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Dec 2018 DOI 10.14302/issn.2377-2549.jndc-18-2430
Introduction: Analysis of 8-oxodG is usually conducted by either chromatography-based methods or by immunochemical methods commonly used based upon their low cost and high-throughput. However, concern regarding the accuracy of ELISA methods has complicated their use. We directly compare the levels of urinary 8-oxodG obtained by HPLC-MS/MS with three commercially available ELISA kits in this report. Methods: In the current study, a total of 9 human urine samples were analyzed by LC-MS/MS and three commonly used commercial available ELISA kits. Results: We found that urinary 8-oxodG levels analyzed by HPLC-MS/MS [1.4 ± 0.3 nmol/mmol creatinine) were 7.6- to 23.5-fold lower than those detected by ELISA. Overall, the correlations between ELISA and HPLC-MS/MS were poor but were improved after SPE purification for kits from ENZO (P = 0.2817 without SPE; P = 0.0086 with SPE) and Abcam (P = 0.0596 without SPE; P = 0.0473 with SPE). Discussion and Conclusion: While we confirmed that SPE purification can improve the correlation between the selected ELISA kits and HPLC-MS/MS, HPLC-MS/MS is still the method of choice to accurately assess the levels of 8-oxodG in human urine.
Oct 2023 DOI 10.14302/issn.2575-1212.jvhc-23-4532
Camels are a significant source of income for nomadic populations in many developing countries, including Ethiopia. Camels are well adapted to dry and semi-dry regions, providing income, food security, and transportation. However, camel production and productivity are constrained by infectious diseases, such as brucellosis, which is a highly infectious bacterial disease that affects camels and humans worldwide. Brucellosis causes significant economic losses due to abortion, low herd fertility, and decreased milk production. In Ethiopia, the prevalence of camel brucellosis varies depending on factors related to the host, agent, climate, and management system, with a reported prevalence ranging from 0.5% to 11.9%. Accurate diagnosis of camel Brucellosis is essential for herd-based screening of animals. Although culturing the pathogen is the preferred method for diagnosis, serological tests such as Rose-Bengal plate test (RBPT), Enzyme-linked immunosorbent assay (ELISA), and Complement fixation test (CFT) and polymerase chain reaction (PCR) assays have been developed. Implementing effective diagnosis and surveillance systems to control the spread of brucellosis in animals and humans is very important, on top of awareness campaigns, vaccination programs, and suitable laboratory establishment recommended. Continued research is essential to maintain the health and productivity of camel populations, particularly in pastoral areas where camels play a significant role in the livelihood of communities. Therefore, the present paper views the seropositive prevalence and potential risk factors associated with camel brucellosis in Ethiopia.
Jun 2023 DOI 10.14302/issn.2377-2549.jndc-23-4615
A literature review was undertaken with a focus on 1) identifying the research gaps regarding CECs, 2) identifying the most common ones, and 3) identifying the typical analytical methods/technologies employed, for their analysis. A total of 214 papers were noted, with a total of 21 review articles (9.8%). Of this total, a surprisingly high number were from South Africa alone: 117 (54.7%), of which 44 (20.6%) reports were associated with South Africa’s Water Research Commission (WRC). The top three CECs research gaps were (decreasing rank: Number of “gaps”, %): 1) Toxicity/Risk/Impact (260, 21.5%), 2) Analysis/Tests/Methods (118, 9.8%) and 2) Future research/studies (118, 9.8%), and 3) Monitoring (89, 7.4%). The common classes of CECs that were reported on, were : (i) Chemical: pharmaceuticals, personal care products, steroids, chlorinated and brominated contaminants, PAHs, PCBs, phthalates, alkyl phenols, herbicides, organochlorine pesticides, engineered nanomaterials and (ii) “Microbiological”: antibiotic resistance genes, human enteric bacteria and viruses, microbial pathogens (e.g., E Coli, rotavirus, Crypto, etc.), infectious biological water contaminants (e.g., E Coli isolates), cyanobacterial blooms (Microcystis). Common test methods used for analysis of the chemical contaminants were found to be chromatography (gas, liquid)-mass spectrometry; for the microbial contaminants, they were culture-based methods, ELISA, fluorescence microscopy, qPCR, RT-qPCR, gel electrophoresis, Raman spectroscopy, and also chromatography (largely liquid)-mass spectrometry, were also used. Some proposals were additionally made to address the very common, significant research gaps noted in CECs research, especially the standardization of analytical chemical test methods, based on chromatography-mass spectrometry, for quantification.
