Highly sensitive and multiplexed platforms for allergy diagnostics

Thesis (Ph.D.)--Boston UniversityAllergy is a disorder of the immune system caused by an immune response to otherwise harmless environmental allergens. Currently 20% of the US population is allergic and 90% of pediatric patients and 60% of adult patients with asthma have allergies. These percentages have increased by 18.5% in the past decade, with predicted similar trends for the future. Here we design sensitive, multiplexed platforms to detect allergen-specific IgE using the Interferometric Reflectance Imaging Sensor (IRIS) for various clinical settings. A microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of patient blood sample. However, conventional fluorescent microarray technology is limited by i) the variation of probe immobilization, which hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics and ii) the use of fluorophore labels, which is not suitable for some clinical applications due to the tendency of fluorophores to stick to blood particulates and require daily calibration methods. This calibrated fluorescence enhancement (CaFE) method integrates the low magnification modality of IRIS with enhanced fluorescence sensing in order to directly correlate immobilized probe (major allergens) density to allergen-specific IgE in patient serum. However, this platform only operates in processed serum samples, which is not ideal for point of care testing. Thus, a high magnification modality of IRIS was adapted as an alternative allergy diagnostic platform to automatically discriminate and size single nanoparticles bound to specific IgE in unprocessed, characterized human blood and serum samples. These features make IRIS an ideal candidate for clinical and diagnostic applications, such a POC testing. The high magnification (nanoparticle counting) modality in conjunction with low magnification of IRIS in a combined instrument offers four significant advantages compared to existing sensing technologies: IRIS i) corrects for any variation in probe immobilization, ii) detects proteins from attomolar to nanomolar concentrations in unprocessed biological samples, iii) unambiguously discriminates nanoparticles tags on a robust and physically large sensor area, iv) detects protein targets with conjugated nanoparticle tags (~40nm diameter), which minimally affect assay kinetics compared to conventional microparticle tagging methods, and v) utilizes components that make the instrument inexpensive, robust, and portable. This platform was successfully validated on patient serum and whole blood samples with documented allergy profiles (ImmunoCAP®, ThermoFisher Scientific)

On-chip food safety monitoring: multi-analyte screening with imaging surface plasmon resonance-based biosensor

