Development of a New Immunosensor for the Detection of Dopamine

2007 ◽  
Vol 62 (7-8) ◽  
pp. 613-618
Author(s):  
Dina Fouad

Graphite immunoelectrodes as immunosensors using indirect immobilization of a hapten were investigated for their applicability to detect dopamine hydrochloride at low levels. Conditions were optimized to achieve the highest sensitivity using the indirect immobilization of dopamine hydrochloride through a polymerized glutaraldehyde network on microtiter plates using ELISA technique. The conditions were later transferred to the graphite rods (ø 0.8 m × 20 mm) and a comparison between the two different sensitivities (IC50 midpoint of test) was carried out. Graphite electrodes showed higher sensitivity towards dopamine than ELISA, since they were able to detect dopamine with a midpoint of test of 0.2 mmol/l while using ELISA they were able to detect dopamine hydrochloride at 2 mmol/l

1994 ◽  
Vol 71 (01) ◽  
pp. 129-133 ◽  
Author(s):  
P J Declerck ◽  
S Vanderschueren ◽  
J Billiet ◽  
H Moreau ◽  
D Collen

SummaryStreptokinase (SK) is a routinely used thrombolytic agent but it is immunogenic and allergenic; staphylokinase (STA) is a potential alternative agent which is under early clinical evaluation. The comparative prevalence of antibodies against recombinant STA (STAR) and against SK was studied in healthy subjects and their induction with intravenous administration in small groups of patients.Enzyme-linked immunosorbent assays, using microtiter plates coated with STAR or SK and calibration with affinospecific human antibodies, revealed 2.1 to 65 μg/ml (median 11 μg/ml) anti-STAR antibodies and 0.9 to 370 μg/ml (median 18 μg/ml) anti-SK antibodies (p <0.001 vs anti-STAR antibodies) in plasma from 100 blood donors, with corresponding values of 0.6 to 100 μg/ml (median 7.1 μg/ml) and 0.4 to 120 μg/ml (median 7.3 μg/ml), respectively, in 104 patients with angina pectoris. Three out of 17 patients with Staphylococcus aureus bacteremia had significantly increased anti-STAR antibody levels (150, 75 and 75 μg/ml), and STAR neutralizing activities (2.2, 3.6 and 4.1 μg STAR neutralized per ml plasma, respectively). In 6 patients with acute myocardial infarction, given 10 mg STAR intravenously over 30 min, median anti-STAR antibody levels were 3.5 μg/ml at baseline, 2.9 μg/ml at 6 to 8 days and 1.2 μg/ml at 2 to 9 weeks, with median corresponding titers of STAR neutralizing activity at 2 to 9 weeks of 42 μg/ml plasma. Conversely, in 5 patients treated with 1,500,000 units SK over 60 min, median anti-SK antibodies increased from 2.9 μg/ml at baseline to 360 μg/ml at 5 to 10 days, with corresponding median SK neutralizing activities of 13 μg/ml. Antibodies against STAR did not cross-react with SK and vice versa.Plasma from human subjects contains low levels of circulating antibodies against recombinant staphylokinase, and intravenous administration of this compound boosts antibody titers. These antibodies do however not cross-react with streptokinase, whereby the use of these two immunogenic thrombolytic agents would not be mutually exclusive.


1996 ◽  
Vol 76 (06) ◽  
pp. 1090-1095 ◽  
Author(s):  
C Ravanat ◽  
M Freund ◽  
S Schuhler ◽  
P Grunert ◽  
L Meyer ◽  
...  

SummaryThe purpose of this study was to develop specific and sensitive immunoassays to detect early indices of hypercoagulability in the rat. Rat platelet factor 4 (rPF4) and rat fibrinopeptide A (rFPA) assays, tools for the detection of activation of platelets and coagulation respectively, were designed using antibodies raised against purified rPF4 and against synthetic rFPA. The relevance of these new assays and of the commercially available ELISA kit for thrombin-antithrombin III (TAT) complexes was demonstrated in a rat model of a prethrombotic state induced by intravenous infusion of varying doses of thromboplastin (90 to 2400 μl/kg/h). In this model, the immunoassays allowed simultaneous detection of low levels of rFPA and rPF4 which were correlated with fibrinogen and platelet consumption and TAT generation and further proved to be of higher sensitivity than the classical methods of platelet count or measurement of fibrinogen levels. Plasma concentrations of rFPA, rPF4 and TAT were dependent on infusion time and thromboplastin dose, while hirudin (1 mg/kg) prevented their appearance. Thus the new specific immunoassays for rPF4 and rFPA and the commercial human TAT assay represent useful tools for pathophysiological studies or the screening of antithrombotic drugs in rats.