May 2023 DOI 10.14302/issn.2574-4488.jna-23-4545
This study aimed to analyze pharmacological actions of phenolic compound luteolin on the renal and cardiac hypertrophy, blood pressure (BP), baroreflex sensitivity (BRS), levels of epoxyeicosatrienoic acids (EETs), prostaglandin E-2 (PGE-2) and endothelin-1 (E1) in plasma in the 2 kidney - 1 clip (2K-1C) model of renovascular hypertension (RVH). All animals, were randomized into 2 groups: control (normal) I - sham-operated, II- RVH male Wistar rats, which after 4 weeks of surgical intervention secondly randomized to control II group, treated 0.1% dimethyl sulphoxide (DMSO) and main group - with luteolin in 15 DMSO, 3 mg/kg body weight, intraperitonially, during 2 weeks. ET-1, EETs and PGE2 levels investigated in carotid artery blood plasma and analyzed using ELISA kits. All data statistically analyzed using the SPSS-10.0 program. In RVH rats BP increased by 32%, cardiac and right kidney hypertrophy and reduction in parasympathetic component of BRS by 40% and sympathetic by 39%. The plasma level of total trans-EETs and PGE2 in RVH rats decreased by 44% and 50% respectively, while the level of ET-1 increased by 67%. Two weeks treatment with luteolin lowered BP, improved parasympathetic, without marked changes in sympathetic component of BRS. Deremodeling of cardiac and renal hypertrophy under prolonged treatment with luteolin accompanied with increasing in the level of EETs by 44%, PGE-2 by 50% and markedly reducing of plasma content of ET-1 (by 60%). Inhibition of EET hydrolase using low doses of luteolin provides beneficial cardio and renoprotective action in experimental model of RVH.
Jan 2022 DOI 10.14302/issn.2372-6601.jhor-22-4061
Background Human malignant cell models which reflect the structural and physiological complexity of tumor tissue are of great importance for preclinical research in oncology. Spheroids/tumoroids derived from solid tumors are of great interest as cellular models mimicking the first vascular-free growth phase of a tumor node. The fact of the identity between artificially created tumor multicellular aggregates and the real tumor tissue, however, needs to be specified, described and validated in order to see how closely the spheroids are biologically similar to the malignized tissues in vivo compared to the monolayer cell cultures traditionally used. We present here a comparison study of the characteristics of solid tumor cells of different histogenesis (melanomas, soft tissue sarcomas and bone sarcomas, epithelial tumors) cultured in two dimensions (monolayer culture) and three dimensional space (spheroid), namely: spatial organization, multiplication, metabolic activity. Patients and Methods For the creation of 2 D and 3D cell models the cells isolated from the patient's solid tumor fragments obtained intraoperatively were used. 15 samples of skin melanoma, 20 samples of soft tissue and osteogenic sarcomas (STBS), and 9 samples of epithelial tumors (ET). The tumor cells were all cultivated for at least 10 passages. We used phase contrast, confocal microscopy, and immunohistochemistry to investigate spheroids and monolayer cultures. The supernatants of tumor cells grown in 2D and 3D cultures were studied using ELISA and multiplex analysis for the production of a spectrum of chemokines and cytokines supporting the immunosuppression, invasion and metastasis processes. Results Tumor specimens received were predominantly of metastatic origin (75%). In 100% of cases 2D cultures were received, in 88.6% of cases (39 out of 44) we succeeded in obtaining spheroids. There was no direct correlation between the efficiency of tumoroid formation and the tumor's histogenetic origin and the stage of the cancer process (primary tumor, recurrence, metastasis). The median size of spheroids by 4-5 days of cultivation with a starting concentration of 10000 cells per well was 657.14 μm for melanoma (min 400 - max 1000 μm), 571.42 μm (min 400 - max 700 μm), 507.14 μm (min 300 - max 600 μm) for soft tissue sarcomas, 650.0 μm (min 400 - max 900 μm) for osteogenic sarcomas. Immunochemical analysis of Ki-67, GLUT1, and Ecadherin markers was carried out for tumor tissue samples, single-layer tumor cultures, and tumoroids of every patient. The distribution of the stained groups in the spheroids was distinct from the monolayer cultures and more in accordance with the distribution of such in the tissue tumor, the number of Ki-67+ cells was increasing in the spheroids. We detected no dependence of Ki-67+ and GLUT1+ cell localization grade on spheroid size. We identified E-cadherin in tumor tissue and tumoroids of breast carcinoma and one melanoma culture. Monolayer cultures did not express it. The increase in secretory cell activity of the solid tumor cells from 2D to 3D system was observed when CCL2, CCL3, CXCL1, CXCL16, MIF, IL10, MICA (p<0.01) were investigated. Conclusion The presence of patient-specific cells of solid tumors in a 3D environment causes activation of the proliferative and metabolic processes as compared to monolayer cultures, which makes these models approximate the real world clinical picture. The production of chemokines that can attract to the tumor various types of immune system cells, to include their immature versions, as well as production of cytokines and Immunosuppression factors that, when present in the tumor microenvironment in the high concentrations, contribute to the formation of immune cells having suppressive capacities occurs in the 3D cell system. Three-dimensional model of the initial tumor nodule formation stage thus demonstrates the forming process of tumor cells favorable for them microenvironment. Construction of three-dimensional models - spheroids of tumor cells of differing histogenesis demands individual approach and more thorough investigation.
