Fluorescent Enzyme-linked Immunosorbent Assay Research Articles

Impact of glucocorticoids and rapamycin on autophagy in Candida glabrata-infected macrophages from BALB/c mice.

In the defense against microorganisms like Candida albicans, macrophages recruit LC3(Microtubule-associated protein 1A/1B-light chain 3) to the periplasm, engaging in the elimination process through the formation of a single-membrane phagosome known as LC3-associated phagocytosis (LAP). Building on this, we propose the hypothesis that glucocorticoids may hinder macrophage phagocytosis of Candida glabrata by suppressing LAP, and rapamycin could potentially reverse this inhibitory effect. RAW264.7 cells were employed for investigating the immune response to Candida glabrata infection. Various reagents, including dexamethasone, rapamycin, and specific antibodies, were utilized in experimental setups. Assays, such as fluorescence microscopy, flow cytometry, ELISA (Enzyme-Linked Immunosorbent Assay), Western blot, and confocal microscopy, were conducted to assess phagocytosis, cytokine levels, protein expression, viability, and autophagy dynamics. Glucocorticoids significantly inhibited macrophage autophagy, impairing the cells' ability to combat Candida glabrata. Conversely, rapamycin exhibited a dual role, initially inhibiting and subsequently promoting phagocytosis of Candida glabrata by macrophages. Glucocorticoids hinder macrophage autophagy in Candida glabrata infection by suppressing the MTOR pathway(mammalian target of rapamycin pathway), while the activation of MTOR pathway by Candida glabrata diminishes over time. Our study elucidates the intricate interplay between glucocorticoids, rapamycin, and macrophage autophagy during Candida glabrata infection. Understanding the implications of these interactions not only sheds light on the host immune response dynamics but also unveils potential therapeutic avenues for managing fungal infections.

Sensitive fluorescence ELISA for the detection of zearalenone based on self-assembly DNA nanocomposites and copper nanoclusters.

Zearalenone (ZEN), produced by Fusarium species, is a potential risk to human health. Traditional enzyme-linked immunosorbent assay (ELISA) is restricted due to low sensitivity for the detection of ZEN. Herein, enzyme nanocomposites (ALP-SA-Bio-ssDNA, ASBD) were prepared with the self-assembly strategy based on streptavidin-labeled alkaline phosphatase (SA-ALP) and dual-biotinylated ssDNA (B2-ssDNA). The enzyme nanocomposites improved the loading amount of ALP and catalyzed more ascorbic acid 2-phosphate to generate ascorbic acid (AA). Subsequently, Cu2+ could be reduced to copper nanoclusters (CuNCs) having strong fluorescence signal by AA with poly T. Benefiting from the high enzyme load of nanocomposites and the strong signal of CuNCs, the fluorescence ELISA was successfully established for the detection of ZEN. The proposed method exhibited lower limit of detection (0.26ngmL-1) than traditional ELISA (1.55ngmL-1). The recovery rates ranged from 92.00% to 108.38% (coefficient of variation < 9.50%) for the detection of zearalenone in corn and wheat samples. In addition, the proposed method exhibited no cross reaction with four other mycotoxins. This proposed method could be used in trace detection for food safety.

Clinical characteristics of children with IgE-mediated cow's milk protein allergy

