Analytical performances of a novel fluorescent immunoassay of anti-Müllerian hormone and establishment of the reference intervals in Chinese children.

Background: AMH is important in child growth and the concentrations change with age and gender. This study aimed to evaluate the performance of the Pylon AMH assays and establish pediatric reference intervals. Methods: The experiments on imprecision, sensitivity, linearity, reportable range, interference and comparison were carried out to evaluate the analytical performance. The AMH reference ranges were calculated in 238 females and 346 males aged 0-18 years using robust methods. Results: The repeatability and the within-laboratory imprecision CVs of the assay were 3.7 % and 6.4 % at 2.25 ng/mL, and 4.6 % and 6.4 % at 15.49 ng/mL, respectively. The sensitivity (LoB = 0.05 ng/mL, LoD = 0.1 ng/mL and LoQ = 0.3 ng/mL) was verified. The linearity was 0.1-19.55 ng/mL and report up to 391 ng/mL with 20x pre-dilution. There was no significant interference from hemoglobin (500 mg/dL), triglyceride (500 mg/dL), bilirubin (10 mg/dL), cholesterol (800 mg/dL) and biotin (3000 ng/mL). The AMH measured by the Pylon assays correlated to those measured by the Elecsys assays. In males, the AMH levels were high at birth (0 d-1 m: median 95.10 ng/mL) and increased to a peak (7 m-1y: median 158.80 ng/mL) before they decreased with age (15-18 y: median 6.31 ng/mL). In females, the AMH concentrations were low at birth (0 d-1 m: median 0.20 ng/mL) and increased with age (15-18 y: median 3.03 ng/mL). Conclusion: The Pylon AMH assays showed good analytical performance and the AMH reference intervals in chinese children determined may provide a basis in clinical diagnosis and treatment of related diseases.

A nanomaterial-independent and fluorescent immunoassay based on Eu-micelles for rapid and sensitive detection of fluoroquinolones in chicken.

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.

Mesoporous Silica Nanoparticles-Enhanced Microarray Technology for Highly Sensitive Simultaneous Detection of Multiplex Foodborne Pathogens.

Ensuring food safety is paramount for the food industry and global health concerns. In this study, we have developed a method for the detection of prevalent foodborne pathogenic bacteria, including Escherichia coli, Salmonella spp., Listeria spp., Shigella spp., Campylobacter spp., Clostridium spp., and Vibrio spp., utilizing antibody-aptamer arrays. To enhance the fluorescence signals on the microarray, the mesoporous silica nanoparticles (MSNs) conjugated with fluorescein, streptavidin, and seven detection antibodies-biotin were employed, forming fluorescein doped mesoporous silica nanoparticles conjugated with detection antibodies (MSNs-Flu-SA-Abs) complexes. The array pattern was designed for easy readability and enabled the simultaneous detection of all seven foodborne pathogens, referred to as the 7FP-biochip. Following the optimization of MSNs-Flu-SA-Abs complexes attachment and enhancement of the detection signal in fluorescent immunoassays, a high level of sensitivity was achieved. The detection limits for the seven pathogens in both buffer and food samples were 102 CFU/mL through visual screening, with fluorescent intensity quantification achieving levels as low as 20-34 CFU/g were achieved on the antibody-aptamer arrays. Our antibody-aptamer array offers several advantages, including significantly reduced nonspecific binding with no cross-reaction between bacteria. Importantly, our platform detection exhibited no cross-reactivity among the tested bacteria in this study. The multiplex detection of foodborne pathogens in canned tuna samples with spiked bacteria was successfully demonstrated in real food measurements. In conclusion, our study presents a promising method for detecting multiple foodborne pathogens simultaneously. With its high sensitivity and specificity, the developed antibody-aptamer array holds great potential for enhancing food safety and public health.

Clinical performance of AMA-M2, anti-gp210 and anti-sp100 antibody levels in primary biliary cholangitis: When detected by multiplex bead-based flow fluorescent immunoassay.