Dec 2021 DOI 10.14302/issn.2575-1212.jvhc-21-4034
In bovine tuberculosis (bTB), cellular, humoral, or both types of immune responses have been observed. The purpose of this study was to examine the immune status of tuberculous cows based on the differential cytokine gene expression associated with Th1 (IFN-γ, IL-2), or Th2 (IL-4, IL-10) responses. Twenty-three (23) cows belonging to a dairy herd located in a rural region of the State of Hidalgo, México, were selected for the study. Single Intradermal Comparative Cervical Tuberculin (SICCT) Test, Interferon-Gamma (IFN-γ) Release Assay (BOVIGAM), and Enzyme-Linked Immunosorbent Assay (ELISA) were used for detection of cattle infected by M. bovis. Thirteen cows were positive to all the tests (Group 1); ten cows were positive only to ELISA (Group 2), and the remaining Group (Group 3, control) included cows negative to all the tests. Peripheral blood mononuclear cells (PBMC) from animals were in vitro stimulated by bovin purified protein derivative (PPD), avian PPD, and Concanavalin A (Con A) mitogen for 72h. Changes in the levels of expression of mRNA of the respective cytokines was measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using β-actin gene as internal control. In group 1, PPD bovis and Con A-stimulated cells exhibited high production of IFN-γ, IL-2 and IL-4, but not IL-10. In contrast, PPD avium-stimulated cells displayed a low production of cytokine transcripts. In group 2, cells showed a significant production of IL-10 in response to bovine PPD (P< 0.001). In the control group, a high production of IFN-γ and IL-2 was observed only in Con A-stimulated cells. Post-mortem examinations in animals of group 1 showed slight and medium lesions in lymph nodes, whereas in group 2, the lesions were more extensive. Results indicate differences on gene expression levels of cytokines considered to determine balance in Th1/Th2 response among the evaluated groups. In addition, high levels of antibodies against M. bovis and high IL-10 expression in PBMC together are indicators of progressive bTB when both tuberculin test and IFN-γ assay are negative in tuberculous anergic cattle. Inclusion of serology and IL-10 cytokine expression in in the diagnosis checklist improves detection of infected cattle to help control bovine tuberculosis.
Aug 2021 DOI 10.14302/issn.2326-0793.jpgr-21-3917
50 years ago the Enzyme Immunoassay Enzyme-Linked Immunosorbent Assay, mostly known as ELISA was developed. This is a powerful but simple method that is very widely used in the diagnostic practice, as well as in biomedical research. During this time a number of ELISA modification were developed that significantly increased its properties, especially the senstivity, such as avidin-biotin assay, immuno-PCR, nano-ELISA and finally, the digital ELISA. This short review describes the principles of ELISA and the evolution from a conventional assay to the modern ultra-sensitive method. Most of the immunological methods have two components: antigen and antibody. The high specificity of their interaction gives a possibility to detect one of them if other one is included in the reaction as a specific partner. The simplest method for antigen detection in the presence of the antibody is immune diffusion (radial immune diffusion in that case), which practically the formation of precipitate of the “antigen-antibody” complex, when the target antigen diffuses from well into agarose containing the specific antibody. Unfortunately, this assay, as well as other traditional methods, like hemagglutination or complement fixation, have a low sensitivity and are unwieldy.
Jul 2021 DOI 10.14302/issn.2766-8681.jcsr-21-3885
Sepsis is a systemic inflammatory response to a confirmed or suspected infection. The transition from sepsis to septic shock causes high rate of mortality. The aim of this experiment was to evaluate the anti-inflammatory potential of the Biofield Energy Treated (Blessed) Proprietary Test Formulation and Biofield Energy Healing (Blessing) Treatment per se to Sprague Dawley rats on Cecal Slurry, LPS, and E. coli-induced systemic inflammatory response syndrome (SIRS) model. In this experiment, various proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, IL-12, 1L-17, and interferon-γ (IFN-γ) were analysed using ELISA. A test formulation was formulated including minerals (magnesium, zinc, calcium, selenium, and iron), vitamins (ascorbic acid, pyridoxine HCl, vitamin E, cyanocobalamin, and cholecalciferol), Panax ginseng extract, β-carotene, and cannabidiol isolate. The constituents of the test formulation were divided into two parts; one section was defined as the untreated test formulation, while the other portion of the test formulation and three group of animals received Biofield Energy Healing Treatment remotely for about 3 minutes by a renowned Biofield Energy Healer Mr. Mahendra Kumar Trivedi. The level of TNF-α was significantly reduced by 40.50%, 85.36% (p≤0.01), 50.66% (p≤0.01), 87.38% (p≤0.01), and 58.63% (p≤0.01) in G5 (Cecal Slurry, LPS, and E. coli + Biofield Energy Treated test formulation), G6 (Cecal Slurry, LPS, and E. coli + Biofield Energy Treatment per se to animals from day -15), G7 (Cecal Slurry, LPS, and E. coli + Biofield Energy Treated test formulation from day -15), G8 (Cecal Slurry, LPS, and E. coli + Biofield Energy Treatment per se + Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS, and E. coli + Biofield Energy Treatment per se animals + untreated test formulation) groups, respectively as compared to the disease control (G2) group. Additionally, the level of IL-1β was decreased by 17.04%, 15.56%, and 12.59% in G6, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group. The level of IL-6 was significantly (p≤0.001) reduced by 36.18%, 50.24%, 43.25%, 52.69%, and 38.23% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the G2 group. The level of IL-10 was altered by 70.53%, 49.25%, 60.18%, 41.54%, and 58.89% in G5, G6, G7, G8, and G9 groups, respectively as compared to the G2 group. Moreover, the level of IL-12 was decreased by 30.24%, 31.67%, 29.82%, 45.77%, and 50.54% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the G2. The level of IL-17 was reduced by 48.75%, 59.61%, 59.28%, 62.49%, and 58.65% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the G2. IFN-γ expression was reduced by 49.56%, 24.09%, 23.7%, 56.98%, and 44.94% in G5, G6, G7, G8, and G9 groups, respectively than G2. Overall, the data suggested anti-inflammatory potentials of the Biofield Energy Treated test formulation and Biofield Energy Treatment per se along with preventive measure on the animal with respect to various inflammatory conditions that might be beneficial various types of systemic inflammatory disorders specially sepsis, trauma, septic shock or any types of injuries. Therefore, the results showed the significant slowdown the inflammation-related disease progression and its complications in preventive treatment groups viz. G6, G7, G8, and G9.
