scholarly journals Blastocysts depict sex-specific signalling of IFNT transcription, translation and activity

Reproduction ◽  
2018 ◽  
Author(s):  
Corina Isabel Schanzenbach ◽  
Sandra Milena Bernal-Ulloa ◽  
Vera Anna van der Weijden ◽  
Michael W. Pfaffl ◽  
Mathias Büttner ◽  
...  
Keyword(s):  
Biological Activity ◽  
Antiviral Activity ◽  
Protein Concentration ◽  
Limit Of Detection ◽  
Activity Level ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Activity ◽  
Interferon Tau ◽  
Endometrial Stroma ◽  
Gene And Protein Expression

Preimplantation bovine blastocyst supernatants exhibit sex-dependent antiviral activity, due to the ruminant pregnancy recognition signal Interferon tau (IFNT). Differing potencies of IFNT variants have been supposed as cause, although evidence remains scarce. Here, we aimed at quantifying the sex-dependent IFNT production on transcriptional, translational, and biological activity level in bovine blastocysts, to elucidate the origin of differences in antiviral activity between male and female blastocysts. Day 8 bovine blastocysts were co-cultured with endometrial stroma cells for 48 hours. The embryonic IFNT mRNA expression was determined by quantitative reverse transcription followed by polymerase chain reaction (RT-qPCR). Additionally, the IFNT protein concentration was determined using a sensitive in-house developed IFNT-specific Enzyme-linked-immunosorbent Assay (ELISA). The biological activity was assessed by quantifying the response of Interferon stimulated gene (ISG) expression in endometrial stroma cells. While the IFNT specific ELISA displayed a limit of detection of 7.3 pg/mL, the stroma cell culture system showed to react to as little as 0.1 pg/mL IFNT in RT-qPCR analysis. The female blastocysts had a significant, 5.6-fold, 3.6-fold, and 5.2-fold higher IFNT production than male blastocysts as determined by transcript abundance, protein concentration and, protein activity, respectively. Additionally, all parameters correlated positively, and therefore, we conclude that female blastocysts most likely have an increased IFNT gene and protein expression rather than expressing more potent IFNT variants.

scholarly journals Pitfalls in the Immunochemical Determination of β‑Lactam Antibiotics in Water

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 298
Author(s):  
Alexander Ecke ◽  
Rudolf J. Schneider
Keyword(s):  
Limit Of Detection ◽  
Cost Effective ◽  
Enzyme Linked Immunosorbent Assay ◽  
Degree Of Hydrolysis ◽  
Site Analysis ◽  
Antibody Recognition ◽  
Immunochemical Determination ◽  
Key Factor ◽  
Lactam Ring

Contamination of waters with pharmaceuticals is an alarming problem as it may support the evolution of antimicrobial resistance. Therefore, fast and cost-effective analytical methods for potential on-site analysis are desired in order to control the water quality and assure the safety of its use as a source of drinking water. Antibody-based methods, such as the enzyme-linked immunosorbent assay (ELISA), can be helpful in this regard but can also have certain pitfalls in store, depending on the analyte. As shown here for the class of β-lactam antibiotics, hydrolysis of the β‑lactam ring is a key factor in the immunochemical analysis as it influences antibody recognition. With the antibody used in this study, the limit of detection (LOD) in the immunoassay could be significantly reduced by hydrolysis for the five tested penicillins, with the lowest LOD for carbenicillin (0.2 nmol/L) and the greatest impact on penicillins G and V (reduction by 85%). In addition to enhanced quantification, our strategy also provides access to information about the degree of hydrolysis in water samples as shown for the most abundant penicillin amoxicillin.

scholarly journals Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Matthew Mannarino ◽  
Hosni Cherif ◽  
Li Li ◽  
Kai Sheng ◽  
Oded Rabau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Back Pain ◽  
Protein Expression ◽  
Disc Degeneration ◽  
Cell Senescence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Matrix Synthesis ◽  
Gene And Protein Expression ◽  
Pain Patients ◽  
In Cells

Abstract Background There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16INK4a, SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

A Simple and Rapid Light-Initiated Chemiluminescence Assay for Quantitation of Artemisia-Specific Immunoglobulin E

