Enzyme-linked Immunosorbent Assay Assay Research Articles

Establishment of a mouse allergy model for Culicoides (Diptera: Ceratopogonidae).

Culicoides is a genus of ubiquitous biting midges (Ceratopogonidae). Female midges have blood-sucking habit. They not only bite and harass humans and animals but also may be an important vector of disease transmission. Therefore, building an animal allergy model caused by Culicoides biting is very beneficial for studying its pathogenesis and exploring the therapeutic methods. Kunming mice were used in this study to build the model and sensitised by two-step injection of midge extracts. Scratching behaviour and histological examination were used to check the immediate and delayed responses. Immunoglobulin E (IgE) and Immunoglobulin G (IgG) were detected using indirect enzyme-linked immunosorbent assay (ELISA) assay. Splenic cell proliferation and cytokine production were determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and ELISA assays. The response of cytokine gene expression to midge stimulation was analysed through quantitative real-time polymerase chain reaction (qPCR). Behavioural results revealed a significant increase in scratching frequency among the midge-sensitised animals (p < 0.05). Histological examination showed more inflammatory cytokine infiltration at the injection site of midge-sensitised mice comparing to the ones in the control group. The serum levels of IgE and IgG1 antibodies in the midge-sensitised group were significantly elevated (p < 0.05). After splenocytes were stimulated in vitro with midge extracts, the midge-sensitised group's splenocyte count significantly increased in comparison to the control group. The midge-sensitised group's qPCR data revealed a down-regulation of tumor necrosis factor alpha (TNF-α) expression and an increase in the expression of interleukin (IL)-4, IL-5, IL-10 and IL-13 but not in the control group (p < 0.05). In this study, an animal model of Culicoides-mouse sensitisation was successfully constructed using a two-step method. The mode of administration of the model was in good agreement with the natural immune pathway, and the immune response induced by the sensitisation of the model was similar to that produced by the bite of a midge.

ELISA assay for the quantification of ipilimumab in human serum, plasma, milk, and cerebrospinal fluid

Ipilimumab is an immune checkpoint inhibitor of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Ipilimumab has become part of the standard of care for different types of cancer. The efficacy of these treatments is limited due to immune-related toxicity and high economic costs. Dose rationalization studies based on pharmacokinetic data may help to address these limitations. For this purpose, more sensitive analytical methods are needed. We report the development and validation of the first enzyme-linked immunosorbent assay (ELISA) for sensitive determination of ipilimumab concentrations in human serum, plasma, cerebrospinal fluid (CSF), and milk. Our assay is based on the specific capture of ipilimumab by immobilized CTLA-4. The lower limit of quantifications of ipilimumab in serum, plasma, and milk are 50 ng/mL and 10 ng/mL in CSF. The ELISA method showed long-term storage stability for at least one year at −80°C and was successfully cross-validated with ultraperformance liquid chromatography coupled with tandem mass spectrometry. The ELISA method is reliable, relatively inexpensive, and can be used in serum, plasma, CSF, and milk from patients treated with ipilimumab, as evidenced by the analysis of real clinical samples.

Prevalence of Powassan Virus Seropositivity Among People with History of Lyme Disease and Non-Lyme Community Controls in the Northeastern United States.

Introduction: Lyme disease (LD) affects ∼476,000 people each year in the United States. Symptoms are variable and include rash and flu-like symptoms. Reasons for the wide variation in disease outcomes are unknown. Powassan virus (POWV) is a tick-borne flavivirus that causes disease ranging from asymptomatic infection to encephalitis, neurologic damage, and death. POWV and LD geographic case distributions overlap, with Ixodes species ticks as the common vectors. Clinical ramifications of coinfection or sequential infection are unknown. Objectives: This study's primary objective was to determine the prevalence of POWV-reactive antibodies in sera samples collected from previously studied cohorts of individuals with self-reported LD history residing in the Northeastern United States. As a secondary objective, we studied clinical differences between people with self-reported LD history and low versus high POWV antibody levels. Methods: We used an enzyme-linked immunosorbent assay (ELISA) to quantify IgG directed at the POWV envelope (E) protein domain III in 538 samples from individuals with self-reported LD history and 16 community controls. The samples were also tested with an ELISA assay to quantify IgG directed at the POWV NS1 protein. Results: The percentage of individuals with LD history and possible evidence of POWV exposure varied depending on the assay utilized. We found no significant difference in clinical symptoms between those with low or high POWV IgG levels in the in-house assay. Congruence of the EDIII and NS1 assays was low with only 12% of those positive in the in-house EDIII ELISA testing positive in the POWV NS1 ELISA. Conclusions: The results highlight the difficulty in flavivirus diagnostic testing, particularly in the retrospective detection of flavivirus exposure. The findings suggest that a prospective study with symptomatic patients using approved clinical testing is necessary to address the incidence and clinical implications of LD and POWV co-infection or sequential infection.