2006 ◽  
Vol 61 (3-4) ◽  
pp. 295-301
Author(s):  
Dina Fouad ◽  
Iris Sound ◽  
Heike Holthues ◽  
Ursula Pfeifer-Fukumura ◽  
Abbas M. Hammam ◽  
...  

An indirect format enzyme-linked immuno-sorbent assay (ELISA) on graphite rods (Ø 0.8 mm x 20 mm) and, for comparison, on microtiter plates has been developed against terbuthylazine. For this purpose, a series of 2-aminoalkyl-4-chloro-6-terbuthyl-s-triazine-2,6- diamine ELISA haptens with alkyl spacer lengths of 2, 4, 6, and 8 CH2 groups has been synthesized. The graphite rods or the microtiter plates were covered by a polymerized glutaraldehyde network, and the ELISA haptens have been coupled by imino coupling to the free aldehyde groups of that network. ε-Aminocaproic acid has been used as an agent to block unspecific binding sites. The ELISA was run in a competitive mode, where the immobilized ELISA hapten and the solute terbuthylazine as a target analyte compete for the solute antibody.


2021 ◽  
Vol 6 (1) ◽  
pp. 6-12
Author(s):  
Ziyad M. Bilal ◽  
◽  
Kedir S. Musa ◽  

Cestode infestations in animals are the most important parasite of livestock and humans because most of these parasites are zoonotic causing cysticercosis and hydatidosis in man and it causes economic and production losses in livestock. Diagnosis of Taenia Spp by microscopic observation lack sensitivity and specificity and detection by enzyme-linked immunosorbent assay (ELISA) technique form cross-reaction. The molecular diagnostic can be best to detect in adult and larval stage in definitive and intermediate host based on the amplification of deoxyribonucleic acid (DNA) of target gene with the primer using a different technique of polymerase chain reaction (PCR) such as multiplex PCR. Conventional PCR, real-time PCR, nested PCR, and PCR-restriction fragment length polymorphism (RFLP) are highly sensitive for the diagnosis of cestode and metacestode. Those diagnoses are used for differentiation of Taenia species and differentiation of Taenia and Echinococcus species. As compared to other diagnostic techniques most molecular methods have higher sensitivity and specificity but due to the relatively higher cost, few are commercially available. Most of the molecular diagnostic tests developed to date are generally applicable for laboratory research purposes. The developments in the genomic and proteomic analysis should be used for further understanding of parasite-animal host interaction to find additional targets for diagnosis.


Biosensors ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 524
Author(s):  
Tania García-Maceira ◽  
Fé I. García-Maceira ◽  
José A. González-Reyes ◽  
Luis A. Torres-Sánchez ◽  
Ana Belén Aragón-Gómez ◽  
...  

Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces.


2017 ◽  
Vol 2017 ◽  
pp. 1-22 ◽  
Author(s):  
Iulia Gabriela David ◽  
Dana-Elena Popa ◽  
Mihaela Buleandra

Due to their electrochemical and economical characteristics, pencil graphite electrodes (PGEs) gained in recent years a large applicability to the analysis of various types of inorganic and organic compounds from very different matrices. The electrode material of this type of working electrodes is constituted by the well-known and easy commercially available graphite pencil leads. Thus, PGEs are cheap and user-friendly and can be employed as disposable electrodes avoiding the time-consuming step of solid electrodes surface cleaning between measurements. When compared to other working electrodes PGEs present lower background currents, higher sensitivity, good reproducibility, and an adjustable electroactive surface area, permitting the analysis of low concentrations and small sample volumes without any deposition/preconcentration step. Therefore, this paper presents a detailed overview of the PGEs characteristics, designs and applications of bare, and electrochemically pretreated and chemically modified PGEs along with the corresponding performance characteristics like linear range and detection limit. Techniques used for bare or modified PGEs surface characterization are also reviewed.


Biosensors ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 36
Author(s):  
Florina Pogacean ◽  
Codruta Varodi ◽  
Maria Coros ◽  
Irina Kacso ◽  
Teodora Radu ◽  
...  

A graphene sample (EGr) was prepared by electrochemical exfoliation of graphite rods in solution containing 0.05 M (NH4)2SO4 + 0.1 M H3BO3 + 0.05 M NaCl. The exfoliation was performed by applying a constant voltage (12 V) between the graphite rods, while the temperature was kept constant (18 °C) with a temperature-controlled cryostat. The structural investigation of the graphene sample, performed by X-ray powder diffraction (XRD), revealed that the sample consists of a mixture of few-layer (69%), multi-layer graphene (14%) and graphene oxide (17%). In addition, XPS analysis proved that the sample was triple-doped with heteroatoms such as nitrogen (1.7 at%), sulfur (2.5 at%), and boron (3 at%). The sample was deposited onto the surface of a clean, glassy carbon electrode (GC) and investigated for the non-enzymatic electrochemical detection of L-tryptophan (TRP). The electrocatalytic properties of the EGr/GC electrode led to a considerable decrease in the oxidation potential from +0.9 V (bare GC) to +0.72 V. In addition, the EGr/GC electrode has higher sensitivity (two times) and a lower detection limit (ten times) in comparison with the bare GC electrode.