Jun 2021 DOI 10.14302/issn.2574-4488.jna-21-3847
The aim of this study was to evaluate the impact of Biofield Energy Treated/Blessed Proprietary Test Formulation and Biofield Energy Treatment/Blessing per se on kidney biomarkers on L-NAME and high fat diet (HFD)-induced cardiovascular disorders in Sprague Dawley rats. In this experiment, the functional kidney biomarkers such as epinephrine/adrenaline, inducible nitric oxide synthase (iNOS), angiotensin-II, C-reactive protein (CRP), and renin were measured using ELISA assay. A test formulation was formulated including minerals (magnesium, zinc, copper, calcium, selenium, and iron), vitamins (vitamin C, vitamin B6, vitamin B12, vitamin B9, and vitamin D3), cannabidiol (CBD) isolate, Panax ginseng extract, and β-carotene. The components of the test item were divided into two; one section was defined as the untreated test formulation, while the other part and three group of animals received Mr. Mahendra Kumar Trivedi’s Biofield Energy healing/Blessing remotely for about 3 minutes. The results showed that the level of adrenaline was reduced by 31.62%, 19.58%, 34.32%, 37.07%, and 29.87% in the G5 (L-NAME + HFD + the Biofield Energy Treated test formulation), G6 (L-NAME + HFD + Biofield Energy Treatment per se to animals from day -15), G7 (L-NAME + HFD + the Biofield Energy Treated test formulation from day-15), and G8 (L-NAME + HFD + Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day-15), and G9 (L-NAME + HFD + Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively as compared to the disease control group (G2). Moreover, the level of iNOS was reduced by 56.76%, 49.51%, 61.79%, 57.63%, and 62.44% in the G5, G6, G7, G8, and G9 groups, respectively, as compared to the disease control group (G2). Additionally, the level of angiotensin-II was decreased by 41.09%, 34.92%, 60.65%, 53.28%, and 60.09% in the G5, G6, G7, G8, and G9 groups, respectively, as compared to the G2 group. The level of CRP was decreased by 47.21%, 38.89%, 59.81%, 55.52%, and 64.02% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the G2 group. Besides, the level of renin was decreased by 20.27%, 20.13%, 12.99%, and 25.73% in the G5, G7, G8, and G9 groups, respectively as compared to the G2 group. Overall, the data suggested significance improvement of vital functional kidney biomarkers of the Biofield Energy Treated/Blessed test formulation and Biofield Energy Treatment per se along with preventive measure on the animal with respect to various pathological conditions that might be beneficial various types of cardiovascular disorders. Therefore, the results showed the significant slowdown the inflammation-related cardiovascular disease progression and its complications/symptoms in the preventive Biofield Energy Treatment group per se and/or Biofield Energy Treated/Blessed Test formulation groups (viz. G6, G7, G8, and G9).
Mar 2021 DOI 10.14302/issn.2575-1212.jvhc-21-3767
Antibodies and antibody fragments, especially single-domain antibodies known as nanobodies, are important tools in diagnostics, research, and therapeutics. In a conventional antibody, light and heavy chains contribute to the formation of the antigen binding site. In addition to conventional antibodies, old and new world camels also have heavy-chain antibodies (hcAbs), which lack the light-chain antibodies that usually bind to the antigen, as well as single domain antibodies, the VHH domain, which are the smallest antigen-binding fragments and have high solubility, stability, and specificity. A VHH library against E. coli lipopolysaccharide (LPS) was produced using the camel immune system. E. coli strains from dead camel calves were isolated to extract the LPS and used to immunize a 2-year-old female camel. After isolating mononuclear lymphocytes for RNA extraction and amplification of the VHH gene, the PCR product was cloned into the pF1AT7 Flexi vector and transformed into JM109 E. coli competent cells by heat shock, resulting in a comprehensive VHHs library with 6.9 × 104 cfu/µg. The VHHs were expressed and screened with ELISA and PCR. Eleven colonies were positive by PCR, six of which were sequenced and submitted to Genbank compared with GenBank data to confirm the production of nanobodies with a similarity >90%.
Oct 2020 DOI 10.14302/issn.2576-6694.jbbs-20-3517
Background of the Study African countries are facing frequent blackout. Thus in sub Saharan region, due to frequent power cut, the laboratory professionals find sometimes difficulty to carry out earlier diverse diagnostic tests. Objective The aim of this work is to evaluate the feasibility of enzyme immunoassay tests in the absence of a conventional source of electricity. Methods We developed a battery-powered experimental device, which was then applied to diagnose measles. The samples included 45 sera randomly selected from non-haemolysed serum samples received and stored at the National Public Health Laboratory of Benin. The experimental device is composed of two devices (Devices 1 and 2). The Device 1 provided an average temperature of 34.47 °C, 20 min after starting. With Device 2 an average temperature of 20.32 °C is obtained 15 min after starting. Results With the experimental device the same rate of measles antibody-positive sera (44.68%) was obtained as recorded from the test using the standard equipment of laboratory. The experimental device detected 18 negative and 8 intermediate results against respectively 19 and 7 by the standard equipment. The analysis of the results of both equipments shows a concordance rate of 93.33% with a kappa reproducibility coefficient of 0.89. Conclusion The device conceived in our study is a simply equipment allowing the realization of the enzyme immunoassay tests, in this case the ELISA anti-measles test. The rate of concordance obtained shows that this device can be used with commercial kits and at temperatures close to those recommended by the manufacturer without altering the results.