Objective: To analyze the clinical characteristics of children with IgE-mediated cow's milk protein allergy (CMPA) and provide a basis for disease management and prevention. Methods: A cross-sectional study was conducted to analyze 142 children aged 0-12 years who were diagnosed with IgE-mediated CMPA in Capital Institute of Pediatrics Affiliated Children's Hospital from 2020 to 2022. There were 79 males (55.6%) and 63 females (44.4%), with an average age of 14 (8, 27) months. 61 cases (43.0%) were in the <1-year-old group, 54 cases (38.0%) in the 1-3-year-old group, and 27 cases (19.0%) in the >3-year-old group. Data on demographic data, clinical manifestations, mean wheel diameter of skin prick test and serum specific IgE level were collected. The serum cow's milk protein sIgE and component sIgE were measured by ImmunoCAP fully automated system of fluorescence enzyme-linked immunosorbent assay, and statistically analyzed using chi-square test, nonparametric tests, correlation. Results: Cutaneous symptoms were the first and most frequent in 142 children (97.9%, 139/142 cases), followed by digestive (29.6%, 42/142 cases) and respiratory symptoms (27.5%, 39/142 cases).The proportion of children with respiratory symptoms after consuming cow's milk was significantly higher in the>3 years age group than those in the infant and toddler groups(66.7% vs 19.7%,χ2=18.396,P<0.01;66.7% vs 16.7%,χ2=20.250,P<0.01), and the symptoms involving ≥3 systems were also significantly higher than those in the other two groups(37.0% vs 13.1%,χ2=6.597,P<0.05;37.0% vs 7.4%,χ2=12.120,P<0.01). The average cow's milk SPT diameter and serum sIgE levels in the>3 years age group were significantly higher than those in the infant and toddler groups (Z=-4.682, P<0.01; Z=-3.498, P<0.01); (Z=-4.463, P<0.01; Z=-6.463, P<0.01). The most common cow's milk component protein were β-lactoglobulin(65.1%,56/86 cases) and casein (57.0%, 49/86 cases). Multiple-sensitization rate of the patients were 54.9%. Egg white (43.7%, 62/142 cases) was the most common co-sensitization food allergen while mold (12.7%, 18/142 cases) and weed pollen (12.7%, 18/142 cases) were the main co-sensitization aeroallergens. The proportion of multiple-sensitization to aeroallergens in the children group was the highest (51.9%, 14/27 cases), followed by the toddler group (29.6%, 16/54 cases), and the infant group was the least (3.3%, 2/61 cases). There was a significant difference among these three groups (χ2=7.476, P<0.05). Conclusion: Skin and mucosal symptoms are the most common in CMPA patients. The proportion of respiratory symptoms and multisystem involvement increased with age as well as the wheal diameter in skin test and serum sIgE level elevated. CMPA patients older than 3 years had the highest proportion of aeroallergen sensitization and airway allergic diseases.

Levels of neutrophil extracellular traps in neonates with acute respiratory distress syndrome

To study the changes in cell free-DNA (cf-DNA), a marker of neutrophil extracellular traps (NETs), in neonates with acute respiratory distress syndrome (ARDS), and to evaluate its relationship with the severity and early diagnosis of ARDS. The neonates diagnosed with ARDS in the Affiliated Hospital of Jiangsu University from January 2021 to June 2022 were enrolled in the prospective study. The neonates were divided into mild, moderate, and severe ARDS groups based on the oxygen index (OI) (4≤OI<8, 8≤OI<16, and OI≥16, respectively). The control group was selected from jaundice neonates who were observed in the neonatal department of the hospital during the same period, and they had no pathological factors causing neonatal jaundice. Peripheral blood samples were collected on day 1, day 3, and day 7 after admission for the ARDS group, and on the day of admission for the control group. Serum cf-DNA levels were measured using a fluorescence enzyme-linked immunosorbent assay. Serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were measured using enzyme-linked immunosorbent assay. A Pearson correlation analysis was used to evaluate the correlation of serum cf-DNA levels with serum IL-6 and TNF-α levels. A total of 50 neonates were enrolled in the ARDS group, including 15 neonates with mild ARDS, 25 with moderate ARDS, and 10 with severe ARDS. Twenty-five neonates were enrolled in the control group. Compared with the control group, the serum levels of cf-DNA, IL-6, and TNF-α in all ARDS groups were significantly increased (P<0.05). Compared with the mild ARDS group, the serum levels of cf-DNA, IL-6, and TNF-α in the moderate and severe ARDS groups were significantly increased (P<0.05), and the increase was more significant in the severe ARDS group (P<0.05). The serum levels of cf-DNA, IL-6, and TNF-α in all ARDS groups were significantly increased on day 3 after admission and significantly decreased on day 7 after admission compared with those on day 1 after admission (P<0.05). The Pearson correlation analysis showed that there was a positive correlation between serum cf-DNA levels and IL-6 levels as well as TNF-α levels in 50 neonates with ARDS (P<0.05). There is an excessive expression of NETs in neonates with ARDS, and dynamic monitoring of serum cf-DNA levels has certain clinical value in evaluating the severity and early diagnosis of ARDS in neonates.