Food safety is an increasing health concern, recognised and promoted by many institutions across the globe. Food products can be contaminated with pathogenic microorganisms, environmental pollutants, veterinary drug residues, allergens and toxins. Public health concerns which have been raised in relation to hazardous agents found in food include, among others, increased cancer risk, endocrine, reproductive and neurobehavioral systems disruption, teratogenesis, antibiotic resistance and even death in cases of allergic reactions and acute poisoning. Some of the food hazardous agents (e.g. pathogenic microorganisms and toxins) can even be used as biological warfare, spread through food and agricultural chains. Thus, an adequate detection of these compounds is also important for biosecurity. In order to safeguard consumers’ health, legislations have been put in place both in the US and the EU. These laws specify for each health threatening compound the maximal acceptable amounts in different food products. Besides health issues, food safety and quality has an economical impact on the food industry, where quality control expenses amount to about 1.5 – 2 % of the total sales. Since more and more food products nowadays contain multiple and processed ingredients, which are often shipped from different parts of the world, and share common production lines and storage spaces, food safety and quality monitoring becomes a challenging task. Traditional analytical methods require dedicated laboratories, equipment and highly trained personnel for detection and identification of each type of hazardous agent (e.g. antibiotics, bacteria, allergens). These techniques are also time-consuming and often expensive. There is a growing need for multi-analyte screening methods, which will enable rapid and simultaneous detection of multiple compounds in complex food samples. In recent years, biosensors have been applied successfully to food analysis, incorporating the same bioassay principals as traditional methods with transducers (optical, electrochemical, etc) in novel, usually miniaturized, integrated analytical devices. However, most of these biosensors still lack the desired level of the multiplexicity. Recent developments in the field of Surface Plasmon Resonance (SPR) technology in the direction of high-throughput systems and multi-analyte measurements present a promising alternative for the existing systems. One of such systems is imaging SPR (iSPR); it enables real-time and label free read-out of spatially modified surfaces (e.g. microarrays). The aim of this study was to develop an iSPR–based biosensor, for simultaneous and quantitative detection of different health-threatening compounds in food. To obtain a comprehensive overview on the analytical applicability of such a system, several points were addressed. The intrinsic sensor properties, such as optical sensitivity and robustness, of the iSPR instrument were studied. Further on, both direct and competitive immunoassay formats for high and low molecular weight compounds detection using the iSPR platform were evaluated. Then, the iSPR-based biosensor was applied for detection of regulated substances in food such as antibiotic residues in milk and allergens in cookies and chocolates. Finally, the most common drawback of using SPR for screening in complex biological matrices, the nonspecific binding to the sensor chip surface, was tackled. The sensitivity of both high and low molecular weight compounds was proven to be sufficient for some of the hazardous agents detection at the maximum residue levels, established in the EU legislation, as was demonstrated by simultaneous detection of seven antibiotic residues in milk and twelve allergens in cookies and dark chocolates. The analysis time takes about 10 minutes and provides quantitative information on multiple targets, producing a fingerprint (allergenic fingerprint for instance) of the tested food. This detailed food profile contributes to the decision making process on the quality and safety of foods, basing it on the total picture of all target compounds present. In order for iSPR-based biosensing to reach its full potential and to become a widely applied routine analytical tool, the instrumental cost needs to be reduced and the analysis further simplified, becoming cost-effective and approachable to non-trained personnel. An additional drawback in analytical applications of a SPR sensor is the nonspecific binding of the matrix components of complex samples to the sensor surface. Many assays based on SPR fail due to inapplicability to measure in “real” samples. As a possible solution to this problem, sensor chip surface engineering was suggested in this thesis. A nanopatterned filter layer covering the sensor chip surface was found to be effective in reducing nonspecific binding when the measurements were performed in “raw” samples by keeping the non-soluble aggregates and big sample matrix components beyond the sensing region of the SPR. With respect to other existing biosensors, iSPR still lags behind in terms of sensitivity and portability. In summary, the results of this study demonstrate that iSPR-based biosensor is a versatile platform, which can be applied for a wide variety of fundamentally different analytes and offers several advantages over already existing methods. SPR detection principle eliminates the need in labelling and the instrumental set-up allows automated analysis. High multiplexing capabilities and short measurement times are obtained with no need for complex and time consuming sample preparation steps. By using iSPR-based biosensor, one can obtain robust and quantitative information on the target analyte concentration, in real time and with high specificity (or broad spectrum, depending on the assay). In conclusion, on-chip screening using iSPR, described here, presents a powerful analytical approach towards food safety and quality monitoring which satisfies the current need in rapid and multi-analytical devices. <br/

IgE allergy diagnostics and other relevant tests in allergy, a World Allergy Organization position paper

Currently, testing for immunoglobulin E (IgE) sensitization is the cornerstone of diagnostic evaluation in suspected allergic conditions. This review provides a thorough and updated critical appraisal of the most frequently used diagnostic tests, both in vivo and in vitro. It discusses skin tests, challenges, and serological and cellular in vitro tests, and provides an overview of indications, advantages and disadvantages of each in conditions such as respiratory, food, venom, drug, and occupational allergy. Skin prick testing remains the first line approach in most instances; the added value of serum specific IgE to whole allergen extracts or components, as well as the role of basophil activation tests, is evaluated. Unproven, non-validated, diagnostic tests are also discussed. Throughout the review, the reader must bear in mind the relevance of differentiating between sensitization and allergy; the latter entails not only allergic sensitization, but also clinically relevant symptoms triggered by the culprit allergen

IgE allergy diagnostics and other relevant tests in allergy, a World Allergy Organization position paper

Correction: Volume14 Issue7 Article Number100557 DOI10.1016/j.waojou.2021.100557Currently, testing for immunoglobulin E (IgE) sensitization is the cornerstone of diagnostic evaluation in suspected allergic conditions. This review provides a thorough and updated critical appraisal of the most frequently used diagnostic tests, both in vivo and in vitro. It discusses skin tests, challenges, and serological and cellular in vitro tests, and provides an overview of indications, advantages and disadvantages of each in conditions such as respiratory, food, venom, drug, and occupational allergy. Skin prick testing remains the first line approach in most instances; the added value of serum specific IgE to whole allergen extracts or components, as well as the role of basophil activation tests, is evaluated. Unproven, non-validated, diagnostic tests are also discussed. Throughout the review, the reader must bear in mind the relevance of differentiating between sensitization and allergy; the latter entails not only allergic sensitization, but also clinically relevant symptoms triggered by the culprit allergen.Peer reviewe

A helminth-derived suppressor of ST2 blocks allergic responses.

