Sensitive detection and quantification of gliadin contamination in gluten-free food with immunomagnetic beads based liposomal fluorescence immunoassay
Graphical abstract
Introduction
Wheat protein is responsible for not only a common food allergy but also a contact allergy, owing to occupational exposure [1]. In clinical diagnosis, wheat allergy (WA) is an IgE-mediated hypersensitivity and the correlation diseases of WA include: atopic dermatitis, baker’s asthma, wheat-dependent exercise-induced anaphylaxis, and contact urticaria [2], [3]. On the other hand, the celiac disease (CD) is the T cell-mediated autoimmune disease and the result of permanent intolerance to gluten [4]. In previous studies, sufferers of WA and CD could be induced by ingestion of prolamins which were the fraction extracted by 40–70% aqueous ethanol, and is known as gliadin in wheat, as hordeins in barley, in oats as avenins, and as secalins in rye [5].
In general, gluten includes two groups of proteins known as glutenins and prolamins, and each group is account for approximately 50% of gluten [6]. In wheat, prolamins are also called as gliadins and can divide into α-, β-, γ- and ω-gliadin. In the midst of gliadin, α-gliadin and ω-5-gliadin have been considered to be the major wheat allergens [7]. In addition to above, the major wheat allergens also include the α-amylase/trypsin inhibitor (12–18 kDa); while the minor wheat allergens contain a 9-kDa lipid transfer protein in the albumin/globulin fraction and the low-molecular-weight glutenin subunits [8]. Patients must comply with a gluten-free diet, should avoid contact with barley and rye or their crossbred varieties and derivatives. The standard of gluten-free diet is described as the gluten level does not exceed 20 parts per million (ppm) in total [1].
Gliadin must be quantified accurately for proper food labeling and consumer protection. Several methods have been developed in recent years to detect gliadin, including quartz crystal microbalance (QCM) [9], mass spectrometry (MS) [10], electronic tongue (E-tongue) [11], real-time PCR [12], electrochemical immunosensor [13], and enzyme-linked immunosorbent assay (ELISA) [14]. Despite their accurate analysis, these methods must still be performed in an analytical laboratory, as well as operated by skilled technicians. Therefore, a simple detection method must be developed, characterized by rapid and sensitive analysis of gliadin in food samples. Among various assays commercially available, enzyme-linked immunosorbent assay (ELISA) is a conventional means of quantifying a target protein in food samples and possesses several advantages, such as high throughput, simplicity, and low cost [15].
Immunomagnetic separation (IMS) is a highly effective diagnostics approach, owing to its high specificity and sensitivity, as well as reduction of assay time. Immunomagnetic bead (IMB), a high-performance material, has been used to extract target analyte from liquid samples [16]. Bead-based immunoassays are generally superior to the conventional one, owing to their sample purification and concentration enabled by applying external magnetic fields. For instance, compared with the quantification of carcinoembryonic antigen (CEA) in human serum using the ELISA and the IMB-based ELISA, the limit of detection of the ELISA assay is 1.6 ng mL−1 which is higher than that of the one with IMB (0.5 ng mL−1) [17]. Additionally, magnetic beads have a three-dimensional structure that increases the surface-to-volume ratio, subsequently providing more binding sites for target molecules than the wells of microtiter plate with a two-dimensional structure. Recently, more magnetic bead assays have been integrated into microfluidic systems because they are further easily manipulated, sorted, and concentrated in the microfluidic systems [18], [19]. Besides, a novel magnetic bead detection method, a competitive immunomagnetic-proximity ligation assay (CIPLA), has been developed for small-molecules detections, which is essential for study related to drug discovery, physiological functions, and chemical residues in foods [20], [21]. By using IMB, a previous study removed allergens from peanut extract by conjugating anti-peanut antibodies to the surface of magnetic beads [22]. To amplify the detection signals, the antibody-tagged liposomal nanovesicle (immunoliposomal nanovesicle; IMLN) has been used as the detection reagent for several assays. Liposomes can be defined as closed spherical vesicles consisting of one or more phospholipid bilayers surrounding an aqueous cavity [23]. The hollow sphere of liposomes can encapsulate hundreds of thousands of dye molecules. Additionally, the release of this large amount of markers can significantly increase the assay signal immediately. To transform a liposome into a functionally labeled reagent, various biological ligands (e.g., peptides, hormones, antibodies, sugars, and nucleic acids) can be conjugated to the liposomal surface to make the targeted liposome. By altering the lipid composition, size, encapsulated materials, and tagged surface biomolecules, liposomes have been used in various applications, including drug delivery, gene transfection, and immunoassays [24], [25], [26].