A high-throughput fluorescence immunoassay based on conformational locking strategy of MOFs to enhance AIE effect of CuNCs-CS for bisphenol S analysis in food samples

Dietary intake is an important exposure pathway of environmental bisphenols including bisphenol S (BPS, a typical emerging organic pollutant). However, available data on the occurrence and risk assessment of BPS in food samples are limited due to the lack of high-throughput analytical methods. Herein, based on H2O2 inducing fluorescence turn-off signals, a sensitive and high-throughput fluorescence enzyme-linked immunosorbent assay (FELISA) was developed for BPS investigation in beverages. In this system, the fluorescence of copper nanoclusters in ZIF-8 (CuNCs-CS@ZIF-8) could be quenched with the presence of H2O2 owing to the strong oxidation, being served as signal amplification in FELISA. Meanwhile, the aggregation-induced emission (AIE) fluorescence enhancement of CuNCs-CS through the conformational locking of ZIF-8 provided an effective strategy to overcome the intrinsic limitations of FELISA including high background, serious fluorescence quenching in the detection of real samples. Thus, the integration of AIE building blocks into porous ZIF-8 showed obvious advantages in enhancing the analytical performance of FELISA. Under optimized conditions, satisfactory results with good accuracy were achieved in this proposed FELISA, which was applied for the systematic investigation of BPS in foods and further health risk assessments. The results after 100 beverages analysis displayed an average detection frequency of 55%, and the detected concentration of BPS ranged from below the limit of quantification (LOQ) to 12.08 ng/mL with a mean value of 1.717 ng/mL. Besides, the estimated daily intake (EDI) of BPS was calculated to be 0.05449–4.483 ng/kg-bw/day, in which milk–tea drinks contributed to the majority of BPS daily intake (mean EDI = 4.483 ng/kg-bw/day). And the hazard quotient (HQ) according to EDI values indicated that BPS exposure in beverages might not make a considerable impact on human health.

Simultaneous Determination of Antibiotics, Mycotoxins, and Hormones in Milk by an 8-17 DNAzyme-Based Enzyme-Linked Immunosorbent Assay.

The simultaneous detection of three kinds of small-molecule contaminants (antibiotics, mycotoxins, and hormones) in milk was realized by using an 8-17 DNAzyme-based fluorescent enzyme-linked immunosorbent assay (ELISA), in which 8-17 DNAzyme was utilized as the catalytic enzyme for amplifying the signal. Compared with the conventional ELISA in which horseradish peroxidase is used as the catalyzing factor, this 8-17 DNAzyme-based ELISA could achieve multicolor signal output with lower detection limits. The linearities for chloramphenicol, 17β-estradiol, and aflatoxin M1 were in the range of 0.3 ng/mL-3 μg/mL, 3 ng/mL-3 μg/mL, and 3 pg/mL-3 ng/mL with quantitation limits of 0.3, 3, and 0.003 ng/mL, respectively. This proposed scheme demonstrated that the 8-17 DNAzyme might be an effective substitute for horseradish peroxidase in ELISA for the development of ultrasensitive and multicolor fluorescence immunoassay, which would stimulate the development of ELISA in a new orientation.

Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has necessitated rapid, easy-to-use, and accurate diagnostic methods to monitor the virus infection. Herein, a ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed using Si-fluorescein isothiocyanate nanoparticles (FITC NPs) for detecting SARS-CoV-2 nucleocapsid (N) protein. Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane (APTES)-FITC as the Si source. This method did not need post-modification and avoided the reduction in quantum yield and stability. The p-nitrophenyl (pNP) produced by the alkaline phosphatase (ALP)-mediated hydrolysis of p-nitrophenyl phosphate (pNPP) could quench Si fluorescence in Si-FITC NPs via the inner filter effect. In ELISA, an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody. ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs. The change in fluorescence intensity ratio could be used for detecting N protein, with a wide linearity range (0.01–10.0 and 50–300 ng/mL) and low detection limit (0.002 ng/mL). The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum. Moreover, this proposed method can accurately distinguish coronavirus disease 2019 (COVID-19) and non-COVID-19 patient samples. Therefore, this simple, sensitive, and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection.Electronic Supplementary MaterialSupplementary material (characterization of Si-FITC NPs (FTIR spectrum, XRD spectra, and synchronous fluorescence spectra); condition optimization of ALP response (fluorescence intensity ratio change); mechanism investigation of ALP response (fluorescence lifetime decay curves and UV—vis absorption spectra); detection of N protein using commercial ELISA Kit; analytical performance of assays for ALP detection or SARS-CoV-2 N protein detection; and determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-4740-5.