2021 ◽  
pp. 1-8
Author(s):  
Dandan Liu ◽  
Bei Zhang ◽  
Lina Zhu ◽  
Lisheng Zheng ◽  
Shaoshen Li ◽  
...  
Keyword(s):  
Food Allergen ◽  
Clinical Guideline ◽  
Immunoglobulin E ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Chemiluminescence Assay ◽  
Limit Of Quantitation ◽  
Specific Immunoglobulin E ◽  
Specific Immunoglobulin

<b><i>Background:</i></b> Light-initiated chemiluminescence assay (LICA) is a homogeneous assay that has been successfully used for the quantitation of food allergen-specific immunoglobulin E (sIgE), but not inhaled allergen-sIgE. Simultaneously, current assays used to detect allergen-sIgE are serum consuming and/or time consuming. Hence, we established a method for the quantitation of <i>Artemisia</i>-sIgE based on LICA and verified its performance according to the clinical guideline documents, laying a foundation for the quantitation of inhaled and food allergen-sIgE in parallel on LICA. <b><i>Methods:</i></b> The assay was established after optimizing the first incubation time and the dilutions of <i>Artemisia</i>-coated chemibeads, biotinylated goat anti-human IgE, and serum. In order to quantitate <i>Artemisia</i>-sIgE, the calibration curve was established with a high positive serum of known concentration. The assay performance was confirmed per the clinical guideline documents. In addition, the correlation between the results of LICA and capture enzyme-linked immunosorbent assay was evaluated. <b><i>Results:</i></b> The developed LICA’s coefficients of variation of repeatability and intermediate precision were 3.20%, 2.14%, and 3.85% and 4.30%, 4.00%, and 4.40%, respectively. The limit of detection was 0.10 kU<sub>A</sub>/L, and the limit of quantitation was 0.11 kU<sub>A</sub>/L. The range of linearity was from 0.27 kU<sub>A</sub>/L to 97.53 kU<sub>A</sub>/L (<i>r</i> = 0.9968). The correlation coefficient (<i>r</i>) for the correlation analysis between results of LICA and capture ELISA was 0.9087. This assay was successfully applied in 64 human serum samples, showing good sensitivity (82.20%) and specificity (100%). <b><i>Conclusion:</i></b> An <i>Artemisia</i>-sIgE quantitation assay based on LICA was successfully established. Its performance satisfied the clinical requirements and could be widely used in clinical laboratories.

scholarly journals Streptavidin-conjugated gold nanoclusters as ultrasensitive fluorescent sensors for early diagnosis of HIV infection

2018 ◽  
Vol 4 (11) ◽  
pp. eaar6280 ◽  
Author(s):  
Aditya Dileep Kurdekar ◽  
L. A. Avinash Chunduri ◽  
C. Sai Manohar ◽  
Mohan Kumar Haleyurgirisetty ◽  
Indira K. Hewlett ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Limit Of Detection ◽  
Gold Nanoclusters ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Metal Nanoclusters ◽  
Detection Range ◽  
In Silico Studies ◽  
P24 Antigen ◽  
Hiv 1

We have engineered streptavidin-labeled fluorescent gold nanoclusters to develop a gold nanocluster immunoassay (GNCIA) for the early and sensitive detection of HIV infection. We performed computational simulations on the mechanism of interaction between the nanoclusters and the streptavidin protein via in silico studies and showed that gold nanoclusters enhance the binding to the protein, by enhancing interaction between the Au atoms and the specific active site residues, compared to other metal nanoclusters. We also evaluated the role of glutathione conjugation in binding to gold nanoclusters with streptavidin. As proof of concept, GNCIA achieved a sensitivity limit of detection of HIV-1 p24 antigen in clinical specimens of 5 pg/ml, with a detection range up to1000 pg/ml in a linear dose-dependent manner. GNCIA demonstrated a threefold higher sensitivity and specificity compared to enzyme-linked immunosorbent assay for the detection of HIV p24 antigen. The specificity of the immunoassay was 100% when tested with plasma samples negative for HIV-1 p24 antigen and positive for viruses such as hepatitis B virus, hepatitis C virus, and dengue. GNCIA could be developed into a universal labeling technology using the relevant capture and detector antibodies for the specific detection of antigens of various pathogens in the future.

scholarly journals Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin

Toxins ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 458 ◽  
Author(s):  
Hisaya Ono ◽  
Nobuaki Hachiya ◽  
Yasunori Suzuki ◽  
Ikunori Naito ◽  
Shouhei Hirose ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Expression System ◽  
Sandwich Elisa ◽  
Food Poisoning ◽  
High Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
E Coli ◽  
Detection Systems ◽  
C Terminus ◽  
Culture Supernatants