May 2020 DOI 10.14302/issn.2575-7881.jdrr-20-3343
Background Anemia of chronic disease is anemia found in certain chronic disease states, is typically marked by the disturbance of iron homeostasis or hypoferremia. Chronic renal failure is currently known as Chronic Kidney Disease (CKD) or Chronic Renal Insufficiency (CRI) implies long-standing, progressive and irreversible renal parenchyma disease resulting in diminished renal function up to 40 to 60%. Often, chronic kidney disease is diagnosed as a result of screening of people known to be at risk of kidney problems, such as those with high blood pressure or diabetes and those with a blood relative with chronic kidney disease. This disease may also be identified when it leads to one of its recognized complications such as cardiovascular disease, anemia, or pericarditis. Methods Sysmex kx21 used to CBC and the Cobase411 used to iron profile. Enzyme-Linked immunoassay (ELISA) was used to determine the level of serum hepcidin. Sample preparation and PCR detection of HAMP DNA Polymorphisms: Restriction digestion of PCR products was done using Fast Digest. (Figure 1). Results Serum hepcidin levels higher in patients with anemia of chronic kidney disease compared with healthy controls mean. The polymorphisms of the hepcidin gene promoter in Sudanese patients with ACKD showed that the hepcidin HAMP AA genotype 70, AG 23, and GG 7 in 100 patients dialysis-dependent and AA 83, AG 17 and GG 0, and the allele A are more frequent in patients affected by ACKD. Significant statistical association observed between the hepcidin level and end-stage kidney disease. Conclusion This study evaluates for the first time the association between anemia of chronic kidney disease and hepcidin genes promoter polymorphisms and show that the hepcidin HAMP AA genotype and the allele A are more frequent in patients affected by ACKD, further investigation is needed, our data support the hypothesis and hepcidin HAMP are important in the pathophysiology of ACKD.
Apr 2020 DOI 10.14302/issn.2372-6601.jhor-20-3189
This article has been retracted on 29 January 2021. VIEW THE RETRACTION NOTICE (https://doi.org/10.14302/issn.2372-6601.jhor-25-5854) In Côte d'Ivoire, sickle cell disease affects 14% of the population. It is responsible for significant morbidity and mortality. Transfusion is a significant element in the management of major sickle cell anemia, which exposes them to post-transfusion hemochromatosis. The biological diagnosis is based on the determination of serum iron and the transferrin saturation coefficient (CST). As the determination of the CST was not available in our exercise context in Côte d'Ivoire, we determined only the ferritinemia. The interest of this work lies in the therapeutic implication linked to the identification of patients at risk of hemochromatosis because chelators are difficult to access for most patients. This was a prospective, descriptive and analytical study, on polytransfused sickle cell patients, followed at the transfusion therapy unit (UTT) of the CNTS of Abidjan, from 2010 to 2018. We included 78 sickle cell patients, all ages and genders who have received at least ten transfusions. The ferritinemia assay was carried out by ELISA. Transfusion exchange, with 59% of cases, was the most used mode of transfusion. The mean ferritinemia was 1719.19 ng / ml. Hyperferritinemia was found in 63% of patients. Most of the patients were on a long-term transfusion program with an average of 27.5 bags of red blood cell concentrates. Thirty-two patients had received at least 20 bags of red blood cell concentrates. We noted 21 patients treated, including 3 with deferoxamine and 18 treated with oral deferasirox. We have identified 33 sickle cell anemia patients at risk for hemochromatosis. The determinants of the risk of hemochromatosis were the high number of blood bags and the method of transfusion.
Jan 2020 DOI 10.14302/issn.2372-6601.jhor-20-3186
Introduction The anti-HCV RIBA test verifies the presence of anti-HCV serum antibodies detected by the Elisa test. In Côte d'Ivoire, screening for hepatitis C is done exclusively by enzyme immunoassays. In order to reduce the number of HCV positive blood donor exclusions on ELISA, we conducted this study which aimed to demonstrate the value of the RIBA test in confirming diagnosis of viral hepatitis C to blood donors. Methods Our study, which took place from 02 to 23 February 2008 in the laboratory of Abidjan NBTC, focused on 200 sera of blood donors anti-HCV positive (Elisa test) selected according to the ratio. The DECISCAN HCV PLUS confirmation test of BIORAD was used. Results Among the 200 HCV samples positive by EIA, 49% (98/200) were confirmed positive. RIBA gave an indeterminate result in 40% of cases (80/200); and negative in 11% of cases (22/200) corresponding to false ELISA devices. In RIBA 96 samples had a low ELISA ratio of which 21% (20/96) were RIBA negative, and 79% (76/96) were indeterminate. RIBA positive samples (98/200) had a high ratio in 82% of cases (80/98). The presence of NS3 (C33) and NS4 (C100) was noted in 100% of cases (98/98, C2 in 37% (36/98) of cases and C1 in 18% of cases (18/98). RIBA indeterminate noted the presence of NS3 in 98% of cases (78/80) and NS4 in 30% of cases (24/80). Proteins C1, C2 and NS4 are essential for the diagnosis of confirmation of viral hepatitis C by RIBA. Conclusion These results attest to the lower specificity of enzyme immunoassays (ELISAs); hence the benefit of using RIBA confirmatory tests. A significant number of donors are excluded from blood donation in Côte d'Ivoire on the basis of false positive results obtained by the ELISA technique.