BACKGROUND AND AIM: Primary biliary cholangitis (PBC) is a chronic autoimmune cholangiopathy, characterized by the presence of some autoantibodies in the serum. This study aimed to evaluate the clinical significance of AMA-M2, anti-gp210 and anti-sp100 antibody levels detected by multiplex bead-based flow fluorescent immunoassay (MBFFI) in PBC. METHODS: This study cohort included 238 PBC patients, 81 autoimmune hepatitis (AIH) patients, 62 systemic lupus erythematosus (SLE) patients, and 118 healthy controls. Serum AMA-M2, anti-gp210 and anti-sp100 antibody were detected by MBFFI and immunoblotting assay (IBT). The relationship between three antibody levels and cirrhosis, liver function, cholestasis markers and therapeutic effect to ursodesoxycholic acid (UDCA) was evaluated in PBC. RESULTS: MBFFI were presented good coincidence rate (87.39%-95.38%) with IBT. The level of AMA-M2, anti-gp210 and anti-sp100 antibodies in PBC patients were higher than other disease group and healthy controls (p ＜ .01). When compared with the healthy controls group, the AUC of AMA-M2, anti-gp210 and anti-sp100 antibodies were 0.9245, 0.7619, and 0.6789, respectively. In addition, gp210 antibody levels have diagnostic value in patients with liver cirrhosis (AUC: 0.7567). We found that when combine detect these three antibodies, the sensitivity was higher than individually detection. High level of serum anti-gp210 antibody could be related to worse liver function and more severe cholestasis in PBC patients. Moreover, serum antibody levels may decrease or remained flat in patients who responded well to UDCA. CONCLUSION: The detection of AMA-M2, anti-gp210 and anti-sp100 antibody levels by MBFFI showed good performance in the diagnosis of PBC. Serum anti-gp210 antibody level is related to cirrhosis, poor liver function and severe cholestasis in PBC.

Development and clinical evaluation of a quantitative fluorescent immunoassay for detecting canine CRP.

Canine C-reactive protein (cCRP) is one of the major positive acute phase proteins in dogs and is commonly measured to detect and monitor systemic inflammation as well as the efficacy of treatment. Traditional methods for testing cCPR, including enzyme-linked immunosorbent assay (ELISA), have some drawbacks, such as a long time for diagnosis and the requirement of well-equipped laboratories. Therefore, there is a need for a rapid and precise diagnostic test for cCRP at point-of-care. This study assessed the accuracy, precision, and validated clinical effectiveness of a diagnostic test based on fluorescent lateral flow immunoassay to detect cCRP. For the standard cCRP concentration ranging from 0 to 200 µg/mL, the cCRP diagnostic test showed strong linearity with R2 of 0.9977 (p < 0.001), and both inter- and intra-assay CVs were <14%. The limit of detection and limit of quantitation were found to be 4.0 µg/mL and 5.0 µg/mL, respectively. The cCRP serum concentration was evaluated in 21 client-owned dogs and the results were compared to a previously validated ELISA. The Pearson Correlation Coefficient between the diagnostic test kit and ELISA was 0.942 [95% confidence interval: 0.859 to 0.976, p < 0.001], and the Bland-Altman plot indicated a bias of 26.82% [95% limits of agreement: -56.03 to 109.67], indicating a significant correlation and the agreement between the data from the cCRP diagnostic test and ELISA. In conclusion, the fluorescent immunoassay based diagnostic test is a suitable option for rapidly and precisely detecting cCRP in dogs, providing a convenient alternative to traditional methods for diagnosing acute inflammation.

Competitive fluorescent immunoassay for the ultrasensitive determination of amyloid beta peptide1-42 based on Ag@SiO2@N, S-GQD nanocomposites.

A competitive fluorescent immunoassay is described for the ultrasensitive determination of amyloid beta peptide1-42 (Aß1-42), a biomarker for early diagnosis of Alzheimer's disease. N, S-doped graphene quantum dots (N, S-GQDs) were freely assembled on the surface of Ag@SiO2 nanoparticles to obtain a composite (Ag@SiO2@N, S-GQD nanocomposite), which was successfully prepared and characterized. By theoretical study, the optical properties of nanocomposites are improved compared with GQDs, due to the advantages of combining N, S co-doping and metal-enhanced fluorescence (MEF) effect of Ag NPs. In addition, Aß1-42 was modified by Ag@SiO2@N, S-GQDs to prepare a probe with high photoluminescence properties (Ag@SiO2@N, S-GQDs-Aß1-42). In the presence of Aß1-42, a competitive reaction towards anti-Aß1-42 fixed on the ELISA plate was proceeded between Aß1-42 and Ag@SiO2@N, S-GQDs-Aß1-42 by specific capture of antigen-antibody. The emission peak of Ag@SiO2@N, S-GQDs-Aß1-42 (400 nm emission) was used for the quantitative determination of Aß1-42. Under the optimal conditions, the fluorescent immunoassay exhibited a linear range of 0.32 pg·mL-1-5 ng·mL-1 with a detection limit of 0.098 pg·mL-1. The results show that the immunoassay has good analytical ability and can provide a new method for the clinical determination of Aß1-42.

