RESUMEN
Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.
Asunto(s)
Antígenos de Histocompatibilidad Clase I , Inmunoglobulina G , Receptores Fc , Concentración de Iones de Hidrógeno , Inmunoglobulina G/metabolismo , Inmunoglobulina G/química , Humanos , Receptores Fc/metabolismo , Receptores Fc/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Ingeniería de Proteínas/métodos , Unión Proteica , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Antígenos/metabolismo , Antígenos/química , Animales , Modelos MolecularesRESUMEN
Bothrops and Lachesis are two of Brazil's medically most relevant snake genera, causing tens of thousands of bites annually. Fortunately, Brazil has good accessibility to high-quality antivenoms at the genus and inter-genus level, enabling the treatment of many of these envenomings. However, the optimal use of these treatments requires that the snake species responsible for the bite is determined. Currently, physicians use a syndromic approach to diagnose snakebite, which can be difficult for medical personnel with limited training in clinical snakebite management. In this work, we have developed a novel monoclonal antibody-based multiplex lateral flow assay for differentiating Bothrops and Lachesis venoms within 15 min. The test can be read by the naked eye or (semi)-quantitatively by a smartphone supported by a 3D-printed attachment for controlling lighting conditions. The LFA can detect Bothrops and Lachesis venoms in spiked plasma and urine matrices at concentrations spanning six orders of magnitude. The LFA has detection limits of 10-50 ng/mL in spiked plasma and urine, and 50-500 ng/mL in spiked sera, for B. atrox and L. muta venoms. This test could potentially support medical personnel in correctly diagnosing snakebite envenomings at the point-of-care in Brazil, which may help improve patient outcomes and save lives.
Asunto(s)
Bothrops , Venenos de Crotálidos , Mordeduras de Serpientes , Animales , Humanos , Mordeduras de Serpientes/tratamiento farmacológico , Venenos de Serpiente/uso terapéutico , Antivenenos/uso terapéutico , Venenos de Crotálidos/uso terapéutico , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
BACKGROUND: The increasing demand for food and feed products is stretching the capacity of the food value chain to its limits. A key step for ensuring food safety is checking for mycotoxin contamination of wheat. However, this analysis is typically performed by rather complex and expensive chromatographic methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). These costly methods require extensive sample preparation that is not easily carried out at different points along the food supply chain. To overcome such challenges in sample processing, an inexpensive and portable sample preparation device was needed, that required low skill, for rapid sample-to-result mycotoxin screening. RESULTS: We describe 3D-printed and interconnectable modules for simple, integrated and on-site sample preparation, including grinding of wheat kernels, and solvent-based extraction. We characterized these 3D-printed modules for mycotoxin screening and benchmarked them against a laboratory mill using commercial lateral flow device(s) (LFD) and in-house validated LC-MS/MS analysis. Different integrated sieve configurations were compared based on grinding efficiency, and we selected a sieve size of 2 mm allowing grinding of 10 g of wheat within 5 min. Moreover, 10 first time-users were able to operate the grinder module with minimal instructions. Screening for deoxynivalenol (DON) in naturally contaminated samples at the regulatory/legal limit (1.25 mg kg-1) was demonstrated using the developed 3D-printed prototype. The whole process only takes 15 min, from sample preparation to screening result. The results showed a clear correlation (R2 = 0.96) between the LFD and LC-MS/MS. SIGNIFICANCE: Our findings demonstrate the potential of 3D-printed sample handling equipment as a valuable extension of existing analytical procedures, facilitating the on-site implementation of rapid methods for the determination of mycotoxins in grains. The presented prototype is inexpensive with material costs of 2.5, relies on biodegradable 3D printing filament and can be produced with consumer-grade printers, making the prototype readily available. As a future perspective, the modular character of our developed tool kit will allow for adaptation to other hard food commodities beyond the determination of DON in wheat.
