuring CLE in food samples with recovery rates ranging from 86.37 % to 103.72 % and coefficient of variations (CVs) ranging from 2.52 % to 9.64 %. Consequently, this work provides qualitative-quantitative point-of-care testing (POCT) approach and has important reference value for other facile, green, and low-cost synthesis of composite signal labels in the LFIA system. [ABSTRACT FROM AUTHOR]

[Display omitted] • A rainbow-like multicolor LFIA based on PRCNPs is developed for detecting acetamiprid. • PRCNPs exhibit advantages in catalytic activity, antibody utilization, and signal readout. • PRCNPs-LFIA achieves single-to-multicolor output via the etching of GNRs. • PRCNPs-LFIA enables qualitative, semi-quantitative, and quantitative analysis. Traditional lateral flow immunoassay (LFIA) using single-color labels has limitations in indistinct color change and restriction to the single-mode readout, which can hardly meet the demands of high-performance detection. Inspired by the multiple colors of the rainbow, we outline a multicolor LFIA using multibranched trimetallic platinum–rhodium-cobalt nanoparticles (PRCNPs) as signal labels to initiate multicolor conversion for diverse and multicolor readouts. The intrinsic properties of PRCNPs including their trimetallic composition, multibranched morphology, and strong absorption in the visible spectrum, confer advantages in catalytic activity, antibody utilization, and signal readout. Through the synergistic integration of the catalytic PRCNPs with the etching of gold nanorods, PRCNPs-LFIA enables multicolor readouts in a rainbow-like manner. PRCNPs-LFIA allows for qualitative analysis of acetamiprid, a widely-used neonicotinoid insecticide, at 2 ng mL−1. It also provides semi-quantitative analysis based on the discrimination of at least 10 colors. Furthermore, quantitative analysis can be conducted across multiple color spaces within the range of 0.01-10 ng mL−1, achieving a lowest detection result of 0.006 ng mL−1. The recoveries of acetamiprid in food samples detected by PRCNPs-LFIA in grayscale and multiple color space modes are 87.35%-103.59%, 85.56%-114.33%, 86.14%-114.77%, and 87.81%-112.01%, respectively, with all coefficients of variation below 15%. These results demonstrate the feasibility and accuracy of PRCNPs-LFIA in practical applications. Overall, this study illustrates the potential of multifunctional PRCNPs in the development of multicolor LFIA and broadens the biosensing strategies for point-of-care testing. [ABSTRACT FROM AUTHOR]

To break through the bottleneck in preparation of nanobody (Nb) for chemical contaminants induced by the difficulties in the synthesis of immunogen, complexity and unexpectable efficiency of immunization, a novel strategy to generate Nbs based on the designed synthetic Nb libraries with final size up to 109 cfu/mL was adopted and succeeded in selection of anti-coumaphos Nb A4. Furthermore, an affinity-matured mutant Nb 3G was obtained from the secondary library. Finally, an ic-ELISA was established with the limit of detection for coumaphos low to 1.90 ng/mL, 6.4-fold improved than the parent Nb A4, and the detection range from 3.06 to 15.77 ng/mL. Meanwhile, the recovery rate of vegetable samples was from 89.9% to 98.5%. Finally, the accuracy was testified by the standard UPLC-MS/MS method with R2 up to 0.99. Overall, fully synthetic Nb libraries constructed in this work provided an alternative possibility to generate the specific Nbs for chemical contaminants. [Display omitted] • A new strategy to generate nanobodies for the chemical contaminants was constructed based on a designed synthetic Nb library. • A specific Nb 3G was selected from the coumaphos-targeted secondary Nb library. • Based on the Nb 3G, an ic-ELISA method showed satisfied sensitivity, accuracy and practicality for detecting coumaphos. [ABSTRACT FROM AUTHOR]