Fluorescence immunoassay based on magnetic separation and ZnCdSe/ZnS quantum dots as a signal marker for intelligent detection of sesame allergen in foods.

To detect sesame allergens, a novel fluorescent immunoassay incorporating a magnetic separation step has been developed via two measurement devices: a fluorescence spectrophotometer and a smartphone equipped with a color reader. Sesame allergen monoclonal antibody (Ab) was covalently bonded with quantum dots (QDs) to act as a signal probe. The capture probes were synthesized as sesame allergen (SA) modified carboxyl-functional magnetic polystyrene microspheres (MPMs). Analytical variables such as the amount of modified SA, the reaction volume of the QDs-Ab, the ionic strength, the pH, and the incubation time of the reaction were optimized. The analytical range and the limit of detection (LOD) of the spectrophotometer-based assay were 80-640 µg/L and 12.75 µg/L, respectively. The analytical range and the LOD with smartphone read-out were 80-640 µg/L and 10.15 µg/L, respectively. Furthermore, the developed assay using the fluorescence spectrophotometer and the smartphone read-out was successfully applied to field testing of SA in solution extracts of biscuits, bread, almond beverage, and energy bars with acceptable results. The recovery rates were in the range 89-108%. It was concluded that the new assay may be used for rapid sesame allergen testing in the laboratory and in the field.

Portable high-throughput multimodal immunoassay platform for rapid on-site COVID-19 diagnostics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a causal agent of Coronavirus Disease 2019 (COVID-19) has led to the global pandemic. Though the real-time reverse transcription polymerase chain reaction (RT-PCR) acting as a gold-standard method has been widely used for COVID-19 diagnostics, it can hardly support rapid on-site applications or monitor the stage of disease development as well as to identify the infection and immune status of rehabilitation patients. To suit rapid on-site COVID-19 diagnostics under various application scenarios with an all-in-one device and simple detection reagents, we propose a high-throughput multimodal immunoassay platform with fluorescent, colorimetric, and chemiluminescent immunoassays on the same portable device and a multimodal reporter probe using quantum dot (QD) microspheres modified with horseradish peroxidase (HRP) coupled with goat anti-human IgG. The recombinant nucleocapsid protein fixed on a 96-well plate works as the capture probe. In the condition with the target under detection, both reporter and capture probes can be bound by such target. When illuminated by excitation light, fluorescence signals from QD microspheres can be collected for target quantification often at a fast speed. Additionally, when pursuing simple detection without using any sensing devices, HRP-catalyzed TMB colorimetric immunoassay is employed; and when pursuing highly sensitive detection, HRP-catalyzed luminol chemiluminescent immunoassay is established. Verified by the anti-SARS-CoV-2 N humanized antibody, the sensitivities of colorimetric, fluorescent, and chemiluminescent immunoassays are respectively 20, 80, and 640 times more sensitive than that of the lateral flow colloidal gold immunoassay strip. Additionally, such a platform can simultaneously detect multiple samples at the same time thus supporting high-throughput sensing; and all these detecting operations can be implemented on-site within 50 min relying on field-operable processing and field-portable devices. Such a high-throughput multimodal immunoassay platform can provide a new all-in-one solution for rapid on-site diagnostics of COVID-19 for different detecting purposes.

Sensitivity and Specificity of Rapid SARS-CoV-2 Antigen Detection Using Different Sampling Methods: A Clinical Unicentral Study.