Comparison of an Enzyme Linked-Immunosorbent Assay and a Chemiluminescent Immunoassay with an Immunofluorescence Assay for Detection of Phase II IgM and IgG Antibodies to Coxiella burnetii.

In this study, we have compared the detection of IgM and IgG against C. burnetii phase II of an enzyme-linked immunosorbent assay (ELISA) (Euroimmun) and a chemiluminescent immunoassay (CLIA) (VIRCLIA, Vircell). In addition, an indirect immunofluorescence assay (IFA) was used as a reference test. One hundred forty-eight sera were used for IgG evaluation, and eighty-eight for IgM. The sensitivity of ELISA and CLIA in detecting phase II IgM was excellent. On the other hand, the CLIA IgM showed better specificity than the ELISA IgM. As for phase II IgG, the specificity of ELISA and CLIA was similar, while the ELISA technique showed a higher sensitivity. In conclusion, the best system to detect phase II IgM antibodies against C. burnetii is the CLIA from Vircell, which is characterized by high sensitivity and specificity. For the detection of phase II IgG, the Euroimmun ELISA and Vircell CLIA assays are suitable for the determination of this marker in the laboratory, although the IgG ELISA has greater sensitivity.

Diagnosis of thrombotic thrombocytopenic purpura: easy-to-use fiber optic surface plasmon resonance immunoassays for automated ADAMTS-13 antigen and conformation evaluation

BackgroundLaboratory diagnosis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) remains challenging when ADAMTS-13 activity ranges between 10% and 20%. To prevent misdiagnosis, open ADAMTS-13 conformation gained clinical attention as a novel biomarker, especially to diagnose acute iTTP in patients with diagnostic undecisive ADAMTS-13 activity. Plasma ADAMTS-13 conformation analysis corrects for ADAMTS-13 antigen, with both parameters being characterized in enzyme-linked immunosorbent assay (ELISA)-based reference assays requiring expert technicians. ObjectivesTo design ADAMTS-13 antigen and conformation assays on automated, easy-to-use fiber optic surface plasmon resonance (FO-SPR) technology to promote assay accessibility and diagnose challenging iTTP patients. MethodsADAMTS-13 antigen and conformation assays were designed on FO-SPR technology. Plasma of 20 healthy donors and 20 acute iTTP patients were quantified, and data from FO-SPR and ELISA reference assays were compared. ResultsFollowing assay design, both antigen and conformation FO-SPR assays were optimized and characterized, presenting strong analytical sensitivity (detection limit of 0.001 μg/mL) and repeatability (interassay variation of 14.4%). Comparative analysis suggested positive correlation (Spearman r of 0.92) and good agreement between FO-SPR and ELISA assays. As expected, FO-SPR assays showed a closed or open ADAMTS-13 conformation in healthy donors and acute iTTP patients, respectively. ConclusionBoth ADAMTS-13 antigen and conformation assays were transferred onto automated, easy-to-use FO-SPR technology, displaying potent analytical sensitivity and reproducibility. ADAMTS-13 antigen and conformation were determined for healthy donors and acute iTTP patients showing strong correlation with ELISA reference. Introducing FO-SPR technology in clinical context could support routine diagnosis of acute iTTP patients, notably when ADAMTS-13 activity fluctuates between 10% and 20%.

Electrochemical Biosensors for the Detection of Viruses: Must-Have Products or Just Science for Publication?

Electrochemical Biosensors for the Detection of Viruses: Must-Have Products or Just Science for Publication?

Seroprevalence of Infection by Borrelia Species Responsible for Lyme Disease in the French Alps: Analysis of 27,360 Serology Tests, 2015-2020.

