Enzyme Linked Immunosorbent Assay
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Tatsuya Moriyama ◽  
Serina Kinugasa ◽  
Shota Hidaka ◽  
Serina Tanaka ◽  
Eri Izumi ◽  

Background: Kiwifruit is a popular fruit consumed worldwide and is also used as a cosmetic ingredient. However, it is known to cause allergic reactions in humans. Recent studies have suggested an association between food allergy and food allergens entering the body via the skin. However, percutaneously sensitizing kiwifruit allergens have not been identified in human studies or in animal models. Objective: This study aimed to identify kiwifruit proteins that percutaneously sensitized mice through the epidermal application of crude extracts from green and gold kiwifruit on the dorsal skin, and serum IgE and IgG1 levels were used as sensitization markers. Design: BALB/c mice were back-shaved and their skin was exposed to crude extracts from green and gold kiwifruit that contained sodium dodecyl sulfate. Specific IgE and IgG1 antibodies generated and secreted in response to antigens were measured using enzyme-linked immunosorbent assay or immunoblotting. Results: Skin exposure to kiwifruit extract induced an increase in the levels of kiwifruit-specific IgE and IgG1, which are helper T cell 2-related allergenic antibodies in mice. These antibodies reacted with 18, 23, and 24 kDa proteins found in both green and gold kiwifruits. Thus, three percutaneously sensitizing allergens were identified and purified. Their amino acid sequences partially matched with that of kiwellin (Act d 5). Discussion and conclusion: Kiwellin has been identified as a plant defense-related protein. Interestingly, many plant allergens are biodefense-related proteins belonging to the pathogenesis-related protein family. Kiwellin, which was discovered to be a transdermal sensitizing antigen, might also be categorized as a biodefense-related protein. This study is the first to identify kiwellin (Act d 5) as a percutaneously sensitizing kiwifruit allergen in a mouse model.

2021 ◽  
Vol 22 (20) ◽  
pp. 11082
Pei-Wei Weng ◽  
Vijesh Kumar Yadav ◽  
Narpati Wesa Pikatan ◽  
Iat-Hang Fong ◽  
I-Hsin Lin ◽  

Osteoarthritis (OA) is a common articular disease manifested by the destruction of cartilage and compromised chondrogenesis in the aging population, with chronic inflammation of synovium, which drives OA progression. Importantly, the activated synovial fibroblast (AF) within the synovium facilitates OA through modulating key molecules, including regulatory microRNAs (miR’s). To understand OA associated pathways, in vitro co-culture system, and in vivo papain-induced OA model were applied for this study. The expression of key inflammatory markers both in tissue and blood plasma were examined by qRT-PCR, western blot, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays. Herein, our result demonstrated, AF-activated human chondrocytes (AC) exhibit elevated NFκB, TNF-α, IL-6, and miR-21 expression as compared to healthy chondrocytes (HC). Importantly, AC induced the apoptosis of HC and inhibited the expression of chondrogenesis inducers, SOX5, TGF-β1, and GDF-5. NFκB is a key inflammatory transcription factor elevated in OA. Therefore, SC75741 (an NFκB inhibitor) therapeutic effect was explored. SC75741 inhibits inflammatory profile, protects AC-educated HC from apoptosis, and inhibits miR-21 expression, which results in the induced expression of GDF-5, SOX5, TGF-β1, BMPR2, and COL4A1. Moreover, ectopic miR-21 expression in fibroblast-like activated chondrocytes promoted osteoblast-mediated differentiation of osteoclasts in RW264.7 cells. Interestingly, in vivo study demonstrated SC75741 protective role, in controlling the destruction of the articular joint, through NFκB, TNF-α, IL-6, and miR-21 inhibition, and inducing GDF-5, SOX5, TGF-β1, BMPR2, and COL4A1 expression. Our study demonstrated the role of NFκB/miR-21 axis in OA progression, and SC75741′s therapeutic potential as a small-molecule inhibitor of miR-21/NFκB-driven OA progression.

2021 ◽  
Vol 8 ◽  
Gian Pietro Pietri ◽  
Marta Tontini ◽  
Barbara Brogioni ◽  
Davide Oldrini ◽  
Stefania Robakiewicz ◽  

Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1–4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment were further detailed at the atomic level by saturation transfer difference nuclear magnetic resonance (NMR) spectroscopy. The NMR results were used for validation of the in silico docking analysis between the X-ray crystal structure of the antibody (Fab fragment) and the modeled hexamer oligosaccharide. The antibody recognizes the MenX fragment by binding all six repeating units of the oligosaccharide via hydrogen bonding, salt bridges, and hydrophobic interactions. In vivo studies demonstrated that conjugates containing five to six repeating units can produce high functional antibody levels. These results provide an insight into the molecular basis of MenX vaccine-induced protection and highlight the requirements for the epitope-based vaccine design.