Periodontitis is one of the most prevalent oral diseases, and the diagnosis and monitoring of periodontitis rely on clinical and imaging examinations. Matrix metalloproteinase-8 (MMP-8) in saliva is a reliable indicator of periodontitis. The traditional detection method is enzyme-linked immunosorbent assay (ELISA), which requires professional expertise and is not sensitive enough to low-abundance protein detection. In this study, we developed a nanoimmunoassay combined with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein (Cas)12a for ultrasensitive detection of MMP-8 in saliva. Antibody-DNA-gold nanoparticles (AuNPs) and trans-cleavage activity of CRISPR/Cas12a were employed for signal amplification. With this protocol, the limit of detection (LoD) for MMP-8 was reduced to the fg/mL, indicating 100-fold higher sensitivity than that of the commercial ELISA kit and its amenability for ultrasensitive detection of trace proteins. The diagnostic sensitivity of the assay was 100 %, and the specificity was 83.3 % based on the testing of 23 clinical saliva samples. The sensitivity and specificity were considerably higher than those for ELISA. Thus, this immuno-CRISPR/Cas12a assay holds promise for both diagnosing and monitoring periodontitis in clinical settings. [Display omitted] • The assay combines the AuNPs with Cas12a for signal amplification, enabling sensitive detection of MMP-8 in saliva. • This assay enables to detect the MMP-8 at fg/mL level, which is 100-fold higher sensitivity than that of ELISA method. • The immuno-CRISPR/Cas12a assay has the potential to be applied for POCT. [ABSTRACT FROM AUTHOR]

A facile approach for direct, sensitive, and on-site identification of respiratory bacteria and viruses is still urgently needed. Here, we developed a dual-mode surface-enhanced Raman spectroscopy (SERS)–lateral flow immunoassay (LFA) based on self-assembling nanogapped SERS tags (Si@Au/Au) for the ultrasensitive and simultaneous quantification of a common respiratory bacterium (Streptococcus pneumoniae) and respiratory virus (influenza A virus) in complex specimens. Two layers of 20 nm AuNPs and an ultranarrow polyethyleneimine inter-layer (0.5 nm) were self-assembled on the surfaces of SiO 2 nanoparticles (170 nm) via electrostatic adsorption to form Si@Au/Au nanogapped tags, which provided higher stability, superior sensitivity, and more intra-particle hotspots for SERS-LFA techniques. The colorimetric mode of the proposed method enabled the rapid determination of the presence of target respiratory pathogens, while the SERS mode based on two different Raman molecules allowed the quantitative detection of a specific bacterium and virus on a single test zone with detection limits of 16 cells/mL and 29 pfu/mL, respectively. The clinical utility of the proposed method was further confirmed through the detection of 31 real clinical respiratory tract specimens. Thereby, the proposed SERS biosensor has massive potential for ultrasensitive monitoring of pathogenic microorganisms. • A self-assembling nanogapped SERS tag was easily fabricated by electrostatic adsorption. • Dual Raman signals were introduced to achieve the simultaneous detection of two microorganisms on one test line. • Simultaneous monitoring of respiratory bacteria and virus on SERS–LFA was first realized. • The dual-signal SERS–LFA greatly improves the detection efficiency of current LFA methods. [ABSTRACT FROM AUTHOR]

In preclinical protein therapeutic development studies, the emergence of anti-drug antibodies (ADA) can potentially impact drug pharmacokinetics and safety. While immunogenicity assessment is not mandatory in preclinical studies, banking samples can be valuable for interpreting unexpected pharmacological responses. Immunoassays that use generic reagents across different drug molecules can simplify ADA assessment and expedite sample evaluations. This work showcases the ability of the Gyrolab automated immunoassay platform to detect and quantify both drug-free and drug-bound (total) ADAs to monoclonal antibody (mAb) therapeutics in cynomolgus monkey preclinical studies. Compared to the previously reported total ADA ELISA, the Gyrolab assay exhibited a wider signal dynamic range and increased drug tolerance. Similar sensitivity, dynamic range and cut point factors were observed for four therapeutic mAbs of different isotypes using the Gyrolab assay. Here we present a comparison of ADA assays using bridging ELISA, total ADA ELISA and total ADA Gyrolab formats in a cynomolgus monkey study where the subjects were treated with a single dose of a mAb therapeutic. We demonstrate that the total ADA assays detected host ADA responses at earlier time points compared to the bridging ELISA. The Gyrolab assay has the best correlation between signal-to-noise (S/N) and titer over a wide ADA concentration range, highlighting the utility of Gyrolab in S/N reporting of ADA response to eliminate the need for secondary titer assays. Collectively, our results demonstrate that the generic ADA Gyrolab assay minimizes the necessity for extensive assay development and optimization for therapeutic mAbs, streamlining preclinical immunogenicity assessment to enable interpretation of pharmacological data. • Development of a plug-and-play anti-drug antibody (ADA) assay on Gyrolab platform. • This assay performs similarly across monoclonal antibodies of different isotypes. • Wider signal dynamic range and higher drug tolerance on Gyrolab as compared to ELISA. • Comparison of bridging ELISA, total ADA ELISA and total ADA Gyrolab was performed. • ADA assay on Gyrolab can potentially eliminate the need for secondary titer assay. [ABSTRACT FROM AUTHOR]