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

scholarly journals Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

Toxins ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 781
Author(s):  
Zhuolin Song ◽  
Lin Feng ◽  
Yuankui Leng ◽  
Mingzhu Huang ◽  
Hao Fang ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Cross Reaction ◽  
Molar Ratio ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enzyme Loading ◽  
M13 Bacteriophage ◽  
Mimic Peptide

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.

scholarly journals Identification and dietary exposure assessment of tetracycline and penicillin residues in fluid milk, yogurt, and labneh: A cross-sectional study in Lebanon

2019 ◽  
Vol 12 (4) ◽  
pp. 527-534 ◽  
Author(s):  
Suzanne Kabrite ◽  
Christelle Bou-Mitri ◽  
Jessy El Hayek Fares ◽  
Hussein F. Hassan ◽  
Jocelyne Matar Boumosleh
Keyword(s):  
Human Health ◽  
Dairy Products ◽  
Detection Rate ◽  
Limit Of Detection ◽  
Human Health Risk ◽  
Daily Intake ◽  
Mean Value ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibiotic Residues ◽  
Mean Values

Background and Aim: The safety and quality of dairy products are considered to be of significant importance to human health. Although antimicrobial drugs are essential for disease treatment in modern medicine, the use of these drugs can have undesired consequences for human and animal health. This study aimed to investigate the presence of tetracycline and penicillin residues in raw, pasteurized, and UHT cow's milk of different fat contents, as well as in the dairy products yogurt and labneh, a traditional Lebanese product. Materials and Methods: A total of 44 samples, 4 raw, 9 UHT, 9 pasteurized milk, 10 yogurt, and 12 labneh samples from common local brands available in the Lebanese market were collected from Keserwan regions in May 2016. Tetracycline and penicillin residues were determined using a competitive enzyme-linked immunosorbent assay (ELISA) technique. Results: The mean values for tetracycline and penicillin were all below the limit of detection (LOD) of the ELISA kit of a maximum standard concentration of 1.80 μg/kg and 4.00 μg/kg, respectively. All samples tested positive for antibiotic residues. The detection rate for tetracycline in milk (n=22) samples was 86.4% with a mean residues value of 1.16±0.70 μg/kg. The detection rate of tetracycline in labneh (n=12) and yogurt (n=10) samples was 50% for each with a mean value of 1.76±0.40 μg/kg and 0.63±0.12 μg/kg, respectively. As for penicillin residues, 90.9% of the milk (n=22) samples tested positive with a mean value of 0.52±0.25 μg/kg. The detection rate in labneh (n=12) and yogurt (n=10) samples was 0% for penicillin residues, where mean values were all below the LOD (<1.25 μg/kg) for these dairy products. None of the samples exceeded the maximum residue levels. The estimated dietary intake (EDI) for tetracycline and penicillin residues for all dairy products is 2.09 ng/kg body weight (BW)/day resulting in 0.007% of the acceptable daily intake (ADI) and 1.83 ng/kg BW/day resulting in 0.006% of the ADI, respectively. Conclusion: All EDI values were below the ADI set for each antibiotic residue and do not exceed relevant toxicological reference values. However, concerns might still be present from consumption of other animal food products containing residues. Moreover, the long-term exposure to such residues is still unknown as a result of bioaccumulation; it is a challenging process to determine the actual dietary consumption of foods containing antibiotic residues; hence, the human health risk cannot be easily predicted.

scholarly journals Characterization and validation of fragment antigen-binding (Fab) antibody-based immunoassay for deoxymiroestrol, a potent phytoestrogen from Pueraria candollei

2021 ◽  
Author(s):  
Worapol Sae-foo ◽  
Supaluk Krittanai ◽  
Wipawee Juengsanguanpornsuk ◽  
Gorawit Yusakul ◽  
Tharita Kitisripanya ◽  
...  
Keyword(s):  
Quantitative Method ◽  
Estrogenic Activity ◽  
Limit Of Detection ◽  
Hormone Replacement ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Binding ◽  
Pueraria Candollei ◽  
Specific Determination ◽  
Fragment Antigen Binding