Objectives: Lyme borreliosis incidence is increasing in several areas; moreover, it has recently gained the public's attention. Apart from erythema migrans, Lyme disease diagnosis relies (among others) on serology test; however, the prevalence of positive enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay has been poorly studied in the general population. We aimed to approach the seroprevalence of infection by Borrelia species responsible for Lyme disease in the French Isere department using city laboratories data. Patients and Methods: We retrieved all serological tests for Borrelia species responsible for Lyme disease performed in the two main networks of city laboratories between 2015 and 2020. All patients with both ELISA and WB IgG were considered seropositive. Results: We analyzed 27,360 tests (ELISA/ELISA+WB). Mean age was 50.9 ± 20.3 years (ranges: 0-101), with 57.1% females. Overall, 11.7% had IgG detected by ELISA, and 4.7% had IgG detected by both ELISA and WB assay. Seropositive status was more frequent in males (7.0% vs. 2.9%, p < 0.001). Seropositivity rate increased with age after a first peak in childhood; men aged 61-70 years had the highest seropositivity rate (10.3%). In addition, seropositivity rate was higher in persons from a rural area. In multivariate analysis, older age, male gender and living in a rural area were independently associated with seropositivity. Seropositivity rate was stable on the 2017-2020 period. Conclusion: The seroprevalence of infection by Borrelia species responsible for Lyme disease is high in Isere; this probably reduces the predictive positive value for Lyme disease of ELISA and WB IgG, suggesting that this serological test should not be performed for nonspecific symptoms.

Three Artemisia pollens trigger the onset of allergic rhinitis via TLR4/MyD88 signaling pathway.

The prevalence of allergic rhinitis is high, making it a relatively common chronic condition. Countless patients suffer from seasonal Allergic rhinitis (AR). The objective of this investigation is to examine the potential involvement of common pollen allergens in seasonal allergic rhinitis, and study the proposed mechanism of Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response gene 88 (MyD88) signaling pathway in the induction of AR. A mouse AR model (sensitized group) was constructed with pollen extracts and ovalbumin(OVA) of Artemisia annua (An), Artemisia argyi (Ar) and Artemisia Sieversiana (Si), and thereafter, AR symptom score was performed. After successful modeling, mouse serum and nasal mucosa tissues were extracted for subsequent experiments. The expression levels of immunoglobulin E (IgE), Interleukin (IL)-4, IL-5, IL-13 and Tumor Necrosis Factor-α (TNF-α) in serum were detected using Enzyme-linked immunosorbent assay (ELISA); Hematoxylin-eosin (H&E) staining methods were used to observe the pathological changes of the nasal mucosal tissue; Utilizing immunohistochemistry (IHC) staining, the expression levels of TLR4, MyD88 and Nuclear factor kappa B (NF-κB) p65 in mouse nasal mucosa were quantified; The mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of sensitized mice were detected with Quantitative reverse transcription PCR (qRT-PCR) and Western Blot. Finally, the in vitro culture of Human nasal mucosal epithelial cells (HNEpC) cells was conducted, and cells were treated with 200µg/ml Artemisia annua pollen extractand OVA for 24h. Western Blot assay was used to detect the expression level of TLR4, MyD88 and NF-κB p65 proteins before and after HNEpC cells were treated with MyD88 inhibitor ST-2825. On the second day after AR stimulation, the mice showed obvious AR symptoms. H&E results showed that compared to the control group, the nasal mucosal tissue in the sensitized group was significantly more inflamed. Furthermore, ELISA assay showed increased expression levels of IgE, IL-4, IL-5, IL-13 and TNF-α in serum of mice induced by OVA and Artemisia annua pollen, Artemisia argyi pollen and Artemisia Sieversiana pollen than those of the control group. However, the expression level of IL-2 was lower than that of the control group (P < 0.05). Using Immunohistochemistry staining visually observed the expression levels of TLR4, MyD88 and NF-κB p65 in mouse nasal mucosa tissues and quantitatively analyzed. The expression levels of TLR4, MyD88 and NF-κB p65 in the sensitized group were higher than those in the control group, and the differences were statistically significant (P < 0.05). The results from qRT-PCR and Western Blot showed that the mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of the sensitized group were significantly higher than those in the control group (P < 0.05). Finally, HNEpC cells were cultured in vitro and analyzed using Western Blot. The expression levels of TLR4, MyD88 and NF-κB p65 in OVA and An groups were significantly increased (P < 0.05). After ST-2825 treatment, TLR4 protein expression was significantly increased (P < 0.05) and MyD88 and NF-κB p65 protein expression were significantly decreased (P < 0.05). To sum up, the occurrence and development of AR induced by OVA and pollen of Artemisia annua, Artemisia argyi and Artemisia Sieversiana were related to TLR4/MyD88 signal pathway.

Suppressed distribution of protein A on the surface of Staphylococcus aureus as a morphological characteristic of erythromycin-resistant strain.