Fluorescence immunoassay based on alkaline phosphatase-induced in situ generation of fluorescent non-conjugated polymer dots

Alkaline phosphatase (ALP) activity assay is not only significant to the clinical diagnosis of some related disease, but also momentous to the construction of ALP-based enzyme-linked immunosorbent assay (ELISA). Herein, for the first time, we have discovered that ascorbic acid (AA) can specially react with N-methylethylenediamine (N-MEDA) to generate fluorescent non-conjugated polymer dots (NCPDs) under mild conditions. On the basis of the AA-responsive emission and ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate (AA2P) to AA, we have exploited a fluorometric ALP activity assay with high sensitivity and selectivity. Furthermore, by means of conventional ALP-based ELISA platform, a conceptual fluorescent ELISA has been constructed and applied in the potential clinical diagnosis, during which cardiac troponin I (cTnI), a well-established biomarker of acute myocardial infarction, has been chosen as the model target. We envision that such original fluorescent NCPDs generation-enabled ELISA could become a versatile tool in biochemical sensing and medical diagnosis in the future.

Characteristics and changes of sensitization patterns of major allergens in children from 2010 to 2020 in a hospital of pediatric in Beijing

Objective: To analyze the sensitization characteristics and change of major allergens in children from 2010 to 2020 in Beijing, so as to provide basis for diagnosis, treatment and prevention of allergic diseases in children. Methods: A retrospective study was conducted to analyze the children who were suspected of allergic diseases and tested for serum immunoglobulin E (total IgE and specific IgE) in the children's Hospital Affiliated to the Capital Institute of Pediatrics from January 2010 to December 2020. A total of 45 746 children aged 0-17 years in Beijing were enrolled with a median (interquartile range) of 3.8 (2.5, 5.3) years. There were 28 918 boys (63.2%) and 16 828 girls (36.8%) and 14 984 cases (32.8%) in the infant group (<3 years old), 22 049 cases (48.2%) in the preschool group (3-6 years old), and 8 713 cases (19.0%) in the school-age group (6 years old and above). Immunocap allergen detection system (fluorescence enzyme-linked immunosorbent assay) was used to detect and the characteristics and change trend of allergens during 11 years were statistically analyzed by Chi square test. Results: The top three positive rates of single food allergens sIgE were egg white 42.6% (2 788/6 577 tests), milk 38.4% (2 606/6 782 tests) and wheat 31.8% (1 417/4 449 tests), and the sequence of single inhaled allergens were Ragweed 42.6% (440/1 034 tests), Artemisia 38.4% (1 045/3 191 tests) and Alternaria alternata 31.3% (3 358/10 725 tests). The positive detection rates of egg white 47.0%(1 904/4 048 tests), milk 41.9%(1 769/4 226 tests) and wheat 33.9%(973/2 870 tests) in the infant group were the highest, and there have statistically significant(χ2=91.495, 73.907, 16.966, P<0.05). The positive rates of fx1 (22.5%, 121/537 tests), peanut (24.9%, 111/446 tests), soybean (20.0%, 74/370 tests) and shrimp (7.48%, 44/588 tests) were the highest in the school-age group(χ²=14.436, 10.751, 11.569, 13.703, P<0.05). The most common inhaled allergens were Alternaria alternata (14.8%, 422/2 859 tests) in the infant group, Ambrosia (34.5%, 143/415 tests) and Alternaria alternata (33.5%, 1 762/5 254 tests) in the preschool group, while in the school-age group were Ambrosia (56.4%, 282/500 tests) and Artemisia (48.2%, 573/1 189 tests). The positive rates of egg white, milk, wheat, sesame seed and peanut varied from 2010 to 2020, showing a trend of falling first and then rising(χ²=10.293,χ²=12.066,χ²=7.402,χ²=32.458,χ2=31.747,P<0.05).The positive detection rates of soybean showed a significant downward trend (21.4%, 173/809 tests in 2010, 15.4%, 70/455 tests in 2020, χ²=6.751,P=0.009), while that of shrimp and crab were at a stable low level(χ²=0.263, 1.346;P>0.05). From 2010 to 2020, mold (26.7%, 1 066/3 998 tests in 2010, 40.2% 1 705/4 243 tests in 2020), grass pollen (19.8%, 259/1 308 tests in 2010, 39.3%, 1 472/3 746 tests in 2020), tree pollen(17%, 180/1 058 tests in 2010, 29.8%, 916/3 075 tests in 2020) and animal dander (18.5%, 111/601 tests in 2010, 26.6%, 672/2 522 tests in 2020) were all showed significant upward trend (χ²=168.600, 163.601, 65.931 and 17.271 respectively, P<0.001) but dust mites (30.7%, 1 270/4 132 tests in 2010, 26.7%, 1 126/4 221 tests in 2020) showed a significant downward trend(χ²=16.822,P<0.001).In 2010, the most common inhaled allergen was dust mite 30.7% (1 270/4 132 tests), followed by mold 26.7% (1 066/3 998 tests), while they were mold 40.2% (1 705/1 243 tests), and grass pollen 39.3% (1 472/3 746 texts) respectively in 2020. Conclusion: In the past 11 years, the main food allergens in children were still egg white and milk, wheat, but the mold and pollen gradually replaced dust mite as the most common inhaled allergen.