Asunto(s)
Micotoxinas , Micotoxinas/análisis , Cromatografía Liquida/métodos , Triticum/química , Espectrometría de Masas en Tándem/métodos , Contaminación de Alimentos/análisisRESUMEN
BACKGROUND: Brazil is home to a multitude of venomous snakes; perhaps the most medically relevant of which belong to the Bothrops genus. Bothrops spp. are responsible for roughly 70% of all snakebites in Brazil, and envenomings caused by their bites can be treated with three types of antivenom: bothropic antivenom, bothro-lachetic antivenom, and bothro-crotalic antivenom. The choice to administer antivenom depends on the severity of the envenoming, while the choice of antivenom depends on availability and on how certain the treating physician is that the patient was bitten by a bothropic snake. The diagnosis of a bothropic envenoming can be made based on expert identification of the dead snake or a photo thereof or based on a syndromic approach wherein the clinician examines the patient for characteristic manifestations of envenoming. This approach can be very effective but requires staff that has been trained in clinical snakebite management, which, unfortunately, far from all relevant staff has. RESULTS: In this article, we describe a prototype of the first lateral flow assay (LFA) capable of detecting venoms from Brazilian Bothrops spp. The monoclonal antibodies for the assay were generated using hybridoma technology and screened in sandwich enzyme-linked immunosorbent assays (ELISAs) to identify Bothrops spp.-specific antibody sandwich pairs. The prototype LFA is able to detect venom from several Bothrops spp. The LFA has a limit of detection (LoD) of 9.5 ng/mL in urine, when read with a commercial reader, and a visual LoD of approximately 25 ng/mL. SIGNIFICANCE: The work presented here serves as a proof of concept for a genus-specific venom detection kit that could support physicians in diagnosing Bothrops envenomings. Although further optimisation and testing is needed before the LFA can find clinical use, such a device could aid in decentralising antivenoms in the Brazilian Amazon and help ensure optimal snakebite management for even more victims of this highly neglected disease.
Asunto(s)
Bothrops , Venenos de Crotálidos , Mordeduras de Serpientes , Animales , Mordeduras de Serpientes/diagnóstico , Mordeduras de Serpientes/tratamiento farmacológico , Antivenenos/uso terapéutico , Venenos de Crotálidos/uso terapéutico , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
Food allergies are hypersensitivity immune responses triggered by (traces of) allergenic compounds in foods and drinks. The recent trend towards plant-based and lactose-free diets has driven an increased consumption of plant-based milks (PBMs) with the risk of cross-contamination of various allergenic plant-based proteins during the food manufacturing process. Conventional allergen screening is usually performed in the laboratory, but portable biosensors for on-site screening of food allergens at the production site could improve quality control and food safety. Here, we developed a portable smartphone imaging surface plasmon resonance (iSPR) biosensor composed of a 3D-printed microfluidic SPR chip for the detection of total hazelnut protein (THP) in commercial PBMs and compared its instrumentation and analytical performance with a conventional benchtop SPR. The smartphone iSPR shows similar characteristic sensorgrams compared with the benchtop SPR and enables the detection of trace levels of THP in spiked PBMs with the lowest tested concentration of 0.625 µg/mL THP. The smartphone iSPR achieved LoDs of 0.53, 0.16, 0.14, 0.06, and 0.04 µg/mL THP in 10x-diluted soy, oat, rice, coconut, and almond PBMs, respectively, with good correlation with the conventional benchtop SPR system (R2 0.950-0.991). The portability and miniaturized characteristics of the smartphone iSPR biosensor platform make it promising for the future on-site detection of food allergens by food producers.
Asunto(s)
Técnicas Biosensibles , Hipersensibilidad a los Alimentos , Humanos , Resonancia por Plasmón de Superficie/métodos , Alérgenos , Teléfono Inteligente , Técnicas Biosensibles/métodos , Límite de Detección , Hipersensibilidad a los Alimentos/diagnósticoRESUMEN
Sandwich lateral flow immunoassays (LFIAs) are limited at high antigen concentrations by the hook effect, leading to a contradictory decrease in the test line (T) intensity and false-negative results. The hook effect is mainly associated with the loss of T, and research focuses on minimizing this effect. Nevertheless, the control line (C) intensity is also affected at higher analyte concentrations, undesirably influencing the T/C ratio in LFIA readers. The main aim of this work is to identify and understand these high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects. Four complementary experiments were performed: performance assessment of three different allergen LFIAs (two for hazelnut, one for peanut) over 0.075-3500 ppm, LFIAs with C only, surface plasmon resonance (SPR) binding experiments on the immobilized control antibody, and smartphone video recording of LFIAs during their development. As antigen concentrations increase, the C signal decreases before the T signal does, suggesting that distinct mechanisms underlie these intensity reductions. Reduced binding at the C occurred even in the absence of T, so the upfront T does not explain the loss of C. SPR confirmed that the C antibody favors binding with free labeled antibody compared with a labeled antibody-analyte complex, indicating that in antigen excess, binding is reduced at C before T. Finally, a smartphone-based video method was developed for dynamically monitoring the LFIA development in real time to distinguish between different concentration-dependent effects. Digitally analyzing the data allows clear differentiation of highly positive samples and false-negative samples and can indicate whether the LFIA is in the dynamic working range or at critically high concentrations. The aim of this work is to identify and understand such high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects.