Sensors ◽  
2019 ◽  
Vol 19 (19) ◽  
pp. 4297 ◽  
Author(s):  
Codruta Varodi ◽  
Florina Pogacean ◽  
Maria Coros ◽  
Marcela-Corina Rosu ◽  
Raluca-Ioana Stefan-van Staden ◽  
...  

In this work we present the preparation of graphene material by exfoliation of graphite rods via pulses of current in electrolyte, containing a mixture of boric acid (0.05 M) and sodium chloride (0.05 M). The material was morphologically and structurally characterized by SEM/TEM/HR-TEM, XRD and FTIR techniques. TEM investigation of graphene flakes deposited onto carbon-coated grids allowed the visualization of thin and transparent regions, attributed to few-layer graphene (FLG), as well as thick and dark regions attributed to multi-layer graphene (MLG). The mixed composition of the material was additionally confirmed by XRD, which further indicated that the amount of FLG within the sample was around 83%, while MLG was around 17%. The performance of a screen-printed electrode (SPE) modified with graphene (SPE-Gr) was tested for 8-hydroxy-2′-deoxyguanosine detection. The graphene-modified electrode had a higher sensitivity in comparison with that of SPE, both in standard laboratory solutions (phosphate buffered saline—PBS) and in human saliva.


Author(s):  
Debashis Dutta ◽  
Naoki Yanagisawa

Enzyme-linked immunosorbent assays (ELISA) are critically important tools in biological research, allowing the presence and concentrations of a wide variety of key biochemical intermediates to be determined. While the signal amplification that is the core advantage of ELISA methods is impressive, it is nevertheless the case that it is insufficient for some particularly demanding challenges in terms of sensitivity, assay time, or sample size. In this paper, we discuss three different approaches developed in our laboratory that can improve the sensitivity of ELISA methods by 2–3 orders of magnitude. Two of these approaches have been shown to reduce the minimum detectable concentration of the target analyte in the system through trapping of the analyte species and the enzyme reaction product around a semi-permeable membrane. The third approach, on the other hand, focuses on reducing the sample volume requirement in these assays by implementing multiplex ELISA methods in a single microfluidic channel using the same enzyme label. This multiplex technique relies on the slow diffusion of the enzyme reaction product across adjacent assay segments for accurate quantitation and has been demonstrated to have a limit of detection substantially better than that of commercial microtiter plates. We believe the combination of these approaches could significantly extend the applicability of the ELISA technique to more challenging assays than is currently possible.


2019 ◽  
Vol 6 (7) ◽  
pp. 3286-3292 ◽  
Author(s):  
Abubakar Shettima ◽  
Muhammad M. Ibrahim

Introduction: Complement Factor H (CFH) is the major soluble regulatory protein that monitors activation and/or amplification of the complement system. Here, we assess serum levels of complement factor H (CFH) among patients, who were diagnosed with bronchiectasis. Methods: 115 subjects of 80 patients and 35 healthy volunteers were recruited for the study. Blood samples were collected and subjected to centrifugation in order to obtain the serum. The sensitive sandwich ELISA technique, specific for CFH was used for evaluation of CFH in the serum. Results: The age and Body Mass Index (BMI) (expressed as Mean+/-SEM (range)) of the observed bronchiectasis patients and healthy volunteers were 66+/-1.13, (30-86) years, 54+/-2.37 (27-84) years and 26.14 kg/m2, 27.4 kg/m2 respectively. CFH was detected in 27.0% of all subjects examined. It was found to be more common among bronchiectasis patients (18.26%) compared to healthy volunteers (8.70%) (F=0.9362; df=1; p<0.05). Mean CFH concentration was 17.0pg/ml and 9.0pg/ml in bronchiectasis patients and healthy volunteers respectively. FEV1% Pre. FEV1/VC% and CFH levels were found to decrease with increasing severity of bronchiectasis. The association between lung function, CFH levels and severity of disease among bronchiectasis patients was negative, strongly linear (r= -0.9316, r2 =0.8678) and statistically significant (p<0.0001). Conclusion: As such, it can be inferred that low level of compliment factor H is related to the progression of bronchiectasis.  


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