Rapid antigen detection of SARS-CoV-2 has been widely used. However, there is no consensus on the best sampling method. This study aimed to determine the level of agreement between SARS-CoV-2 fluorescent detection and a real-time reverse-transcriptase polymerase chain reaction (rRT-PCR), using different swab methods. Fifty COVID-19 and twenty-six healthy patients were confirmed via rRT-PCR, and each patient was sampled via four swab methods: oropharyngeal (O), nasal (N), spit saliva (S), and combined O/N/S swabs. Each swab was analyzed using an immunofluorescent Quidel system. The combined O/N/S swab provided the highest sensitivity (86%; Kappa = 0.8), followed by nasal (76%; Kappa = 0.68), whereas the saliva revealed the lowest sensitivity (66%; kappa = 0.57). Further, when considering positive detection in any of the O, N, and S samples, excellent agreements with rRT-PCR were achieved (Kappa = 0.91 and 0.97, respectively). Finally, among multiple factors, only patient age revealed a significant negative association with antigenic detection in the saliva. It is concluded that immunofluorescent detection of SARS-CoV-2 antigen is a reliable method for rapid diagnosis under circumstances where at least two swabs, one nasal and one oropharyngeal, are analyzed. Alternatively, a single combined O/N/S swab would improve the sensitivity in contrast to each site swabbed alone.

Fluorescent Immunoassay with a Copper Polymer as the Signal Label for Catalytic Oxidation of O-Phenylenediamine.

This work suggested that Cu2+ ion coordinated by the peptide with a histidine (His or H) residue in the first position from the free N-terminal reveals oxidase-mimicking activity. A biotinylated polymer was prepared by modifying His residues on the side chain amino groups of lysine residues (denoted as KH) to chelate multiple Cu2+ ions. The resulting biotin-poly-(KH-Cu)20 polymer with multiple catalytic sites was employed as the signal label for immunoassay. Prostate specific antigen (PSA) was determined as the model target. The captured biotin-poly-(KH-Cu)20 polymer could catalyze the oxidation of o-phenylenediamine (OPD) to produce fluorescent 2,3-diaminophenazine (OPDox). The signal was proportional to PSA concentration from 0.01 to 2 ng/mL, and the detection limit was found to be eight pg/mL. The high sensitivity of the method enabled the assays of PSA in real serum samples. The work should be valuable for the design of novel biosensors for clinical diagnosis.

Analytical and clinical performance of different platforms simultaneously detecting 15 antinuclear antibodies.

BACKGROUND: Antinuclear antibodies (ANAs) are invaluable biomarkers for the diagnosis of autoimmune diseases (AIDs). This study aims to compare the performances of line immunoassay (LIA), multiplex bead-based flow fluorescent immunoassay (MBFFI), and magnetic bar code immunofluorescence assay (MBC-IF) to detect ANA-Profile-15S. METHODS: In total, 184 samples from AID patients and 50 healthy controls (HCs) were collected. Fifteen ANAs (anti-dsDNA, nucleosome, histone, Sm, PCNA, ribosomal-P, SS-A/Ro52, SS-A/Ro60, SS-B/La, centromere B [CENP-B], Scl-70, U1-snRNP, AMA-M2, Jo-1, and Pm/Scl) were subjected to parallel detection by the LIA, MBFFI, and MBC-IF. The consistency between assays was analyzed. The discrepant results were further examined by chemiluminescent immunoassay (CLIA). RESULTS: Anti-SS-A/Ro52 and SS-A/Ro60 autoantibodies were the most common autoantibodies in ANA positive-profiles, and were detected with equal efficiency by the LIA, MBFFI, and MBC-IF (p = 0.101 and p = 0.732, respectively). The three assays showed excellent agreement (consistency range: 66.5%-97.5%), and total consistency was 85.8%. The MBFFI and MBC-IF assays were in good agreement in terms of ANA-Profile-15S determination; the kappa coefficient ranged from 0.59 to 0.95, except for the PCNA and PM-Scl. Of the 262 re-assessed divergent results, 124 (47.33%) were positive on CLIA; the various autoantibodies exhibited variable patterns. More importantly, the ANA-Profile-15S results of the MBFFI and MBC-IF accurately identified patients with AID; the area under the curves ranged from 0.642 to 0.919. CONCLUSIONS: The novel MBFFI and MBC-IF assay performed well in detecting ANA-Profile-15S. The application of MBFFI and MBC-IF play important roles in laboratory diagnosis of AIDs.