Relationship and effect of miR-1-3p expression and BDNF level in patients with primary hypertension complicated with depression.

This experiment was designed to explore the relationship and effect of miR-1-3p expression and BDNF level in patients with primary hypertension complicated with depression. The subjects of the study were 145 patients with hypertension with a small fluctuation range of blood pressure in recent three months. Within 48 hours after admission, patients were evaluated with the Hospital Anxiety and Depression Scale (HADS) and Hamilton Depression Rating Scale (HAMD). After fasting for 12 hours, enrolled subjects were subject to blood collection (5 ml) in the morning for detecting blood lipid levels, miR-1-3p expression and BDNF by using an automatic biochemical analyzer, real-time fluorescence quantitative PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Results showed that compared with the normal control group, while miR-1-3p expression increased obviously in patients with hypertension, while the level of BDNF decreased significantly; and compared with patients with simple hypertension, the expression of miR-1-3p in hypertension patients with depression was significantly increased, while BDNF level was decreased evidently (All P < 0.05). miR-1-3p expression in patients with hypertension complicated with depression was negatively correlated with serum BDNF level (r=-0.302, P < 0.05). In relative to the normal control population, the area under the curve (AUC) of ROC produced by serum miR-1-3p and BDNF in patients with primary hypertension complicated with depression was 0.971 (95% CI = 0.945-0.998, P < 0.0001) and 0.875 (95% CI = 0.808-0.942, P < 0.0001); and in relative to primary hypertension patients without depression, the AUC of ROC produced by serum miR-1-3p and BDNF in patients with primary hypertension with depression was 0.957 (95% CI = 0.925-0.989, P < 0.0001) and 0.883 (95% CI = 0.821-0.944, P < 0.0001), respectively. HADS-D score, HAMD score, course of the disease, miR-1-3p expression and BDNF level showed statistical differences in primary hypertension patients with and without depression (All P < 0.05). It was concluded that there are high miR-1-3p expression and low serum BDNF levels in patients with primary hypertension complicated with depression. miR-1-3p has a negative correlation with BDNF, and it may play a role by negatively regulating the expression of BDNF. Detecting miR-1-3p and BDNF in patients with primary hypertension can indicate the occurrence of depression to some extent.

Bovine brucellosis serological survey in small dairy herds in Lushnja district, Albania