Jan 2020 DOI 10.14302/issn.2474-9273.jbtm-19-3157
Sleep biomarkers in brain such as melatonin, BDNF (Brain-derived neurotrophic factor), PGD2 (Prostaglandin D2), leptin, orexin-A, and acetylcholine were evaluated in the unpredictable chronic stress (UCS) rodent model in the presence of Consciousness Energy Healing Treated (the Trivedi Effect®) novel test formulation in male Sprague Dawley (SD) rats using ELISA assay. The test formulation was consisted of minerals (Zn, Fe, Cu, Se, Ca, Mg), vitamins (C, E, B6, B12, D3), β-carotene, ginseng, and cannabidiol isolate. The test formulation constituents were divided into two parts, one part of each ingredient was distinct as the untreated test formulation, while the other portion of the test formulation and a group of animals received Biofield Energy Healing Treatment by a renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi. The level of melatonin in groups viz. G5 (Biofield Energy Treated Test formulation) and G7 (15-days pre-treatment of Biofield Energy Treated Test formulation) was significantly increased by 17.6% (p≤0.01) and 16%, respectively as compared with the disease control group (G2). Brain-derived neurotrophic factor (BDNF) level in brain was increased by 5.2% in G7 group as compared with the G4. Prostaglandins D2 (PGD2) level was significantly (p≤0.001) increased by 12.7%, 18.1%, 23.7%, and 30.7% in the G6, G7, G8 (15 days pre-treatment of Biofield Energy Treated Test formulation to the Biofield Energy Treatment per se rats), and G9 (untreated test formulation to the Biofield Energy Treatment per se to the rats) groups, respectively as compared with the G2. The level of leptin after Biofield Energy Treatment and with the test formulation was altered. However, orexin-A level was significantly decreased by 37.1% (p≤0.05), 32.6%, 40.5% (p≤0.05), 44.4% (p≤0.05), and 28.2% in the G5, G6, G7, G8, and G9 groups respectively, as compared with the G2. Similarly, acetylcholine (Ach) level was significantly (p≤0.001) decreased by 42.5%, 49.2%, 40.1%, 47.9%, and 45% in the G5, G6, G7, G8, and G9 groups, respectively as compared with the G2. Overall, the results showed the significant slowdown the stress-related disease progression and its complications/symptoms in the preventive in the Biofield Energy Treatment group per se and/or Biofield Energy Treated Test formulation groups (viz. G6, G7, G8, and G9) comparatively with the disease group. The Trivedi Effect® showed increased level of melatonin and decreased levels of insomnia related brain biomarkers which might be helpful to induce better sleep in human.
Mar 2019 DOI 10.14302/issn.2379-7835.ijn-19-2639
Nine healthy individuals with a mean ± SD BMI of 22.0 ± 0.7 kg/m² and age of 20 ± 0.2 years, participated in this single-blind randomised, crossover trial investigating the impact of ingesting two different honeys (1) Tropical Forest Honey (TFH) and (2) Manuka Honey; strength 12+ (MAN) on circulating levels of plasma interferon gamma following ex-vivo lipopolysaccharide (LPS) stimulation. Blood samples were prepared into duplicate aliquots of whole blood (800 μl) and 100 μg/l of LPS was added to samples to give a final volume of 1 ml. Levels of IFN-γ in plasma fractions were measured via commercially available sandwich ELISA and all comparisons were made with paired data using the Wilcoxon Signed Rank test taking a significance level of 5%. Whilst significant intra-and-interpersonal variation was observed, IFN-γ concentrations remained statistically unchanged 48 hours after the ingestion of either honey (p=0.15). Thus, in this instance the type of honey did not influence the IFN-γ response to plasma samples spiked with LPS.
Dec 2018 DOI 10.14302/issn.2575-1212.jvhc-18-2487
Parasitic infection by the Fasciola hepatica (F. hepatica) promotes susceptibility towards other infections, such as Mycobacterium bovis. As consequence, could affect diagnostic tests for this disease. Hence, the objective of this study was to assess the impact of F. hepatica coinfection on the most commonly used immunodiagnostic bovine tuberculosis (bTB) tests in field conditions in an enzootic area for both diseases. Thus, from a dairy herd located in Hidalgo State, México, displaying a 59.2% and 28% prevalence of fascioliasis and bTB, respectively. Sixty-one cows were analyzed based on their response towards bTB immunodiagnostic tests, such as Single Intradermal Comparative Tuberculin Test (SICTT), gamma-interferon test (BOVIGAM) and enzyme-linked immunosorbent assay (ELISA), along with the assessment of the F. hepatica parasite load and serodiagnosis by ELISA. Three study groups were formed according to test results. Group 1: coinfected (n=22). Group 2: non-parasitized cows, and positive for bTB tests (n=13) and Group 3: parasitized cows without tuberculosis (n=26). In addition, a group of cows kept in fascioliasis - and tuberculosis-free zones were included (Group 4, n=10). A non-parametric Kruskal-Wallis test and a Dunn test were applied to analyze the results. In Group 1, significant differences were observed regarding IFN-γ production, but not for antibody levels to M. bovis or reactivity towards bovine PPD in relation Group 2. While, Groups 1 and 3 did not display difference in antibody levels against F. hepatica. Differences were observed regarding tuberculosis and Fasciola diagnostic tests when both coinfected and infected groups were compared to controls. It is concluded that F. hepatica coinfection in tuberculous animals studied, depressed the production of IFN-γ towards bovine PPD under in vitro conditions, but its reactivity to the SICTT not show to be altered.