To identify a new morphological phenotype of erythromycin (EM)-resistant Staphylococcus aureus (S. aureus) were isolated in vitro from EM-sensitive parent strain, and the distribution of staphylococcus specific protein A (SpA) on the surface of these strains was examined morphologically by using applied immunoelectron microscopy. The isolated EM-resistant strains had thickened cell walls, and the distribution of SpA on the surfaces of these strains was demonstrated to be lower than that of the parent strain. The SpA suppression was confirmed by enzyme-linked immunosorbent assay (ELISA) using fixed EM-resistant cells. Moreover, the spa gene of EM-resistant cells was detected by polymerase chain reaction (PCR) and confirmed by quantitative real-time PCR assay, showing that the expression of SpA was repressed at the transcriptional level in these strains. Furthermore, ELISA assay showed that whole EM-resistant cell SpA content was significantly decreased. Therefore, it was considered that the suppression of surface SpA on the EM-resistant strain was due to regulated SpA production, and not dependent on the conformational change in SpA molecule expression through cell wall thickening. These results strongly suggest that suppressed SpA distribution on the EM-resistant S. aureus is a phenotypical characteristic in these strains.

Atractylenolide I Suppresses A1 Astrocyte Activation to Improve Depression in Mice.

This work aimed to investigate the role of atractylenolide I (ATR) in resisting depression and its mechanism of action. The mouse model of depression was constructed through chronic unpredictable mild stress (CUMS) method. After ATR intervention, changes in the depression-related behaviors of mice were detected through open field test and elevated plus maze. In addition, enzyme-linked immunosorbent assay (ELISA) was conducted to detect inflammatory factor levels. Real-time fluorescence quantitative PCR (RT-qPCR) was performed to measure the mRNA levels of A1/A2 astrocyte markers. Furthermore, primary astrocytes were induced in vitro, and the A1 differentiation level was detected by ELISA and RT-qPCR assays. ATR improved the behaviors of CUMS mice and alleviated the depression symptoms. Moreover, it reduced tissue inflammation, inhibited the A1 differentiation of astrocytes, and decreased the mRNA levels of A1 markers. After NLRP3 knockout, the effects of ATR were suppressed. Similarly, in vitro experimental results also revealed that ATR suppressed the A1 differentiation of astrocytes. Based on molecular dynamics and small molecule-protein docking results, ATR mainly targeted NLRP3 and suppressed the NLRP3-mediated A1 differentiation. We discover that ATR can target NLRP3 to suppress A1 differentiation of astrocytes, restrain tissue inflammation, and improve the depression symptoms in mice.

(069) Markers of Neovascularization and Neurogenesis May Change After Low-Intensity Extracorporeal Shockwave Therapy for Erectile Dysfunction

Abstract Introduction Low-intensity extracorporeal shockwave therapy (LiESWT) is thought to treat erectile dysfunction (ED) but the mechanism remains elusive. It is believed that LiESWT may work by stimulating neovascularization and nerve regeneration as demonstrated in animal models by histologically increased angiogenesis-related growth factors and neuronal factors including vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS) and brain derived-neurotropic factor (BDNF). Given its prolific use, a thorough understanding of the mechanism of action of this therapy at a cellular level is critical prior to recommendation of this therapy on a larger scale. Objective Objective data post LiESWT is sparse. In this study, we aimed to determine whether markers for neovascularization and nerve regeneration can be detected in the serum of penile tissue in men following LiESWT treatment. We hypothesized that LiESWT results in appreciable increases in vascular and neuronal factors post therapy. Methods Patients were prospectively enrolled in a clinical trial of LiESWT for ED. Patients received 12 bi-weekly LiESWT treatments of 0.2mJ/mm2 at 5Hz, 1500 shocks delivered per treatment, with follow up at 1-2 weeks, 4-6 weeks, 3 months and 6 months post-treatment. Cavernosal penile blood samples were obtained prior to treatment and at each visit post-treatment. The concentrations of eNOS, nNOS, VEGF, and BDNF in penile plasma samples were measured using Enzyme-Linked Immunosorbent Assay (ELISA) with specific commercial kits, following the protocols provided by the manufacturer. Results Twenty-five patients completed all five study visits. Mean patient age was 63. Mean baseline IIEF-EF score prior to treatment was 14.24 (±1.21). At the time of data analysis, plasma samples from 9 men were analyzed for BDNF, eNOS, and nNOS levels using the ELISA assay, and samples from all 25 men were analyzed for VEGF. BDNF levels demonstrated a sustained decrease after the first treatment (P = 0.0001, Figure 1A). Levels of eNOS were noted to trend upward, but no significant changes in eNOS, nNOS or VEGF levels were noted. Conclusions To our knowledge, this is the first study in human tissues to attempt to quantify objective measures of neurogenesis and neovascularization in penile tissue following LiESWT for ED. Though our N is small, our results suggest that LiESWT may decrease BDNF levels after initial LiESWT treatment while VEGF, eNOS and nNOS levels remain unchanged. The clinical significance of these findings is currently unknown, as our findings are not consistent with histological studies in animals which demonstrate upregulation of these markers in penile tissues. However, we believe this represents a promising first step in attempting to understand the effect of LiESWT at a tissue level in men undergoing LiESWT. Disclosure No.