A spatial-resolved and self-calibrated photoelectrochemical (PEC) biosensor has been fabricated by a multifunctional CeO 2 /CdS heterostructure, achieving portable and sensitive detection of carcinoembryonic antigen (CEA) using a homemade 3D printing device. The CeO 2 /CdS heterostructure with matched band structure is prepared to construct the dual-photoelectrodes to improve the PEC response of CeO 2. In particular, as the photoactive nanomaterial, the CeO 2 also plays the role of peroxidase mimetic nanozymes. Therefore, the catalytic performance of CeO 2 with different morphologies (e.g., nano-cubes, nano-rods and nano-octahedra) have been studied, and CeO 2 nano-cubes (c-CeO 2) achieve the optimal catalytic activity. Upon introducing CEA, the sandwich-type immunocomplex is formed in the microplate using GOx-AuNPs-labeled second antibody as detection antibody. As a result, H 2 O 2 can be produced from the catalytic oxidization of glucose substrate by GOx, which is further catalyzed by CeO 2 to form •OH, thus in situ etching CdS and decreasing the photocurrents. The self-calibration is achieved by the dual-channel photoelectrodes on the homemade 3D printing device to obtain the photocurrents ratio, thus effectively normalizing the fluctuations of external factors to enhance the accuracy. This integrated biosensor with a detection limit as low as 0.057 ng mL−1 provides a promising way for ultrasensitive immunoassay in clinic application in complex environments. [ABSTRACT FROM AUTHOR]

The multiple-readout capability of multimodal detection enhances the flexibility, reliability, and accuracy of lateral flow immunoassay (LFIA). The conjugation of two different metal-organic frameworks (MOFs) as a new-generation composite material offers extraordinary opportunities for developing multimodal LFIA. It is anticipated to compensate limitations of traditional single colorimetric signal LFIA and improve the analysis performance. Herein, an ultra-bright fluorescent AIE-MOF was proposed and coupled with an in-situ growth of Prussian blue (PB) nanoparticles strategy to obtain a novel multimodal signal tracer (AIE-MOF@PB). Thereafter, it was successfully applied to develop the multimodal LFIA platform for the detection of nitrofurazone metabolites. The synergy of AIE-MOF and PB endows AIE-MOF@PB with superb water dispersibility, robust fluorescence emission, brilliant colorimetric signal, marvelous photothermal conversion, and enhanced antibody coupling efficiency, all of which facilitate a highly sensitive triple-readout LFIA platform. The detection sensitivity improved by at least 5-fold compared with the colloidal gold-based LFIA. This work not only inspires the rational design of aggregation-induced emission luminogens (AIEgen)-based complex materials but also highlights the promising potential in flexible point-of-care applications. • AIE-MOF with boosted fluorescent emission was fabricated. • PB modification increases biocompatibility and water dispersibility. • Synergy endows tracer with fluorescence, colorimetric, and photothermal signals. • Triple-readout LFIA improves detection performance and flexibility. • Compared to AuNP-LFIA, assay sensitivity is improved by 17 times (FL mode). [ABSTRACT FROM AUTHOR]