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258541
Yonggang Zhang ◽  
Yipeng Zhang ◽  
Limin Zhao ◽  
Junzhu Shi ◽  
Hongling Yang

Object This study aimed to combine plasma protein SerpinA5 with uterine artery doppler ultrasound and clinical risk factor during the first trimester for prediction of preeclampsia. Methods and materials This study was a nested cohort study and was divided into the screening set and developing set. The plasma was collected during the first trimester (11+0–13+6 weeks), at the same time, UtA-PI was detected and recorded with four-dimensional color Doppler ultrasound. These pregnancies were followed up until after delivery. The plasma proteins were examined using ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) and enzyme linked immunosorbent assay (ELISA). Placental samples preserved after delivery were analysed by immunohistochemistry. Clinical risk factors were obtained from medical records or antenatal questionnaires. Upregulation or downregulation of SerpinA5 expression in TEV-1 cells was performed to investigate the role of SerpinA5 in trophoblasts invasion. Results We demonstrated that SerpinA5 levels were greater not only in preeclampsia placental tissue but also in plasma (both p<0.05), and we found that SerpinA5 may interfere with trophoblastic cell invasion by inhibiting MSP. SerpinA5 may be a potential predictor of preeclampsia. What is more, the sensitivity and specificity of predictive power were strengthened when plasma SerpinA5 was combined with UtA-PI and pre-pregnancy BMI & family history of PE for prediction of preeclampsia. Conclusion These findings showed that placenta-derived plasma SerpinA5 may be a novel biomarker for preeclampsia, which together with uterine artery Doppler ultrasound and clinical risk factor can more effectively predict preeclampsia.

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Jing Zhou ◽  
Hao Wu ◽  
Quan-Xin Su ◽  
Xiao-Kai Shi ◽  
Bo-Wen Tang ◽  

Background. The CXC chemokines belong to a unique family of cytokines that participates in the progression and development of many malignant tumors. Evidence for the relationship between chemokine (C-X-C motif) receptor 2 (CXCR2) C1208T polymorphism and susceptibility to cancer remains inconsistent. Methods. Odds ratios (ORs), 95% confidence intervals (CIs), and combined analysis were used to investigate the effect of CXCR2 variation on cancer risk. Gene Set Enrichment Analysis (GSEA) and enzyme-linked immunosorbent assay (ELISA) were also used to evaluate the expression of CXCR2 in prostate cancer (PCA). Results. Across 11 case-control studies, 4,909 cases and 5,884 controls were involved in the current analysis. Individuals with a TT genotype were associated with increased risk of digestive cancer, compared to those with a TC+CC genotype ( OR = 1.16 , 95 % CI = 1.02 -1.31, P = 0.025 ). Individuals carrying the TT genotype had a 39% higher risk of urinary cancer than those carrying CC genotype ( OR = 1.39 , 95 % CI = 1.04 -1.87, P = 0.025 ). Individuals with a TT genotype showed a 56% augmented breast cancer risk, compared to those with a CC genotype ( OR = 1.56 , 95 % CI = 1.03 -2.35, P = 0.034 ). It was found that CXCR2 expression was downregulated in PCA. Compared with PCA subjects carrying the CC genotype, the expression of CXCR2 was decreased in patients with the TT genotype. Conclusions. The CXCR2 C1208T variation was associated with elevated risk of urinary, breast, and digestive cancer. However, the C1208T polymorphism was correlated with attenuated risk of lung cancer.

Foods ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2429
Weipeng Tong ◽  
Hao Fang ◽  
Hanpeng Xiong ◽  
Daixian Wei ◽  
Yuankui Leng ◽  

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.

J Kyunguk ◽  
L So-Yeon ◽  
J Se-Ah ◽  
G Purevsan ◽  
N Jin Young ◽  

Background: Perilla seeds are known to cause immediate allergic reactions. However, reports on perilla seed allergies are limited to a few case reports worldwide, and there is currently no diagnostic test for such allergies. Objective: Our objective was to analyze the clinical and immunological characteristics of perilla seed allergy and to identify allergens for the development of diagnostic methods. Methods: Twenty-one children with clinical perilla seed allergy were enrolled from two tertiary hospitals between September 2016 and June 2019. Using perilla seed extract, we developed a skin prick test (SPT) reagent and an IgE enzyme-linked immunosorbent assay (ELISA) for perilla seed allergy diagnosis. IgE immunoblotting was performed for identifying putative allergenic components, and amino acid composition analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: The median age of children with perilla seed allergy was 3 years; the proportion of children with anaphylaxis was 28.6%. Perilla seed SPT was performed in 15 of 21 children, all of whom tested positive. On ELISA, 85.7% of children tested positive for perilla seed-specific IgE. Proteins with molecular weights of 50, 31–35, and 14–16 kDa showed binding with the sera of >50% of children with perilla seed allergy. LC-MS/MS analysis of these three protein fractions indicated 8 putative proteins, including perilla oleosin (Accession No. 9963891), to be allergens. Conclusion: This study documented the clinical characteristics and immunological profiles of 21 children with perilla seed allergy. Our results suggest that oleosin is one of the major allergens in perilla seeds.