Bovine brucellosis is an important zoonotic disease in Albania. Both B. abortus and B. melitensis have been isolated from ruminants. National control and eradication programs for brucellosis are applied on sheep and goat farms and larger dairy cattle farms. However, the current control programs for brucellosis do not cover small dairy cattle farms, and there is no valid data on the prevalence of the disease in this category; this prevents formulating evidence-based and effective strategies for control of the disease in this population subset. Therefore, the current study aimed to assess the herd and within-herd prevalence of bovine brucellosis in small bovine herds and provide scientific evidence for establishing an evidence-based approach to control the disease in this subset of the population not previously included in the national eradication program. To achieve this objective, a statistical survey was designed and implemented in small dairy herds in the Lushnja district, where samples from statistically selected herds were serologically tested in parallel with Rose Bengal Test (RBT), Fluorescence Polarization Assay (FPA) and Enzyme-Linked Immunosorbent Assay (ELISA). In total, 120 dairy herds were randomly selected from a list of 1,955 registered herds: from these selected herds, 368 blood samples were collected from all animals older than 12 months, and their sera were tested using RBT, FPA, and c-ELISA. The test results revealed no positive or suspect cases. Based on these results, we are confident (P0.05) that Brucella spp is not circulating in this subpopulation of cattle in the Lushja district. This deduction is supported by analyses of the main risk factors, other epidemiological data, and the perceptions of official and private veterinarians. This is the first structured survey of bovine brucellosis in small dairy herds in Albania. In conclusion, our study results and our findings show that the epidemiological status of bovine brucellosis in the Lushnja district is encouraging. Therefore, a test and slaughter control program appears appropriate in smaller herds. Furthermore, the approach used in this pilot study could be extended to establish the prevalence of brucellosis in other districts, the result of which would establish the basis for rational control measures in the smaller herds of cattle.

Risk factors associated with Toxoplasma gondii seroprevalence in domestic pig farms in Argentina.

Toxoplasmosis is a worldwide parasitic zoonosis caused by Toxoplasma gondii. Pigs can become infected by consuming water or food contaminated with sporulated oocysts, or by carnivorism (like the consumption of infected rodents). In pigs most infections are asymptomatic. In certain countries, pig meat containing tissue cysts is a major source of infection for human beings. The aims of this study were to estimate the seroprevalence of toxoplasmosis and to identify which factors were related with the increase of the risk of infection in Argentina. The seroprevalence of T. gondii was determined in 240 pigs from 27 farms in the central-western area of Buenos Aires province, Argentina. Serum samples were analyzed using indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) techniques. Prevalence determined was 53.33% and 32.08% by IFAT and ELISA, respectively. Results showed that 81.5% (22/27) of the farms were seropositive to T. gondii. Seropositivity for T. gondii was related with the following risk factors (p value ≤0.05): presence of felids and rodents in the farms, feeding with waste of human food and storage of food outdoors with free access to felids and to the reservoirs when applying both serological techniques. Our results strongly suggest that the risk of infection with T. gondii in pigs is related to the outdoor/extensive type of production system with low infrastructure conditions, which allows both felids and rodents to have free access to pigs and stored food. Also, the high seroprevalence detected in the present study could indicate a potential role of pork in human infections in the region.

Dual-wavelength ratiometric immunosensor for Bacillus cereus: Oxidase-like MnO2-Au trigged “OFF-ON” detection strategy

As an opportunistic pathogen, Bacillus cereus (B. cereus) has caused increasing emetic and diarrheal food poisoning, even fulminant hepatic failure and death case. Herein, a mimetic oxidase-mediated ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed to detect B. cereus. In this system, 3D MnO2-Au nanoflowers with layered crystalline structure can oxidize o-phenylenediamine (OPD) to generate a fluorescence peak at 570 nm (I570, response signal), while blue carbon quantum dots (bCDs) display a fluorescence peak at 455 nm (I455, reference signal). Under the effect of fluorescence resonance energy transfer (FRET), the response signal increased while the reference signal was weakened. By using MnO2-Au as the mimetic oxidase to replace the horseradish peroxidase and associate with antibody in traditional ELISA, a linear relationship was established between the ratiometric fluorescence signal (I570/I455) and the logarithm of B. cereus concentrations in the range from 1.7 × 102 to 3.3 × 106 CFU/mL. Comparing with conventional sandwich ELISA, the detection time was shortened by 43 min and the sensitivity was improved by 5-folds, indicating that the proposed ratiometric immunoassay holds great potential as a rapid and convenient tool for sensitive screening of B. cereus.