Clinical trial and performance evaluation of the Wantai HBsAg (CMIA) diagnostic kit for screening blood donors in China

In China, according to the ‘Technical Operating Procedures for Blood Stations (2019 Edition),’ blood stations are authorized to utilize Chemiluminescence Immunoassay (CLIA) to detect pathogen markers linked with transfusion-transmissible infections. However, currently, there is no approved CLIA reagent for the screening of blood-borne diseases in China, specifically for the detection of Hepatitis B surface antigen. The objective of this research project is to conduct a comprehensive evaluation of the performance of the Wantai Chemiluminescent Microparticle Hepatitis B surface antigen reagent. This study evaluates the performance of the Wantai Chemiluminescent Microparticle Immunoassay (CMIA) on the Wan200 + analyzer in screening for Hepatitis B Surface Antigen (HBsAg) in blood samples. The clinical trial component of this evaluation is included as part of the documentation submitted to the National Medical Products Administration (NMPA) of China for the approval of blood screening reagents. The evaluation plan of this study encompasses two main components: clinical trials and performance assessment. We adopted a controlled trial design, utilizing the WanTai Chemiluminescent Microparticle Immunoassay (CMIA) on the Wan200 + analyzer and the Enzyme-Linked Immunosorbent Assay (ELISA) to screen for Hepatitis B Surface Antigen (HBsAg) in routine blood donor samples and reference serum panel samples. To ensure the accuracy of the screening, we additionally employed Abbott's ELISA reagents and HBV DNA for validation. The assessment primarily focused on key performance indicators such as sensitivity, specificity, and analytical sensitivity. Moreover, this clinical trial data has been included as part of the submission to China's National Medical Products Administration (NMPA). In the clinical trials of this study, a total of 10,470 blood donor samples underwent simultaneous testing using both CMIA and ELISA methods. Across two clinical trials, there was remarkable concordance between CMIA and the two ELISA reagents, with Kappa values exceeding 0.82. Among the 269 samples that were double-reactive in the enzyme immunoassay (ELISA) tests, CMIA exhibited a 100% reactivity detection rate. However, CMIA produced 14 and 6 false-positive results in the respective clinical trials, resulting in specificities of 99.73% and 99.89%. In contrast, the specificities for Wantai ELISA and Xin Chuang ELISA were both greater than 99.94%.When testing samples in the gray zone serum plates, CMIA's detection limit significantly exceeded that of the two ELISA assays. CMIA had a detection cutoff of 0.05 IU/mL, while the two ELISA reagents had cutoffs of 0.1 IU/mL and 0.09 IU/mL, respectively. CMIA's detection limits for the adr and adw subtypes were 0.05 IU/mL, and for the ay subtype, it was 0.1 U/mL. The detection limit for 10 HBV mutant samples was 0.5 U/mL. In 165 cases where ELISA tested negative but HBV DNA tested positive, CMIA detected 5 HBsAg-positive samples. This study evaluated the performance of the Wantai CMIA in screening for HBsAg among blood donors. The results demonstrate outstanding performance of CMIA in both clinical trials and performance assessments, detecting all true positive samples with a sensitivity of 100%. It exhibits excellent concordance with the two ELISA assays. Of particular note is its superiority in early detection of HBsAg in the screening of early-stage hepatitis B infections, reducing the window period compared to ELISA. CMIA achieves a specificity exceeding 99.73% for negative blood donors, aligning with the European Union's standards for blood screening assay specificity. In summary, Wantai's CMIA displays high sensitivity and specificity in blood donor screening, making it suitable for screening blood donors in China.