This work develops a rapid and sensitive method by using IMBs as the capture reagent to isolate gliadin from food, followed by using anti-gliadin antibody tagged IMLNs as the detection reagent to quantitatively detect the contamination of gliadin in food. Moreover, these detection results are compared with an AOAC approved gliadin-ELISA kit to validate the precision and feasibility of this developed assay.
Section snippets
Materials
Two different sized magnetic beads, 2.8 and 2–10 μm coupled with carboxyl acid, were purchased from Dynal Invitrogen (CA, USA) and Abnova (Taipei, Taiwan), separately. Dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidylcholine (DPPC), and dipalmitoyl phosphatidylglycerol (DPPG) were purchased from NOF Corp. (Tokyo, Japan). The Mini Extruder was purchased from Avanti Polar Lipid, Inc. (AL, USA), and the 0.2 and 0.4 μm pore size polycarbonate membranes were purchased from Whatman
Antibody specificity
The performance of an immunoassay is heavily dependent on the affinity and specificity of the antigen-antibody interaction, however, the cross-reactivity of antibody may reduce the specificity of immunoassays. Since the anti-gliadin antibodies from yolk used in this developed assay are polyclonal, the specificity of antibodies was evaluated. Western blot and quartz crystal microbalance (QCM) in our previous study has confirmed the specificity of the anti-gliadin IgY used in this work [9].
Conclusions
This work presents an immunoliposomal nanovesicle (IMLN) based on immunomagnetic bead (IMB) assay for detecting gliadin in foods which could produce a specific and selective immune response to the target allergen. Importantly, the developed assay could detect levels of gliadin as low as 0.6 μg mL−1. Moreover, the sample preparation method in this work also provides excellent recovery rates of spiked gliadin in gliadin-free and gliadin-content food samples, ranging from 83.5 to 102.6%.
Acknowledgements
The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under Contract No. NSC98-2313-B005-037-MY3. This work was also supported by Dr. C. F. Chen at Department of Animal Science of National Chung Hsing University in Taiwan, who provided excellent animal handling services. Ted Knoy is appreciated for his editorial assistance.
References (55)
- et al.
Food Chem. Toxicol.
(2000) - et al.
J. Cereal. Sci.
(2005) J. Am. Diet Assoc.
(2008)- et al.
Trends. Food. Sci. Tech.
(2004) - et al.
Talanta
(2011) - et al.
J. Pharm. Biomed. Anal.
(2012) - et al.
Anal. Chim. Acta
(2004) J. Biol. Chem.
(1959)- et al.
J. Allergy Clin. Immunol.
(2004) - et al.
J. Chromatogr A
(2008)
Sens. Actuat. B. Chem.
Anal. Biochem.
Biosens. Bioelectron.
FEBS. Lett.
J. Am. Diet Assoc.
J. Pharm. Biomed. Anal.
Spill. Sci. Technol. Bull.
Talanta
J. Am. Diet Assoc.
Aliment Pharmacol. Ther.
Anal. Bioanal. Chem.
Clin. Exp. Allergy
Int. Arch. Allergy Immunol.
J. Agric. Food. Chem.
J. Agric. Food. Chem.
Eur. Food. Res. Technol.
Anal. Chem.
Cited by (39)
A sensitive dual mode turn-on fluorescence and colorimetric nanosensor for ultrasensitive detection of trace amount of gluten proteins in bread products based on crystalline nano cellulose and gold nanoparticles
2023, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyLiposomes as biosensors in the food sector
2022, Liposomal Encapsulation in Food Science and TechnologyIn vitro selection of anti-gliadin single-domain antibodies from a naïve library for cDNA-display mediated immuno-PCR: Anti-gliadin VHH by cDNA display
2020, Analytical BiochemistryCitation Excerpt :Gliadin is the storage protein in wheat grain that is soluble in aqueous alcohols but is insoluble in water or neutral salt solutions. In general, gliadin is extracted with aqueous alcohol (60%–70%) [6,12,37]. In the present study, gliadin was extracted with 70% alcohol (with or without 10% DMSO) in PBS buffer.