Comparison of the Automated Fluorescent Immunoassay System With Roche Elecsys and Beckman Coulter Access 2 Assays for Anti-Müllerian Hormone Measurement.

BACKGROUND: Since 2017, automated assays have been used in most clinical laboratories for anti-Müllerian hormone (AMH) level measurement. We evaluated the analytical performance of the newly developed automated fluorescent immunoassay system (AFIAS) AMH assay (Boditech Med, Gangwon-do, Korea) in comparison with the Roche Elecsys and Beckman Coulter Access 2 AMH assays. METHODS: Analytical performance of the AFIAS AMH assay was assessed in terms of linearity, repeatability, and within-laboratory precision (CV%) using human recombinant AMH samples according to the Clinical and Laboratory Standards Institute (CLSI) guidelines EP05 and EP06. Using 293 serum samples collected from an infertility clinic, the AMH levels were compared across AFIAS, Elecsys, and Access 2 AMH assays according to the CLSI EP09 guidelines. RESULTS: The AFIAS AMH assay results were linear across the measurement range of 0.420-72.386 pmol/L AMH, with repeatability of 6.341%. CV% of the AFIAS AMH assay for three levels of control, 1.786, 7.143, and 56.857 pmol/L, were 5.801%, 5.714%, and 6.228%, respectively. The results of the three AMH assays showed strong correlation: AFIAS and Elecsys [slope, 1.055 (95% confidence interval (CI), 1.022-1.088) and Spearman's rho, 0.978 (95% CI, 0.973-0.983)], Elecsys and Access 2 [slope, 0.813 (95% CI, 0.791-0.834) and Spearman's rho, 0.986 (95% CI, 0.983-0.989)], and AFIAS and Access 2 [slope, 0.836 (95% CI, 0.821-0.853) and Spearman's rho, 0.984 (95% CI, 0.980-0.988)]. CONCLUSIONS: The AFIAS AMH assay may be an alternative to the Roche Elecsys and Beckman Coulter Access 2 AMH assays.

Horseradish peroxidase-triggered direct in situ fluorescent immunoassay platform for sensing cardiac troponin I and SARS-CoV-2 nucleocapsid protein in serum.

Direct in situ fluorescent enzyme-linked immunosorbent assay (ELISA) is rarely investigated and reported. Herein, a direct in situ high-performance HRP-labeled fluorescent immunoassay platform was constructed. The platform was developed based on a rapid in situ fluorogenic reaction between Polyethyleneimine (PEI) and p-Phenylenediamine (PPD) analogues to generate fluorescent copolymer nanoparticles (FCNPs). The formation mechanism of FCNPs was found to be the oxidation of •OH radicals, which was further proved by nitrogen protection and scavenger of •OH radicals. Meantime, the fluorescence wavelength of FCNPs could be adjusted from 471 to 512 nm by introducing various substitution groups into the PPD structure. Using cardiac troponin I (cTnI) and SARS-CoV-2 nucleocapsid protein (N-protein) as the model antigens, the proposed fluorescent ELISA exhibited a wide dynamic range of 5-180 ng/mL and a low limit of detection (LOD) of 0.19 ng/mL for cTnI, and dynamic range of 0-120 ng/mL and a LOD of 0.33 ng/mL for SARS-CoV-2 N protein, respectively. Noteworthy, the proposed method was successful applied to evaluate the cTnI and SARS-CoV-2 N protein levels in serum with satisfied results. Therefore, the proposed platform paved ways for developing novel fluorescence-based HRP-labeled ELISA technologies and broadening biomarker related clinical diagnostics.

Eco-Friendly Fluorescent ELISA Based on Bifunctional Phage for Ultrasensitive Detection of Ochratoxin A in Corn.

