57 research outputs found
Development of a new hazelnut sandwich ELISA based on detection of Cor a 9, a major hazelnut allergen
The emerging health problem of food-induced allergic reactions, including life-threatening anaphylaxis, presents an important challenge to the food. In the framework of the current ALLERRISK project, analytical approaches for allergen detection are evaluated and developed. Cor a 9 is a major hazelnut protein with nutrient reservoir function, and that also known as a major food allergen. Presence of Cor a 9 indicates contamination of a product with hazelnut and, consequently, potential risk of allergenicity.
Crude extract of hazelnut was obtained from milled mixture of hazelnuts from 10 commercially available brands. Then the extract was concentrated and applied to gel filtration and Con A affinity chromatography columns for Cor a 9 isolation and purification. The final product was characterized by analytical gel permeation chromatography, SDS PAGE and MALDI mass spectrometry. The purified Cor a 9 was used as an antigen for the production of polyclonal antibodies in chickens according to the protocol developed in our laboratory. A part of the most active antibodies was further purified by gel permeation chromatography and characterized by SDS PAGE before further use. The prepared antibodies were applied for development of a sandwich ELISA using polyclonal antibody-enzyme adducts as a tracer. Optimization of reagent concentrations led us to achieve detection limit ~10 ng/ml. Influence of ionic strength, pH and buffer composition on analytical parameters of the developed ELISA were thoroughly studied. The developed ELISA can be successfully applied for quantitative detection of Cor a 9 in large pH (pH = 5 – 10) and ionic strength (0.05 – 1.5 M salts solution) ranges. Cross-reactivity with series of nuts, ovalbumin, whey, and wheat proteins was also investigated (Figure 2) and showed that the developed assay was very specific. Moreover, presence of several other proteins (peanut, cashew, ovalbumin, whey and wheat) at high concentrations (1 mg/ml) in the presence of Cor a 9 did not influence the calibration curve of Cor a 9. Detection of hazelnut protein in cookies with known amount of hazelnut inside initial dough showed good recovery (35-40%) and absence of significant signal in blank samples (i.e. without spiking of hazelnut)
Age-dependent sensitization to the 7S-vicilin-like protein Cor a 11 from hazelnut (Corylus avellana) in a birch-endemic region
Background: Hazelnut (Corylus avellana) allergy exhibits age and geographically distinct sensitization patterns that have not yet been fully resolved.
Objective: To study sensitization to Cor a 11 in different age groups of hazelnut-allergic patients and infants with atopic dermatitis (AD) sensitized to hazelnut in a birch-endemic region.
Methods: Sera from 80 hazelnut-allergic patients, 33 infants under 1 year of age with AD (24 sensitized and 9 not sensitized to hazelnut), 32 healthy control individuals, and 29 birch pollen–allergic but hazelnut-tolerant individuals were tested for immunoglobulin (Ig) E reactivity to Cor a 11 by ImmunoCAP. IgE reactivity to Cor a 1.01, Cor a 1.04, Cor a 8, and Cor a 9 was studied by ISAC microarray.
Results: Forty patients (22 preschool children, 10 schoolchildren, and 8 adults) with systemic reactions on consumption of hazelnut were sensitized to Cor a 11 (respective rates of 36%, 40%, and 12.5%). Forty patients (6 preschool children, 10 schoolchildren, and 24 adults) reported oral allergy syndrome but only 2 of them (of preschool age) were sensitized to Cor a 11. Two (8%) of the AD infants sensitized to hazelnut showed IgE reactivity to Cor a 11. This reactivity was not observed in any of the AD infants without sensitization to hazelnut, in any of the birch-pollen allergic patients without hazelnut allergy, or in any of the healthy control individuals.
Conclusion: Sensitization to Cor a 11 in a birch-endemic region is predominantly found in children with severe hazelnut allergy, a finding that is consistent with observations concerning sensitization to Cor a 9
Development of a nanobody-based amperometric immunocapturing assay for sensitive and specific detection of Toxocara canis excretory-secretory antigen
Introduction Human Toxocariasis (HT) is a zoonosis that,
despite of its wide distribution around the world, remains poorly
diagnosed. The identification of specific IgG immunoglobulins
against the Toxocara canis Excretory-Secretory antigen (TES), a
mix of glycoproteins that the parasite releases during its
migration to the target organs in infected patients, is currently
the only laboratory tool to detect the disease. The main
drawbacks of this test are the inability to distinguish past and
active infections together with lack of specificity. These factors
seriously hamper the diagnosis, follow-up and control of the
disease.
Aim To develop an amperometric immunocapturing diagnostic
assay based on single domain immunoglobulins from camelids
(nanobodies) for specific and sensitive detection of TES.
Methods After immunization of an alpaca (Vicugna pacos)
with TES, RNA from peripheral blood lymphocytes was used as
template for cDNA amplification with oligo dT primers and
library construction. Isolation and screening of TES-specific
nanobodies were carried out by biopanning and the resulting
nanobodies were expressed in Escherichia coli. Two-epitopes
amperometric immunocapturing assay was designed using
paramagnetic beads coated with streptavidin and bivalent
nanobodies. Detection of the system was carried out with
nanobodies chemically coupled to horseradish peroxidase. The
reaction was measured by amperometry and the limit of
detection (LOD) was compared to conventional sandwich
ELISA.