Jan 2018 DOI 10.14302/issn.2578-8590.ipj-17-1910
Following ocular trauma and retinal detachment, gliotic changes in the retina may develop over the subsequent month, a process known as PVR (proliferative vitreoretinopathy). There have been no successful therapeutic interventions to inhibit PVR. The protein CTGF (Connective Tissue Growth Factor) has been associated with retinal PVR and other fibrotic diseases of the retina in clinical studies but the mechanistic link between different pathologies and retinal gliosis has not been determined. In addition, CTGF has been previously noted to be associated, in some cases, with YAP/TAZ (Yes-associated protein and Tafazzin protein complex), transcriptional regulatory proteins that change subcellular localization in response to mechanical cues, such as the stiffness of the underlying material. We have previously shown that the mRNA for CTGF is markedly (100-fold) upregulated in retinal Müller cells grown on soft substrates. In order to evaluate if the mechanism by which mechanotransduction modulating CTGF production in retinal Müller cells involves the YAP/TAZ complex, this study tests the influence of substrate stiffness on the time dependence of CTGF protein expression, as well as subcellular localization of YAP/TAZ using a conditionally-immortalized mouse retinal Müller cell line plated on laminin-coated, polyacrylamide substrates of varying elastic modulus. Changes were assayed using immunohistochemistry and ELISA (Enzyme-Linked ImmunoSorbent Assay). In retinal Müller cells, the relationship between elastic modulus and the pattern of CTGF protein expression was bimodal, with CTGF levels rising more rapidly for cells on hard substrates and more slowly for cells grown on soft substrates. In addition, nuclear localization of YAP/TAZ corresponded directly to the maximum CTGF expression.
Sep 2017 DOI 10.14302/issn.2577-137X.ji-17-1736
Some strains of Foot and mouth disease virus (FMDV) are endemic in Egypt. The present study was performed on cattle and buffaloes (ages: 3 months up to 1.5 years old, of years 2015 and 2016), which were suffering foot and mouth disease (FMD). Sera and tissues samples were tested by different techniques including serum and virus neutralization tests (SNT, VNT), virus isolation and identification by tissue culture methods, Enzyme linked immune-Sorbent Assays (ELISA); and by the pathological and hematology techniques. The results showed the predominance of FMDV serotype O with the presence of serotypes SAT2 and A. The results showed the pathologic picture of FMD was similar regardless its specific subtypes, as apparently the studied strains produces same pathological and hematological changes. Microscopic examination reveals severe hydropic degenerations and necrosis in most affected organs, accompanied by significant changes in blood parameters which indicate severity and direct effects of FMDV on the hematopoietic system. These findings indicates the mode of pathogenesis of FMD virus in its way to exhibits the characteristic symptoms of illness. However, the investigation showed the presence of FMDV type O, A and SAT2 in the studied areas of delta governorates. It is important to focus on producing of vaccines which have only these serotypes as solution to get rid of the endemic behavior of FMDV in delta of Egypt.
Aug 2017 DOI 10.14302/issn.2575-1212.jvhc-17-1661
Serum samples from wild and domestic South American Camelids (SAC) from Argentina, collected before (2008), during (2009) and after (2010) the 2009 H1N1 influenza pandemic were tested by hemagglutination-inhibition assay (HIA) to evaluate the seroprevalence of antibodies (Ab) against different subtypes of influenza A viruses: A(H1N1)pdm09, A/sw/Argentina/SIV/2009(H3N2) and A/eq/Argentina/97(H3N8). For A(H1N1), an ELISA using a recombinant H1-hemmaglutinin from a reference strain (HA0 PuertoRico/8/1934) was also conducted. Serum samples from Guanacos (126), vicugnas (21) and llamas (100) from Jujuy, Mendoza and Río Negro provinces were analyzed; no clinical signs of respiratory disease were detected, reason for which no nasal swabs were obtained. No seropositive reactors to H3N2 nor H3N8 variants were detected, nevertheless high incidence of Ab reactive to A(H1N1)pdm09 were found by HIA; results which were confirmed by ELISA. The Ab seropositive animals to H1-like IAV found in llamas from Jujuy, and Mendoza (2009) were 78% and 86% by HIA and ELISA, respectively. Thirty-seven samples taken over the three years from guanacos kept in captivity in Rio Negro showed 62% of seropositive animals, while wild guanacos from Mendoza sampled in 2010 showed 36% seropositive animals to H1-like IAV, by both techniques. Finally, wild vicugnas from Jujuy, sampled in 2008 showed 38% and 52% seropositive animals to H1-like IAV by HIA and ELISA, respectively. Our results could indicate the potential role of these species as a reservoir of this zoonotic viral agent of high impact in Public Health, and may suggest that SAC populations might have been infected with an influenza strain antigenically related to H1 IAV. . Surprisingly, for llama and guanaco populations sampled over time in Jujuy and Río Negro, respectively, the HIA and ELISA geometric mean Ab titers (GMT) for 2008 were significantly higher than the ones of 2010. In addition, HIA and ELISA Ab titers found in domestic llamas were significantly higher than those detected in wild vicugnas sampled during that year (2008) in Jujuy. New field campaigns are in progress to collect serum samples and nasal swabs in order to isolate and characterize the virus responsible for triggering H1 reactive Abs. These findings remark the need to better understand the dynamics and ecology of influenza A virus within Sacs populations.