Inhibitory effect of concomitantly administered rabies immunoglobulins on the immunogenicity of commercial and candidate human rabies vaccines in hamsters

The World Health Organization protocol for rabies post-exposure prophylaxis (PEP) recommends extensive wound washing, immediate vaccination, and administration of rabies immunoglobulin (RIG) in severe category III exposures. Some studies have shown that RIG can interfere with rabies vaccine immunogenicity to some extent. We investigated the interference of RIG on a next generation highly purified Vero cell rabies vaccine candidate (PVRV-NG) versus standard-of-care vaccines in a previously described hamster model. The interference of either human (h) or equine (e) RIG on the immune response elicited by PVRV-NG, Verorab® (purified Vero cell rabies vaccine, PVRV), and Imovax® Rabies (human diploid cell rabies vaccine; HDCV) was evaluated using the 4-dose Essen PEP regimen. The anti-rabies seroneutralizing titers and specific serum IgM titers were measured by fluorescent antibody virus neutralization test and enzyme-linked immunosorbent assay, respectively, for the vaccines administered with or without RIG. The RIG interference on PVRV-NG, observed transiently at Day 7, was similar to that on PVRV and tended to be lower than that on HDCV using both read-outs. In summary, the results generated in the hamster model showed that RIG induced similar or less interference on PVRV-NG than the standard-of-care vaccines.

Filter Papers to Collect Blood Samples from Dogs: An Easier Way to Monitor the Mass Vaccination Campaigns against Rabies?

Rabies is a deadly viral disease present mainly in low-income countries of Africa and Asia. Dogs are the main reservoir and the source of human deaths. Mass vaccination campaigns of dogs are pivotal to achieve rabies elimination. The monitoring of the immune response of the dog population is necessary to evaluate the effectiveness of these campaigns, taking into account field conditions. This study explores the feasibility and the performance of a new tool using filter papers (FPs) to collect blood samples associated with an Enzyme-Linked ImmunoSorbent Assay (ELISA) titration of rabies antibodies in dogs. A total of 216 eluates from FP samples were collected from 111 dogs kept in experimental facilities in France and 29 dogs from the field in Tunisia. Sera were also analyzed using both the Fluorescence Antibody Virus Neutralization test (FAVNt) and ELISA. A high specificity (98.0%) was obtained by testing FP blood eluates from 51 unvaccinated dogs, with the results compared with those of FAVNt and ELISA on serum samples. The coefficients of concordance between FP eluates and tested sera were 88.9% for FAVNt and 88.0% for ELISA. Blood filter papers coupled with the titration of rabies antibodies by ELISA provide a reliable, simple, and effective solution to overcome the issues of the logistics and transport of samples, especially in low-income countries.

In vitro behaviour of human gingival fibroblasts cultured on 3D-printed titanium alloy with hydrogenated TiO2 nanotubes

Selective laser melting (SLM), as one of the most common 3D-printed technologies, can form personalized implants, which after further surface modification can obtain excellent osseointegration. To study the surface properties of SLM titanium alloy (Ti6Al4V) with hydrogenated titanium dioxide (TiO2)nanotubes (TNTs) and its influence on the biological behaviour of human gingival fibroblasts (HGFs), we used SLM to prepare 3D-printed titanium alloy samples (3D-Ti), which were electrochemically anodizing to fabricate 3D-TNTs and then further hydrogenated at high temperature to obtain 3D-H2-TNTs. Polished cast titanium alloy (MP-Ti) was used as the control group. The surface morphology, hydrophilicity and roughness of MP-Ti, 3D-Ti, 3D-TNTs and 3D-H2-TNTs were measured and analysed by scanning electron microscopy (SEM), contact angle metre, surface roughness measuring instrument and atomic force microscope, respectively. HGFs were cultured on the four groups of samples, and the cell morphology was observed by SEM. Fluorescence staining (DAPI) was used to observe the number of adhered cell nuclei, while a cell counting kit (CCK-8) was used to detect the early adhesion and proliferation of HGFs. Fluorescence quantitative real time polymerase chain reaction (RT–qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of adhesion-related genes and fibronectin (FN), respectively. The results of this in vitro comparison study indicated that electrochemical anodic oxidation and high-temperature hydrogenation can form a superhydrophilic micro-nano composite morphology on the surface of SLM titanium alloy, which can promote both the early adhesion and proliferation of human gingival fibroblasts and improve the expression of cell adhesion-related genes and fibronectin.Graphical abstract

Ratiometric fluorescence enzyme-linked immunosorbent assay based on carbon dots@SiO2@CdTe quantum dots with dual functionalities for alpha-fetoprotein

A ratiometric fluorescence enzyme-linked immunosorbent assay was constructed for the screening of alpha-fetoprotein using carbon dots@SiO2@CdTe quantum dots.

Astragaloside IV Reduces OxLDL-Induced BNP Overexpression by Regulating HDAC.