Foods ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2426
Yanwei Ji ◽  
Lili Chen ◽  
Yingying Wang ◽  
Kaihui Zhang ◽  
Haofen Wu ◽  

Staphylococcal enterotoxins (SEs) represent the leading reason for staphylococcal food poisoning (SFP) and various other diseases. Reports often indicate Staphylococcal enterotoxin C (SEC) as the most frequently found enterotoxin in dairy products. To minimize consumer exposure to SEC, this paper aimed to create a sandwich enzyme-linked immunosorbent assay (ELISA) based on nanobodies (sandwich Nbs-ELISA) to accurately detect SEC in dairy products without the influence of staphylococcal protein A (SpA). Therefore, after inoculating a Bactrian camel with SEC, a phage display Nb library was created. Eleven Nbs against SEC were identified in three biopanning steps. Based on their affinity and pairing level, a sandwich Nbs-ELISA was developed using the C6 anti-SEC Nb as the capture antibody, while the detection antibody was represented by the C11 phage display anti-SEC Nb. In optimal conditions, the quantitative range of the present sandwich ELISA was 4-250 ng/mL with a detection limit (LOD) of 2.47 ng/mL, obtained according to the blank value plus three standard deviations. The developed technique was subjected to specific measurements, revealing minimal cross-reactivity with Staphylococcus aureus (S. aureus), Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin B (SEB), and SpA. The proposed method exhibited high specificity and an excellent recovery rate of 84.52~108.06% in dairy products. Therefore, the sandwich Nbs-ELISA showed significant potential for developing a specific, sensitive technique for SEC detection in dairy products.

Salim Aziz ◽  
Shailendra Sharma ◽  
Jenna Aziz ◽  
James Gould ◽  
Xiomara Fernandez

Management of patients with acute heparin-induced thrombocytopenia (HIT) with cavo-atrial renal cancer requiring surgery on cardiopulmonary bypass (CPB) and possible deep hypothermia circulatory arrest is a challenge. A staged approach using Bivalirudin, plasmapheresis, and intravenous immunoglobulin (IVIG) was used to preoperatively de-escalate HIT guided by enzyme-linked immunosorbent assay (ELISA) and serotonin release assay (SRA). Intraoperatively heparin was used as the anticoagulant for CPB as DHCA was likely to be used to remove the atrio-caval tumor. Heparin is effective in preventing clots in the circuit during DHCA. To prevent HIT upon re-exposure to heparin during CPB, a bolus of a Cangrelor (reversible P2Y12 platelet receptor inhibitor) was given before heparin and during CPB whilst platelet activity was monitored using platelet reactivity units (PRU). Postoperatively, to prevent recurrence of HIT, plasmapheresis was used until SRA and optical density (OD) resulted. The patient had a successful outcome.

2021 ◽  
Vol 11 (1) ◽  
Yunjoo Im ◽  
Hongseok Yoo ◽  
Ryoung-Eun Ko ◽  
Jin Young Lee ◽  
Junseon Park ◽  

AbstractCD63 is one of the tetraspanin protein family members that is ubiquitously expressed on exosomes and is involved in the signal transduction of various types of immune cells. It may thus contribute to immunometabolic mechanisms of cellular and organ dysfunction in sepsis. Nonetheless, the association of exosomal CD63 with the severity and mortality of sepsis is not well known. Therefore, in the present study, the overall levels of exosomal CD63 were evaluated to ascertain whether they were associated with organ failure and mortality in patients with sepsis. Exosomal CD63 was measured from prospectively enrolled critically-ill patients with sepsis (n = 217) and healthy control (n = 20). To detect and quantify exosomes in plasma, a commercially available enzyme-linked immunosorbent assay kit was used according to the manufacturer’s protocol. The total number of exosomal CD63 was determined by quantifying the immunoreactive CD63. The association between plasma levels of exosomal CD63 and sequential organ failure assessment (SOFA) score was assessed by a linear regression method. The best cut-off level of exosomal CD63 for 28-day mortality prediction was determined by Youden’s index. Among 217 patients with sepsis, 143 (66%) patients were diagnosed with septic shock. Trends of increased exosomal CD63 levels were observed in control, sepsis, and septic-shock groups (6.6 µg/mL vs. 42 µg/mL vs. 90 µg/mL, p < 0.001). A positive correlation between exosomal CD63 and SOFA scores was observed in patients with sepsis (r value = 0.35). When patients were divided into two groups according to the best cut-off level, the group with higher exosomal CD63 levels (more than 126 µg/mL) was significantly associated with 28-day and in-hospital mortality. Moreover, the Kaplan–Meier survival method showed a significant difference in 90-day survival between patients with high- and low-exosomal CD63 levels (log-rank p = 0.005). Elevated levels of exosomal CD63 were associated with the severity of organ failure and predictive of mortality in critically ill patients with sepsis.

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