Asunto(s)
Alérgenos/análisis , Inmunoensayo/métodos , Alérgenos/inmunología , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Arachis/inmunología , Corylus/inmunología , Inmunoensayo/instrumentación , Límite de Detección , Teléfono Inteligente , Propiedades de SuperficieRESUMEN
While consumer-focused food analysis is upcoming, the need for multiple sample preparation and handling steps is limiting. On-site and consumer-friendly analysis paradoxically still requires laboratory-based and skill-intensive sample preparation methods. Here, we present a compact, inexpensive, and novel prototype immunosensor combining sample preparation and on-chip reagent storage for multiplex allergen lateral flow immunosensing. Our comprehensive approach paves the way for personalized consumer diagnostics. The prototype allows for handheld solid-liquid extraction, pipette-free on-chip dilution, and adjustment of sample concentrations into the appropriate assay dynamic working range. The disposable and interconnectable homogenizer unit allows for the extraction and 3D-sieve based filtration of allergenic proteins from solid bakery products in 1 min. The homogenizer interconnects with a 3D-printed unibody lab-on-a-chip (ULOC) microdevice, which is used to deliver precise volumes of sample extract to a reagent reservoir. The reagent reservoir is implemented for on-chip storage of carbon nanoparticle labeled antibodies and running buffer for dilution. The handheld prototype allows for total homogenization of solid samples, solid-liquid protein extraction, 3D-printed sieve based filtration, ULOC-enabled dilution, mixing, transport, and smartphone-based detection of hazelnut and peanut allergens in solid bakery products with limited operational complexity. The multiplex lateral flow immunoassay (LFIA) detects allergens as low as 0.1 ppm in real bakery products, and the system is already consumer-operable, demonstrating its potential for future citizen science approaches. The designed system is suitable for a wide range of analytical applications outside of food safety, provided an LFIA is available.
Asunto(s)
Técnicas Biosensibles , Alérgenos , Inmunoensayo , Dispositivos Laboratorio en un Chip , Teléfono InteligenteRESUMEN
(1) Background: The lack of globally standardized allergen labeling legislation necessitates consumer-focused multiplexed testing devices. These should be easy to operate, fast, sensitive and robust. (2) Methods: Herein, we describe the development of three different formats for multiplexed food allergen detection, namely active and passive flow-through assays, and lateral flow immunoassays with different test line configurations. (3) Results: The fastest assay time was 1 min, whereas even the slowest assay was within 10 min. With the passive flow approach, the limits of detection (LOD) of 0.1 and 0.5 ppm for total hazelnut protein (THP) and total peanut protein (TPP) in spiked buffer were reached, or 1 and 5 ppm of THP and TPP spiked into matrix. In comparison, the active flow approach reached LODs of 0.05 ppm for both analytes in buffer and 0.5 and 1 ppm of THP and TPP spiked into matrix. The optimized LFIA configuration reached LODs of 0.1 and 0.5 ppm of THP and TPP spiked into buffer or 0.5 ppm for both analytes spiked into matrix. The optimized LFIA was validated by testing in 20 different blank and spiked matrices. Using device-independent color space for smartphone analysis, two different smartphone models were used for the analysis of optimized assays.
Asunto(s)
Alérgenos/análisis , Técnicas Biosensibles , Hipersensibilidad a los Alimentos/diagnóstico , Inmunoensayo , Teléfono Inteligente , Hipersensibilidad a los Alimentos/inmunología , HumanosRESUMEN
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 10â»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.
Asunto(s)
Anticuerpos Monoclonales/análisis , Corylus/inmunología , Resonancia por Plasmón de Superficie/métodos , Antígenos de Plantas/metabolismo , Carbono/química , Hipersensibilidad a los Alimentos/diagnóstico , Humanos , Inmunoensayo , Límite de Detección , NanopartículasRESUMEN
In this critical review, we provide a comprehensive overview of immunochemical food allergen assays and detectors in the context of their user-friendliness, through their connection to smartphones. Smartphone-based analysis is centered around citizen science, putting analysis into the hands of the consumer. Food allergies represent a significant worldwide health concern and consumers should be able to analyze their foods, whenever and wherever they are, for allergen presence. Owing to the need for a scientific background, traditional laboratory-based detection methods are generally unsuitable for the consumer. Therefore, it is important to develop simple, safe, and rapid assays that can be linked with smartphones as detectors to improve user accessibility. Smartphones make excellent detection systems because of their cameras, embedded flash functions, portability, connectivity, and affordability. Therefore, this review has summarized traditional laboratory-based methods for food allergen detection such as enzyme-linked-immunosorbent assay, flow cytometry, and surface plasmon resonance, and the potential to modernize these methods by interfacing them with a smartphone readout system, based on the aforementioned smartphone characteristics. This is the first review focusing on smartphone-based food-allergen detection methods designed with the intention of being consumer-friendly. Graphical abstract A smartphone-based food allergen detection system in three easy steps (1) sample preparation, (2) allergen detection on a smartphone using antibodies, which then transmits the data wirelessly, (3) analytical results sent straight to smartphone.