Effective drug intervention is the most important method to improve the prognosis, improve the quality of life, and prolong the life of patients with heart failure. This study aimed to explore the protective effect of astragaloside IV on myocardial cell injury induced by oxidized low-density lipoprotein (OxLDL) and its regulatory mechanism on the increase of brain natriuretic peptide (BNP) caused by myocardial cell injury. The model of myocardial cell injury, protection, and histone deacetylase (HDAC) inhibition in HL-1 mice was established by OxLDL treatment, astragaloside IV intervention, and UF010 coincubation. The effects of OxLDL and astragaloside IV on apoptosis were detected by flow cytometry. The expression level of BNP mRNA and protein in cells was investigated by real-time fluorescence quantification, western blot, and enzyme-linked immunosorbent assay. HDAC activity in nucleus was calibrated by fluorescence absorption intensity. Enzyme-linked immunosorbent assay (ELISA) was applied to test eNOS level in myocardial cells. OxLDL significantly promoted apoptosis, upregulated BNP mRNA, increased BNP protein level inside and outside cells, and decreased eNOS level. Compared with OxLDL treatment group, apoptosis decreased, BNP mRNA expression level decreased, BNP protein concentration decreased, and eNOS level increased significantly combined with low and high concentration astragaloside IV treatment group. HDAC activity significantly increased in OxLDL treatment group and significantly decreased after combined incubation with low and high concentrations of astragaloside IV. Inhibition of HDAC significantly increased eNOS level and decreased BNP protein level. In conclusion, astragaloside IV can reverse the low level of eNOS caused by OxLDL by regulating HDAC activity to protect myocardial cells from oxide damage, which is manifested by the decrease of BNP concentration.

Comparative Study of Indirect Fluorescent Antibody, ELISA, and Immunochromatography Tests for Serological Diagnosis of Bovine Babesiosis Caused by Babesia bovis.

Simple SummaryCurrently serological diagnosis of bovine babesiosis is based on the detection of Babesia-specific antibodies (immunoglobulin-G). Antibody detection is commonly used in seroepidemiological studies or in the assessment of antibabesial antibody titers after cattle vaccination. The indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) are the most widely used diagnostic tests, although there their implementation has some drawbacks, principally due to the requirements for trained personnel, specific materials, and special laboratory equipment. This study compared a newly designed rapid immunochromatography test (ICT), which has been reported recently and used for Babesia bovis-specific antibody detection with promising results, with an in-house ELISA for the serological diagnosis of cattle exposed to B. bovis (Babesia bovis) in Mexico. Higher sensitivity and specificity values were found by ICT, proving its effectiveness over ELISA. ICT also had better concordance than ELISA when IFAT was used as the “gold standard”. The rapid ICT was shown to have diagnostic utility for the detection of antibodies against B. bovis and could be used as a field test in Mexico due to its practicality, as it does not need laboratory equipment for implementation and interpretation of results.The indirect fluorescent antibody test (IFAT) is the most frequently used test to conduct seroepidemiological studies so far, and it is regarded as the "gold standard" test for the serological diagnosis of bovine babesiosis. The aim of the present study was to compare the enzyme-linked immunosorbent assay (ELISA) and the rapid immunochromatography test (ICT) for use in the serological diagnosis of cattle exposed to B. bovis in Mexico. The evaluation of test performance was carried out with 30 positive and 30 negative reference sera. A total of 72 bovine sera samples collected from cattle in a region with endemic bovine babesiosis were analyzed by ELISA and ICT, and the results were compared with those of IFAT. Kappa value (k) was also calculated to determine the agreement between tests. The sensitivity and specificity of ELISA for detecting antibodies against B. bovis were 87% (26/30) and 80% (24/30), respectively. The sensitivity and specificity of ICT for detecting antibodies against B. bovis were 90% (27/30) and 83.3% (25/30), respectively. The overall concordance determined for ELISA and ICT was 94.4% (68/72) and 98.6% (71/72), respectively, when the results were compared with those of IFAT. ICT was more sensitive and specific in this comparative study, showing good strength of agreement (k = 0.79) with respect to IFAT. ICT combines a strip-based assay system that is fast, practical, and sensitive for detection of antibodies to B. bovis, which suggests that it could be applied in the field without requiring any laboratory equipment for its use and interpretation of test results.

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.