Conventional enzyme-linked immunosorbent assay (ELISA) is commonly used for Ochratoxin A (OTA) screening, but it is limited by low sensitivity and harmful competing antigens of enzyme-OTA conjugates. Herein, a bifunctional M13 bacteriophage with OTA mimotopes fused on the p3 protein and biotin modified on major p8 proteins was introduced as an eco-friendly competing antigen and enzyme container for enhanced sensitivity. Mercaptopropionic acid-modified quantum dots (MPA-QDs), which are extremely sensitive to hydrogen peroxide, were chosen as fluorescent signal transducers that could manifest glucose oxidase-induced fluorescence quenching in the presence of glucose. On these bases, a highly sensitive and eco-friendly fluorescent immunoassay for OTA sensing was developed. Under optimized conditions, the proposed method demonstrates a good linear detection of OTA from 4.8 to 625 pg/mL and a limit of detection (LOD) of 5.39 pg/mL. The LOD is approximately 26-fold lower than that of a conventional horse radish peroxidase (HRP) based ELISA and six-fold lower than that of a GOx-OTA conjugate-based fluorescent ELISA. The proposed method also shows great specificity and accepted accuracy for analyzing OTA in real corn samples. The detection results are highly consistent with those obtained using the ultra-performance liquid chromatography-fluorescence detection method, indicating the high reliability of the proposed method for OTA detection. In conclusion, the proposed method is an excellent OTA screening platform over a conventional ELISA and can be easily extended for sensing other analytes by altering specific mimic peptide sequences in phages.

Development and Evaluation of a Quantitative Fluorescent Lateral Flow Immunoassay for Cystatin-C, a Renal Dysfunction Biomarker.

The diagnosis, prognosis, and control of chronic kidney disease rely on an understanding of the glomerular filtration rate (GFR). The renal clearance of the cystatin-C is closely associated with the GFR. Cystatin-C is a more suitable GFR marker than the commonly used creatinine. General techniques for cystatin-C calculation, such as particle-enhanced turbidimetric and nephelometric assay, are time-consuming and tedious. Here, we propose a rapid, quantitative immunoassay for the detection of cystatin-C. A fluorescence-based lateral-flow kit was developed in a sandwich format by using a monoclonal antibody. A Linear calibration was obtained over the clinical diagnostic range of 0.023-32 µg/mL and the limit of detection (LOD) was 0.023 µg/mL and the limit of quantification (LOQ) was 0.029 µg/mL. Average recoveries from spiked urine samples ranged from 96-100% and the coefficient of variation was less than 4% for both intra and inter-day assays with excellent repeatability. With the comparison with an ELISA kit, the developed kit is highly sensitive, performs well over the detection range, provides repeatable results in a short time, and can easily be used at point-of-care (POC), making it an ideal candidate for rapid testing in early detection, community screening for renal function disorders.

Simultaneous determination of ochratoxin A and enterotoxin A in milk by magnetic nanoparticles based fluorescent immunoassay.

Ochratoxin A (OTA) and staphylococcus enterotoxin A (SEA) are highly toxic contaminants and have induced human health problems. They commonly occur in milk and milk products. A competitive fluorescent immunoassay was developed for rapid and simultaneous determination of these toxins in milk samples. The procedure was based on the competitive immunoreactions between antigens in sample and antigen-fluorescent dye conjugates with immobilised antibodies on magnetic nanoparticles (MNPs). Each monoclonal antibody specifically recognises its corresponding toxin (antigen), and there is no cross-reactivity in the assay. First, monoclonal antibodies against OTA and SEA were produced. The activity of the obtained antibodies was determined by fluorescent-linked immunosorbent assay. Then, the monoclonal antibodies were immobilised on MNPs. The amounts of immobilised anti-OTA antibody and anti-SEA antibody were determined to be 20 and 22 µg mL-1, respectively. The antigen-fluorescent dye conjugates OTA-OVA-ATTO620 and SEA-FITC were prepared. The optimal amount of immobilised antibodies for competitive immunoassay was determined. It was found that the linear range of OTA in buffer was larger (0.001-100 ng mL-1) than the linear range of SEA (0.001-20 ng mL-1). The results for simultaneous determination of OTA and SEA in sixfold diluted milk were almost the same in buffer; the linear range for OTA was from 0.005  to 100 ng mL-1 and for SEA from 0.005  to 20 ng mL-1. The detection limit for both OTA and SEA in milk was 0.004 ng mL-1. The developed method took half the time of the individual assays (20 min). The assay was evaluated using spiked milk samples. The influences of somatic cell count, fat, pH and protein concentration in milk on immunoassay were studied. In summary, this developed immunoassay could provide an effective and rapid approach for detecting multi-toxins in milk samples.