PDK4 inhibits osteoarthritis progression by activating the PPAR pathway

BackgroundOsteoarthritis (OA) is a degenerative joint disease caused by the deterioration of cartilage. However, the underlying mechanisms of OA pathogenesis remain elusive.MethodsHub genes were screened by bioinformatics analysis based on the GSE114007 and GSE169077 datasets. The Sprague–Dawley (SD) rat model of OA was constructed by intra-articular injection of a mixture of papain and L-cysteine. Hematoxylin–eosin (HE) staining was used to detect pathological changes in OA rat models. Inflammatory cytokine levels in serum were measured employing the enzyme-linked immunosorbent assay (ELISA). The reverse transcription quantitative PCR (RT-qPCR) was implemented to assess the hub gene expressions in OA rat models. The roles of PDK4 and the mechanism regulating the PPAR pathway were evaluated through western blot, cell counting kit-8 (CCK-8), ELISA, and flow cytometry assays in C28/I2 chondrocytes induced by IL-1β.ResultsSix hub genes were identified, of which COL1A1, POSTN, FAP, and CDH11 expressions were elevated, while PDK4 and ANGPTL4 were reduced in OA. Overexpression of PDK4 inhibited apoptosis, inflammatory cytokine levels (TNF-α, IL-8, and IL-6), and extracellular matrix (ECM) degradation protein expressions (MMP-3, MMP-13, and ADAMTS-4) in IL-1β-induced chondrocytes. Further investigation revealed that PDK4 promoted the expression of PPAR signaling pathway-related proteins: PPARA, PPARD, and ACSL1. Additionally, GW9662, an inhibitor of the PPAR pathway, significantly counteracted the inhibitory effect of PDK4 overexpression on IL-1β-induced chondrocytes.ConclusionPDK4 inhibits OA development by activating the PPAR pathway, which provides new insights into the OA management.

Dementia exacerbates periodontal bone loss in females.

Periodontitis is a chronic inflammatory disease defined by the pathologic loss of the periodontal ligament and alveolar bone in relation to aging. Although clinical cohort studies reported that periodontitis is significantly elevated in males compared to females, emerging evidence indicates that females with dementia are at a greater risk for periodontitis and decreased alveolar bone. This study aimed to evaluate whether dementia is a potential sex-dependent risk factor for periodontal bone loss using an experimental model of periodontitis induced in the triple transgenic (3x-Tg) dementia-like mice and clinical samples collected from senior 65 plus age patients with diagnosed dementia. We induced periodontitis in dementia-like triple-transgenic (3x-Tg) male and female mice and age-matched wild-type (WT) control mice by ligature placement. Then, alveolar bone loss and osteoclast activity were evaluated using micro-CT and insitu imaging assays. In addition, we performed dental examinations on patients with diagnosed dementia. Finally, dementia-associated Aβ42 and p-Tau (T181) and osteoclastogenic receptor activator of nuclear factor kappa-Β ligand (RANKL) in gingival crevicular fluid (GCF) collected from mice and clinical samples were measured by enzyme-linked immunosorbent assay (ELISA). Alveolar bone loss and insitu osteoclast activity were significantly elevated in periodontal lesions of 3x-Tg females but not males, compared to wild-type control mice. In addition, we also observed that the probing pocket depth (PPD) was also significantly elevated in female patients with dementia. Using ELISA assay, we observed that females had elevated levels of osteoclastogenic RANKL and dementia-associated Aβ42 and p-Tau (T181) in the GCF collected from experimental periodontitis lesions and clinical samples. Altogether, we demonstrate that females with dementia have an increased risk for periodontal bone loss compared to males.

Robust Gold Electrode Fabrication in a Microfluidic System

Electrode fabrication in microfluidic devices is often avoided due to the smaller footprint of the platform, associated complexity in the process, and absence of a well-established method. In this article a glass microchip with chemically modified and vapor deposited gold internal electrode was fabricated and used for biomolecule immobilization and electrochemical applications. The silanol (-OH) on glass surface of the microdevice was modified with (3-mercaptopropyl)trimethoxysilane prior to gold deposition to achieve a strong attachment of the metal film using the gold-thiol binding. The practical utility of the microdevice with gold surface was demonstrated by performing quantitative enzyme linked immunosorbent assay (ELISA) of human interferon gamma (IFN-γ) antigen, yielding a limit of detection (LOD) of 0.15 pg/mL compared to 4 pg/mL in the microplate assay. Additionally, the functionality of the internal electrode was established by performing cyclic voltammetry (CV) and linear sweep voltammetry (LSV) of potassium ferricyanide (III) in potassium chloride (KCl) solution using the proposed gold patterned electrode in the microchip, offering limits of detection of 0.14 and 0.11 mM for CV and LSV respectively. Both the ELISA and electrochemical assays were validated by generating calibration curves and using a control sample for the electroanalytical sensing applications. The measurements yielded experimental results that agreed with control concentration with 7% relative standard deviation (RSD). The promising reproducibility of the electrode in fabrication with no/minimum failure rate and its outstanding performance in immunosensor applications as well as an electron exchange surface makes our technique of gold electrode fabrication in microchips a state of the art.