Results We obtained three nanobodies that specifically
recognize TES with no-cross reactivity to antigens of Ascaris
lumbricoides and A. suum. The LOD of the assay using PBST20
0.05% as diluent was 100 pg/ml, 10 times more sensitive than
sandwich ELISA.
Conclusion Sensitive and specific detection of TES for
discrimination of active and past infections is one of the most
difficult challenges of T. canis diagnosis. The main advantage of
our system is the use of two different nanobodies that
specifically recognize two different epitopes in TES with a highly
sensitive and straightforward readout. Considering that the
amounts of TES available for detection in clinical samples are in
the range of picograms or a few nanograms maximum, the LOD
found in our experiments suggests that the test is potentially
useful for the detection of clinically relevant cases of HT
Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.
peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration
of the larval stage of the roundworm Toxocara canis in the human host.
Despite of being the most cosmopolitan helminthiasis worldwide, its
diagnosis is elusive. Currently, the detection of specific immunoglobulins
IgG against the Toxocara Excretory-Secretory Antigens (TES), combined
with clinical and epidemiological criteria is the only strategy to diagnose
HT. Cross-reactivity with other parasites and the inability to distinguish
between past and active infections are the main limitations of this
approach. Here, we present a sensitive and specific novel strategy to
detect and quantify TES, aiming to identify active cases of HT. High
specificity is achieved by making use of nanobodies (Nbs), recombinant
single variable domain antibodies obtained from camelids, that due to
their small molecular size (15kDa) can recognize hidden epitopes not
accessible to conventional antibodies. High sensitivity is attained by the
design of an electrochemical magnetosensor with an amperometric readout
with all components of the assay mixed in one single step. Through
this strategy, 10-fold higher sensitivity than a conventional sandwich
ELISA was achieved. The assay reached a limit of detection of 2 and15
pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These
limits of detection are sufficient to detect clinically relevant toxocaral
infections. Furthermore, our nanobodies showed no cross-reactivity
with antigens from Ascaris lumbricoides or Ascaris suum. This is to our
knowledge, the most sensitive method to detect and quantify TES so far,
and has great potential to significantly improve diagnosis of HT. Moreover,
the characteristics of our electrochemical assay are promising for the
development of point of care diagnostic systems using nanobodies as a
versatile and innovative alternative to antibodies. The next step will be the
validation of the assay in clinical and epidemiological contexts
Nanobody-Based Immunosensor Detection Enhanced by Photocatalytic-Electrochemical Redox Cycling
peer reviewedDetection of antigenic biomarkers present in trace
amounts is of crucial importance for medical diagnosis. A parasitic
disease, human toxocariasis, lacks an adequate diagnostic method
despite its worldwide occurrence. The currently used serology tests
may stay positive even years after a possibly unnoticed infection,
whereas the direct detection of a re-infection or a still active infection
remains a diagnostic challenge due to the low concentration of
circulating parasitic antigens. We report a time-efficient sandwich
immunosensor using small recombinant single-domain antibodies
(nanobodies) derived from camelid heavy-chain antibodies specific
to Toxocara canis antigens. An enhanced sensitivity to pg/mL levels is achieved by using a redox cycle consisting of a photocatalytic oxidation and electrochemical reduction steps. The photocatalytic oxidation is achieved by a photosensitizer generating singlet oxygen (1O2) that, in turn, readily reacts with p-nitrophenol enzymatically produced under alkaline conditions. The photooxidation produces benzoquinone that is electrochemically reduced to hydroquinone, generating an amperometric response. The light-driven process could be easily separated from the background, thus making amperometric detection more reliable. The proposed method for detection of the toxocariasis antigen marker shows superior performances compared to other detection schemes with the same nanobodies and outperforms by at least two orders of magnitude the assays based on regular antibodies, thus suggesting new opportunities for electrochemical immunoassays of challenging low levels of antigens
Corrosion protection of steel by electropolymerized lignins
Electropolymerization of water soluble lignin derivatives (lignosulfonates) on steel surface is reported. It was noticed from both an appearance of anodic current of lignin oxidation and a subsequent considerable decrease in current at high anodic potentials, and confirmed by the knowledge of lignin oxidative polymerization. Ionic strength affects a potential of lignin oxidation: an increase of KCl concentration ten times (from 0.1 to 1Â M) causes a decrease in oxidation potential for 1Â V. Lignosulfonate electropolymerization is resulted in corrosion protection of carbon steel, which was more effective compared to the standard commercially available corrosion inhibitor COREXIT SXT-1003. Keywords: Lignosulfonate, Electropolymerization, Carbon steel, Corrosion protectio
Label-free detection of DNA hybridization at a liquid|liquid interface
A novel electrochemical approach for label-free detection of DNA primary sequence has been proposed. The flow of nonelectroactive ions across a liquid|liquid interface was used as an electrochemical probe for detection of DNA hybridization. Disposable graphite screen-printed electrodes shielded with a thin layer of inert polymer plasticized with water-immiscible polar organic solvent were modified by probe oligonucleotide and used as a DNA sensor. The specific DNA coupling has been detected with impedance spectroscopy by decrease of ion-transfer resistance. The detection limit was of 10(-8) M of target oligonucleotide. The reported sensor was suitable for discrimination of a single mismatch oligonucleotide from the full complementary one. The reported DNA sensor was advantageous over known physicochemical approaches, providing the most significant changes in the measured parameters
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