The IL-33-ST2 pathway is an important initiator of type 2 immune responses. We previously characterised the HpARI protein secreted by the model intestinal nematode Heligmosomoides polygyrus, which binds and blocks IL-33. Here, we identify H. polygyrus Binds Alarmin Receptor and Inhibits (HpBARI) and HpBARI_Hom2, both of which consist of complement control protein (CCP) domains, similarly to the immunomodulatory HpARI and Hp-TGM proteins. HpBARI binds murine ST2, inhibiting cell surface detection of ST2, preventing IL-33-ST2 interactions, and inhibiting IL-33 responses in vitro and in an in vivo mouse model of asthma. In H. polygyrus infection, ST2 detection is abrogated in the peritoneal cavity and lung, consistent with systemic effects of HpBARI. HpBARI_Hom2 also binds human ST2 with high affinity, and effectively blocks human PBMC responses to IL-33. Thus, we show that H. polygyrus blocks the IL-33 pathway via both HpARI which blocks the cytokine, and also HpBARI which blocks the receptor

IgE allergy diagnostics and other relevant tests in allergy, a World Allergy Organization position paper

Currently, testing for immunoglobulin E (IgE) sensitization is the cornerstone of diagnostic evaluation in suspected allergic conditions. This review provides a thorough and updated critical appraisal of the most frequently used diagnostic tests, both in vivo and in vitro. It discusses skin tests, challenges, and serological and cellular in vitro tests, and provides an overview of indications, advantages and disadvantages of each in conditions such as respiratory, food, venom, drug, and occupational allergy. Skin prick testing remains the first line approach in most instances; the added value of serum specific IgE to whole allergen extracts or components, as well as the role of basophil activation tests, is evaluated. Unproven, non-validated, diagnostic tests are also discussed. Throughout the review, the reader must bear in mind the relevance of differentiating between sensitization and allergy; the latter entails not only allergic sensitization, but also clinically relevant symptoms triggered by the culprit allergen.info:eu-repo/semantics/publishedVersio

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Rapid antibody selection using surface plasmon resonance for high-speed and sensitive hazelnut lateral flow prototypes

Lateral Flow Immunoassays (LFIAs) allow for rapid, low-cost, screening of many biomolecules such as food allergens. Despite being classified as rapid tests, many LFIAs take 10–20 min to complete. For a really high-speed LFIA, it is necessary to assess antibody association kinetics. By using a label-free optical technique such as Surface Plasmon Resonance (SPR), it is possible to screen crude monoclonal antibody (mAb) preparations for their association rates against a target. Herein, we describe an SPR-based method for screening and selecting crude anti-hazelnut antibodies based on their relative association rates, cross reactivity and sandwich pairing capabilities, for subsequent application in a rapid ligand binding assay. Thanks to the SPR selection process, only the fast mAb (F-50-6B12) and the slow (S-50-5H9) mAb needed purification for labelling with carbon nanoparticles to exploit high-speed LFIA prototypes. The kinetics observed in SPR were reflected in LFIA, with the test line appearing within 30 s, almost two times faster when F-50-6B12 was used, compared with S-50-5H9. Additionally, the LFIAs have demonstrated their future applicability to real life samples by detecting hazelnut in the sub-ppm range in a cookie matrix. Finally, these LFIAs not only provide a qualitative result when read visually, but also generate semi-quantitative data when exploiting freely downloadable smartphone apps.</p

Mass Barcode Signal Amplification for Multiplex Allergy Diagnosis by MALDI-MS

A highly sensitive method based on mass-barcoded gold nanoparticles (AuNPs) and immunomagnetic separation has been developed for multiplex allergy diagnosis by MALDI mass spectrometry in a component-resolved manner. Different analytical probes were prepared by coating AuNPs with individual allergenic proteins and mass barcode, represented by polyethylene glycol molecules of various chain lengths. Magnetic beads (MBs) functionalized with antihuman IgE antibodies (Abs) were used as immunomagnetic capture probes. IgE Abs were extracted from a patient’s blood serum by the formation of a sandwich structure between the AuNPs and MBs. Multiple specific IgE Abs were simultaneously identified by mass spectrometry detection of the mass barcodes, providing an efficient component-resolved allergy diagnosis. Because of the signal amplification provided by the mass barcodes, the developed diagnosis method is very sensitive, with a limit of detection down to picograms per milliliter level for specific IgE Abs. The method can be potentially useful when the sample amount is highly limited and a multiplex diagnostic procedure is required