Integrated double signal amplification systems with ELISA assay for sensitive detection of tylosin in food

ABSTRACT Tylosin (TYL) is a kind of antibiotics which promotes the development of animal husbandry, but it poses potential threats to food safety and human health. Therefore, it is necessary to establish a highly sensitive method for the detection of TYL. Here, an enzyme-linked immunosorbent assay (ELISA) combined with biotin–streptavidin (Bio-SA) system and hybridization chain reaction (HCR) technique (Bio-SA-HCR-ELISA) was developed. In the absence of the TYL, a strong fluorescence was generated through double signal amplification as signal output, while in the presence of TYL, the fluorescence intensity decreased. The results showed that the limit of detection for TYL was 0.39 ng/mL, and this method had a good specificity for 9 common antiboics. Compared to the traditional indirect competitive ELISA, the sensitivity of this method was 6.8 times higher. Additionally, the established double signal amplification Bio-SA-HCR-ELISA was used for detecting TYL in milk and honey, the LOD of this method were 4.9 and 2.5 ng/mL, respectively.

Immunomagnetic Assay Using Photon-Upconversion Nanoparticles for the Detection of Cancer Biomarkers

Prostatic carcinoma is the most frequently diagnosed type of cancer within the male population worldwide. The most important prostate cancer biomarker is the prostate-specific antigen (PSA). Changes in PSA concentration may occur in very narrow ranges, making ultrasensitive analytical methods necessary. Enzyme-linked immunosorbent assay (ELISA) is considered the gold standard of analytical methods. It utilizes microtiter plates as solid phase and enzyme-based labels. However, ELISAs are often not sensitive enough for early-stage detection of subclinical levels of PSA. To enhance the sensitivity, it is possible to introduce suitable alternatives for both the solid phase and the label. Our work focuses on the use of magnetic microparticles (MBs) and photon-upconversion nanoparticles (MBs) as alternatives for solid phase and the label, respectively. The superparamagnetic properties and higher specific surface of MBs allows for analyte preconcentration and simple magnetic separation of the immunocomplex. UCNPs, on the other hand, are nanocrystals exhibiting anti-Stokes luminescence (converting light in the NIR region to visible light). After surface modification, it is possible to utilize UCNPs as labels in the upconversion-linked immunosorbent assay (ULISA), significantly enhancing the sensitivity. We have developed and optimized ELISA and ULISA assays for the detection of PSA and compared their parameters with the equivalent assays utilizing MBs as solid phase. Moreover, we have developed a novel ULISA method with an additional magnetic preconcentration step to further enhance sensitivity, providing a limit of detection of 0.46 pg mL–1.

Performance of a New Rapid Point-of-Care Test for Infliximab Levels in Patients with Inflammatory Bowel Disease: A Comparison to ELISA

BackgroundTherapeutic drug monitoring of infliximab levels in patients with inflammatory bowel disease (IBD) optimizes patients’ treatment. The reference technique is based on enzyme-linked immunosorbent assay (ELISA) although point of care (POC) assays are being developed.AimsTo assess the performance of a new rapid immunochromatographic POC assay (Promonitor Quick IFX) compared with ELISA technique to measure infliximab levels in patients with IBD.MethodsA prospective, observational, unicentric study was performed on capillary blood samples from patients with IBD before infliximab infusion (trough levels). Infliximab levels and anti-infliximab antibodies were measured using the ELISA technique (Promonitor IFX) and the POC assay. Correlation between both techniques was assessed by Pearson’s coefficient. Quantitative differences were evaluated by Bland–Altman analysis. Samples were stratified according to infliximab therapeutic ranges (< 3 μg/mL, 3–8 μg/mL, and > 8 μg/mL).ResultsA total of 135 experimental samples were assessed. Infliximab levels showed a high correlation between POC and ELISA tests (r = 0.84, P < 0.001). The mean difference between tests was 1.46 μg/mL (P < 0.001), being minimal for concentrations < 8 μg/mL. POC and ELISA assays showed an overall concordance of 87.4%. Most samples were in the same therapeutic range, which lead to equivalent therapeutic decisions. POC and ELISA assays detected the presence of anti-infliximab antibodies in 2.2% and 3.7% of the samples, respectively.ConclusionsPOC assay results in blood samples from patients with IBD were comparable to those obtained with the reference ELISA technique. The POC assay could be considered for routine testing based on its ease of use and rapidity.