Deoxymiroestrol is the most potent phytoestrogen in chromenes group that has been found in Pueraria candollei, Thai name known as Kwao Krua Khao. Several studies reported estrogenic activity of P. candollei in order to using as hormone replacement therapy for postmenopausal women. Previously, specific determination of deoxymiroestrol content by enzyme-linked immunosorbent assay (ELISA) using polyclonal antibody (pAb) have been reported. However, production of pAb has limitation and variability from different batches. Therefore, in this study, we established quantitative method for determination of deoxymiroestrol using fragment antigen-binding (Fab) antibody-based immunoassay. The developed immunoassay has specificity to deoxymiroestrol with a calibration range of 15.6-1000 ng mL-1. Precision including intra-assay and inter-assay are 1.48-7.11 and 0.58-9.31%, respectively. Accuracy of the assay showed in recovery between 99.77-101.61% when spike deoxymiroestrol standard into the samples. The limit of detection (LOD) is 30.80 ng mL-1. Comparation antibody-based immunoassay for determination of deoxymiroestrol using Fab with pAb was represented consistency (R2 = 0.9807) when analysis roots bark of Pueraria candollei from difference areas. Therefore, this development assay can apply to determine deoxymiroestrol content in the plant samples.

scholarly journals Plasma Level and Expression of Visfatin in the Porcine Hypothalamus During the Estrous Cycle and Early Pregnancy

2020 ◽  
Author(s):  
Tadeusz Kaminski ◽  
Marta Kiezun ◽  
Ewa Zaobidna ◽  
Kamil Dobrzyń ◽  
Barbara Wasilewska ◽  
...  
Keyword(s):  
Protein Expression ◽  
Blood Plasma ◽  
Estrous Cycle ◽  
Early Pregnancy ◽  
Energy Homeostasis ◽  
Plasma Concentrations ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tissue Samples ◽  
Hormone Synthesis ◽  
Gene And Protein Expression

Abstract BackgroundVisfatin exists in two forms: the intracellular form which is a rate limiting enzyme engaged in nicotinamide adenine dinucleotide biosynthesis and the extracellular form considered as an adipokine, produced mainly by the adipose tissue. Visfatin could be an energy sensor involved in regulating female fertility, which creates a hormonal link integrating the control of energy homeostasis and reproduction. MethodsThe study compares the expression levels of visfatin gene (quantitative real time PCR) and protein (Western blotting and fluorescent immunohistochemistry) in the selected areas of the porcine hypothalamus responsible for gonadotropin releasing hormone synthesis: the mediobasal hypothalamus (MBH) and preoptic area (POA), and visfatin concentrations in the blood plasma (enzyme-linked immunosorbent assay). The tissue samples were harvested from gilts on days 2-3, 10-12, 14-16 and 17-19 of the estrous cycle, and on days 10-11, 12-13, 15-16, 27-28 of pregnancy. Differences between groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ResultsDuring the estrous cycle, visfatin mRNA expression in the MBH was higher on days 2-3 and 17-19, while the visfatin protein concentration on days 17-19. During early pregnancy, the most pronounced gene and protein expression in the MBH was found on days 15-16 and 10-11, respectively. In the POA during the estrous cycle, visfatin gene expression was the highest on days 17-19, and the protein level of visfatin on days 14-16. During early pregnancy, the highest expression of visfatin gene in the POA was observed on days 15-16, whereas the protein concentrations on days 27-28. Blood plasma concentrations of visfatin during the estrous cycle were higher on days 2-3 in relation to other studied phases of the cycle. During early pregnancy, the highest visfatin contents in the blood plasma were observed on days 12-13. Visfatin gene and protein expression in MBH and POA, and visfatin plasma concentrations differed during early pregnancy in relation to days 10-12 of the cycle. ConclusionsThis study demonstrated visfatin expression in the porcine hypothalamus and its dependence on hormonal milieu related to the estrous cycle and early pregnancy.

Simulation model for predicting limit of detection and range of quantitation of competitive enzyme-linked immunosorbent assay

2007 ◽  
Vol 103 (5) ◽  
pp. 427-431 ◽  
Author(s):  
Dong Hwan Choi ◽  
Yoshio Katakura ◽  
Rieko Matsuda ◽  
Yuzuru Hayashi ◽  
Kazuaki Ninomiya ◽  
...  
Keyword(s):  
Simulation Model ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay
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