Prostate cancer is the most prevalent cancer in men. At present, the diagnosis and screening of prostate cancer rely on the essential biomarker known as prostate-specific antigen (PSA). The main purpose of this study was to develop a novel immunoassay for the detection of PSA based on a tri-part split-nanoluciferase system and a nanobody targeting PSA. In our approach, two small components of the split-nanoluciferase, referred to as β9 and β10, were individually fused to two anti-PSA nanobodies, N7 and N23. When these proteins bind to PSA and in the presence of the third nanoluciferase component, called Δ11S, the split-nanoluciferase components are brought into close proximity, facilitating the reassembly of the active nanoluciferase and activation of luminescence. These proteins were expressed in a bacterial expression system, purified, and employed for the intended immunoassay. The developed immunoassay demonstrated the capability to sensitively detect PSA within a linear range from 1.0 to 20.0 ng/mL with LOD of 0.4 ng/mL, and the results obtained through this immunoassay agreed with those derived from the ELISA. Our study indicates that the homogeneous immunoassay developed with nanobodies exhibits remarkable specificity for PSA and can serve as a reliable, fast, and user-friendly test for detecting PSA. • A bioluminescent-based immunoassay was created to detect prostate-specific antigen. • The new immunoassay relies on two nanobodies and a tri-part split nanoluciferase. • Recombinant proteins were expressed, purified, and used for detecting PSA. • The new test provides a quick and user-friendly in vitro assay for PSA. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cauliflower-like core-shell ReS 2 @Pt NPs are first used in lateral flow immunoassay. • Synergy of rhenium and platinum enhanced the peroxidase activity of the nanoenzymes. • The colorimetric-catalytic dual-modal LFIA enables signal amplification on demand. • ReS 2 @Pt NPs, as signal labels, exhibit higher antibody affinity and stability. Deoxynivalenol (DON) is a highly toxic secondary metabolite that poses a substantial threat to global human and animal health. However, existing detection technologies are often constrained by limited sensitivity, poor selectivity, or the high costs associated with instrumentation. Consequently, the development of rapid, low-cost, and highly sensitive biosensors for the detection of DON is of paramount importance for ensuring food safety. In this study, we synthesized the precursor of ReS₂ nanospheres using a relatively simple one-step hydrothermal method. Subsequently, Platinum (Pt) was grown in situ on the surface of the ReS₂ nanospheres via hydrothermal reduction, resulting in the formation of cauliflower-like ReS₂@Pt nanospheres. These spheres offer notable advantages, including strong colorimetric signal brightness and high anti-body loading efficiency. Surface modification with platinum significantly enhances peroxidase-like activity, broadening the detection range and improving sensitivity. Leveraging this innovation, we developed Colorimetric-LFIA (CR-LFIA) and Catalytic-LFIA (CL-LFIA) assays. Under optimized conditions, the limit of detection (LOD) for DON was determined to be 0.018 ng/mL for CR-LFIA and 6.5 pg/mL for CL-LFIA, representing 5 times and 16 times improvement over AuNPs-based LFIA (0.111 ng/mL), respectively. Moreover, the detection linear range of CL-LFIA is expanded by 2.5 times compared to CR-LFIA. Validation with real samples demonstrated recovery rates between 90.33% and 110.64%, affirming method stability and repeatability. The developed dual-modal LFIA detection system (D-LFIA) employing ReS 2 @Pt NPs holds promise for ultra-sensitive detection, with potential applications in expanding the detection range of various substances. [ABSTRACT FROM AUTHOR]

This study presents a new multiplex lateral flow immunoassay (LFIA) for the detection of aflatoxin B 1 (AFB 1), deoxynivalenol (DON), and zearalenone (ZEN). The multi-LFIA utilized phycocyanin (PC), a protein with excellent photophysical properties, as a fluorescent indicator for quantifying the three mycotoxins on a lateral flow strip platform. PC and monoclonal antibodies (mAbs) against AFB 1 , DON, and ZEN were immobilized onto carboxylated latex nanospheres (LNS) through a one-step coupling process. The resulting conjugates with strong red fluorescence were then used for multiplex sensing of AFB 1 , DON, and ZEN in wheat, corn, and animal feed. The visual results of the assay were obtained using LED UV lights coupled with a smartphone in less than 25 min. Under the optimal conditions, the new multi-LFIA achieved minimum detection limits of 1.04, 1.6, and 2.08 ng/mL for AFB 1 , 2.2, 6.45, and 2.9 ng/mL for DON, and 1.74, 1.67, and 2.11 ng/mL for ZEN in wheat, corn, and feed, respectively. The proposed method demonstrated high selectivity for AFB 1 , DON, and ZEN and showed strong correlation with LC-MS/MS results in spiked samples. In summary, the proposed method is feasible, rapid, and could serve as a highly sensitive platform for detecting AFB 1 , DON, and ZEN and other analytes in a single format. • A new method for multi-target mycotoxin detection was developed using PC as a source of fluorescence signal. • PC and anti-AFB1, DON, or ZEN mAbs were immobilized on the surface of latex nanospheres. • Conjugates with strong signal were developed and used to detect AFB1, DON, and ZEN in wheat, corn, and animal feed. • The natural fluorescent protein PC demonstrated promising potential for multiplex mycotoxin detection. [ABSTRACT FROM AUTHOR]

Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields. [ABSTRACT FROM AUTHOR]