Development and validation of enzyme-linked immunosorbent assays for the measurement of infliximab and anti-drug antibody levels

Infliximab and its anti-drug antibody (ADA) serum concentrations exhibit a strong correlation with clinical response and loss of response. The use of therapeutic drug monitoring to measure the concentration of infliximab and ADA can facilitate clinical decision-making, helping patients attain optimal therapeutic effects. However, there are still limitations to the existing infliximab and its ADA detection methods. Therefore, this study aimed to develop and validate enzyme-linked immunosorbent assay (ELISA)-based methods for measuring infliximab and its ADA levels in human plasma according to the general recommendations for immunoassays. Free infliximab is bound by recombinant TNF-α and detected using HRP-labeled anti-human antibody. The ADA is captured by on-plate-coated infliximab and recognized by biotin-labeled infliximab. Two bridging ELISA assays were developed and after assay optimization and validation, these assays have been applied in ten patients with inflammatory bowel disease (IBD). In infliximab detection assay, a standard curve ranging from 0.10 μg/mL to 8.0 μg/mL with great precision and accuracy has been established. Drug tolerance of the ADA assay was that 100 ng/mL ADA could tolerate at least 5.0 μg/mL infliximab in the plasma using a commercially available monoclonal anti-infliximab antibody as the positive control. The ADA screening and confirmatory assays achieved a sensitivity of 36.74 ng/mL and 37.15 ng/mL, respectively. All other assay characteristics met the requirements. The mean concentration of infliximab in eight patients with IBD was 7.88 (1.87–21.1) μg/mL, and the ADA levels were all negative. Moreover, the concentrations of infliximab in the remaining two patients were below the LLOQ and the ADAs were positive. Thus, accurate and sensitive ELISA methods have been developed and validated for the detection of infliximab and its ADA concentrations and have been successfully applied to clinical therapeutic drug monitoring.

Prickly Ash Seeds improve immunity of Hu sheep by changing the diversity and structure of gut microbiota.

Prickly Ash Seeds (PAS), as a traditional Chinese medicinal herb, have pharmacological effects such as anti-asthma, anti-thrombotic, and anti-bacterial, but their impact on gut microbiota is still unclear. This study used a full-length 16 s rRNA gene sequencing technique to determine the effect of adding PAS to the diet on the structure and distribution of gut microbiota in Hu sheep. All lambs were randomly divided into two groups, the CK group was fed with a basal ration, and the LZS group was given a basal diet with 3% of PAS added to the ration. The levels of inflammatory factors (IL-10, IL-1β, and TNF-α) in intestinal tissues were measured by enzyme-linked immunosorbent assay (ELISA) for Hu sheep in the CK and LZS group. The results indicate that PAS can increase the diversity and richness of gut microbiota, and can affect the community composition of gut microbiota. LEfSe analysis revealed that Verrucomicrobiota, Kiritimatiella, WCHB 41, and uncultured_rumen_bacterium were significantly enriched in the LZS group. KEGG pathway analysis found that LZS was significantly higher than the CK group in the Excretory system, Folding, sorting and degradation, and Immune system pathways (p < 0.05). The results of ELISA assay showed that the level of IL-10 was significantly higher in the LZS group than in the CK group (p < 0.05), and the levels of TNF-α and IL-1β were significantly higher in the CK group than in the LZS group (p < 0.05). LEfSe analysis revealed that the dominant flora in the large intestine segment changed from Bacteroidota and Gammaproteobacteria to Akkermansiaceae and Verrucomicrobiae after PAS addition to Hu sheep lambs; the dominant flora in the small intestine segment changed from Lactobacillales and Aeriscardovia to Kiritimatiellae and WCHB1 41. In conclusion, the addition of PAS to sheep diets can increase the number and types of beneficial bacteria in the intestinal tract, improve lamb immunity, and reduce intestinal inflammation. It provides new insights into healthy sheep production.