Aug 2017 DOI 10.14302/issn.2638-4469.japb-17-1563
GAGA-binding proteins in plants are encoded by the BARLEY B-RECOMBINANT / BASIC PENTACYSTEINE (BBR/BPC) family, which can be spilt into several groups on the basis of sequence divergence. The proteins of the different groups share an evolutionary conserved BASIC PENTACYSTEINE (BPC) domain at their very C-terminus that is important for DNA binding. Hallmark of this domain are five Cysteines at defined positions and spacing, which are considered to form a zinc-finger like structure that is involved in GAGA-motif recognition. Here, we report the formation of stabile homodimers between Arabidopsis thaliana group I member BPC1 or between group II member BPC6 in SDS-PAGE. Serial mutations of the highly conserved five Cysteines in the BPC domain of Arabidopsis thaliana BPC1 were tested for their capacity to bind to GAGA-motifs by DPI-ELISA. Our results do not support the idea of a direct involvement of these residues in making physical contact with the DNA, e.g. by formation of a zinc-finger structure. Instead, the data implies an indispensable function for the five Cysteines in homodimerization and stabilization of the protein structure by disulfide bonds. Accordingly, protein folding and structure prediction suggests the formation of a scaffold for dimerization that is supported by three intermolecular and one intramolecular S-S bond. The high degree of conservation between the BPC domains from the different groups and from different species denotes that this role for the five Cysteines might be evolutionary retained.
Jan 2016 DOI 10.14302/issn.2379-8572.joa-15-814
Background and Objective: The etiological factors for nasal polyps include infection, inflammation or an imbalance of a metabolic pathway. This study was designed to compare serum Helicobacter pyloriantibodies and H. pylori–DNAs between cases of nasal polyp and controls (nasal fracture). Patients and Methods: This case control study was carried out in ENT Department of Rasul Hospital in Tehran (2007-2008), upon nasal polyp tissues in 62 cases and inferior nasal turbinate mucosa in 25 controls. H. pylori–DNAs were searched by qualitative polymerase chain reaction (PCR) and serum specific H. pylori antibodies (ELISA IgG and IgA). Comparative tests were performed for the 2 groups, and P value < 0.05 was considered as statistically significant. Results: The mean age of cases and controls were 37.5 ± 13.7 and 31 ± 11.5 years, respectively. H. pylori–DNA was found in 32.3% (20/62) of the cases and 4% (1/25) of the controls (P value = 0.005). Serum H. pylori antibody (IgA) was found in 14.5% (9/62) of the cases and 4% (1/25) of the controls (P value = 0.27). However, previous immunity (IgG) was higher in 71% of the cases and 32% of the controls (P = 0.001). Conclusion: H. pylori infection may play a key role in the formation of nasal polyps. We recommend the PCR as the best method of searching for H. pylori infection. However, from the data obtained in this investigation it could not be determined whether or not H. pylori play a pathogenic role. Long-term antibiotics treatment in cases with nasal polyp, especially in cases with severe chronic rhinosinusitis where patients do not respond to surgery or steroids, may be useful. More randomized controlled trial (RCT) studies are necessary to validate the role of H. pylori infection in nasal polyp and the effect of antibiotics for eradication of H. pylori infection.
Apr 2014 DOI 10.14302/issn.2377-2549.jndc-13-329
The aim of the study was to synthesize sub-100nm poly-ε-caprolactone nanoparticles (PCL NP), load them with the mycobacterial protein, ESAT 6 and study the resulting immune responses in CD4+ and CD8+ T cells when incubated with human peripheral blood monocyte derived macrophages that had internalized the PCL NP. The synthesized PCL NP were characterized for size, shape and charge. They were found to be about 60nm in size with spherical shape. MTT assay revealed that the particles were perfectly biocompatible when tested in vitro on THP1 human monocytic cell line. The particles had a slow protein release kinetics and did not degrade appreciably even after 30 days in buffer solution. ELISA was used to quantify the cytokine response of CD4+ and CD8+ T cells when incubated with the monocyte derived macrophages as antigen presenting cells. The result of antigen presentation assay revealed that the antigen loaded PCL NP enhanced Th1 and CD8+ T cell responses significantly compared to the pure antigen. Thus we conclude that PCL NP of 60nm size can be effectively tested as a vaccine adjuvant with resulting activation of Th1/Th2 immunity as well as cytotoxic T cell response.