Magnetic assisted fluorescence immunoassay for sensitive chloramphenicol detection using carbon dots@CaCO3 nanocomposites.

Analytical methods with high sensitivities and short assay times are urgently required for the screening of "zero tolerance" hazardous substances in food. Herein, we propose a fluorescent immunoassay for the highly sensitive and rapid analysis of chloramphenicol (CAP) based on carbon dots (CDs)-encapsulated CaCO3 nanospheres and magnetic nanoparticles (MNPs). The fluorescent immunoprobes were prepared by coupling the anti-CAP antibodies to carboxymethyl cellulose-functional CDs@CaCO3 nanospheres. Chitosan-modified MNPs with "core-shell" structures were prepared and then conjugated to the CAP hapten, acting as the nano-carrier and interface for the immunoreaction. With the assistance of MNPs, the established fluorescent immunoassay achieved the sensitive detection of CAP in chicken with a limit of detection of 0.03 µg kg-1 and recoveries ranging from 83.7%-105.0%. The analysis results of the fluorescent immunoassay were evaluated by the enzyme-linked immunosorbent assay, having a correlation coefficient of 0.981. Our work provides a rapid, facile, and reliable strategy for the highly sensitive analysis of food contaminants based on "green" fluorescent nanoprobes.

Evaluation of a novel high sensitivity cardiac troponin I assay with whole blood.

BACKGROUND: High-sensitivity cardiac troponin (hs-cTn) assays provide high sensitivity detection of myocardial injury. Although an assay using whole blood can reduce turn-around-time and labour, hs-cTn assays using whole blood samples are novel requiring characterization of their analytical performance. METHODS: The imprecision of Pylon hs-cTnI assay was evaluated with whole blood, plasma and commercial quality control samples. The limit of quantitation (LOQ) of whole blood samples and plasma were determined for the Pylon hs-cTnI assay. The correlation between the Pylon hs-cTnI assay and the Abbott Architect hs-cTnI assay was evaluated using whole blood samples and plasma. RESULTS: The average concentrations of pooled patient plasma were 8.3, 15.0 and 396.9 ng/l, while the corresponding CVs of repeatability and within-laboratory CVs were calculated respectively as 7.6% and 9.9%, 4.3% and 4.5%, and 3.3% and 4.5%. LOQ (20% CV) was 1.2 ng/l in plasma and 2.0 ng/l in whole blood. The lowest concentrations to reach 10% CV were 4.8 ng/l with plasma and 9.4 ng/l with whole blood. Quantification of whole blood and corresponding plasma samples correlated with no effect by hematocrits ranging from 25 to 44%. CONCLUSION: The analytical performance of the Pylon hs-cTnI assay with whole blood is comparable to that of a clinical lab instrument.

A sensitive immunoassay for simultaneous detection of foodborne pathogens using MnO2 nanoflowers-assisted loading and release of quantum dots.

In this study, a sensitive immunoassay using immunomagnetic nanobeads (MNBs), manganese dioxide nanoflowers (MnO2 NFs) and quantum dots (QDs) was developed for simultaneous detection of E. coli O157: H7 and Salmonella typhimurium. MnO2 NFs were synthesized, functionalized and incubated with QDs to obtain QDs@MnO2 nanocomposites, followed by modification with antibodies (pAbs) to obtain pAb-QDs@MnO2 nanocomposites (QM NCs). Target bacteria were first conjugated with MNBs and QM NCs to form MNB-bacteria-QM complexes. Then, QDs were quickly released from the complexes using glutathione to reduce MnO2 to Mn2+. Finally, fluorescent intensity at characteristic wavelength was measured by optical detector to determine target bacteria. This immunoassay could simultaneously and quantitatively detect E. coli from 1.5 × 101 to 1.5 × 106 CFU/mL with detection limit of 15 CFU/mL and Salmonella from 4.0 × 101 to 4.0 × 106 CFU/mL with detection limit of 40 CFU/mL in 2 h. The mean recovery for both bacteria in spiked chicken samples was ∼96%.