ANTIGEN TESTING

Abstract

The invention provides methods of testing food for antigens that are more stable to food processing than more clinically problematic allergens from the same food. When clinically-significant allergens are disproportionately broken down by common preparation methods, the presence of certain food ingredients may be “masked” to some tests yet may still be allergenic. To prevent false negative results due to food preparation, the invention provides tests that test for specific food antigens that are selected on the basis of their stability under processing. Antigens are selected for inclusion in the test not because they are the most clinically relevant allergens, but rather because they are robust to processing (e.g., and do not denature during cooking). Tests of the invention may also test for the most clinically relevant allergens, but importantly, by testing for stable protein products/antigens, the tests report the presence of food residues even after commercial processing.

Claims

1. A method of testing for an allergen, the method comprising: introducing a sample from a food product into a test device; conducting an assay in the test device to detect an antigen that is substantially resistant to degradation during food processing, and the presence of which is indicative of a potential allergic reaction; and reporting the presence of an allergen in the food product as a result of detection of the antigen.

2. The method of claim 1, wherein the antigen is an isotype of a second antigen that is degraded during food processing.

3. The method of claim 1, wherein the antigen is Ara h 2, Ara h 6 or a combination of Ara h 2 and Ara h6.

4. The method of claim 2, wherein the antigen that is degraded is Ara h 1,Ara h 3, or a combination of Ara h 1 and Ara h 3.

5. The method of claim 1, wherein the test device further tests for a plurality of antigens that occur naturally in the food product.

6. The method of claim 1, wherein the test device tests for a plurality of antigens from a food item, wherein each antigen is selected because it is relatively more stable than other allergens found in said food item.

7. The method of claim 1, wherein the test device includes a plurality of molecular binders attached to a surface that bind to the antigen when the antigen is introduced into the test device.

8. The method of claim 1, wherein the test device is adapted to perform an enzyme-linked immunosorbent assay or a lateral flow assay for the antigen.

9. The method of claim 1, wherein the test device detects the antigen and the second antigen that is degraded during food processing.

10. The method of claim 2, wherein the food product is milk, the antigen is from a casein protein, and the second antigen that is degraded during food processing is selected from the group consisting of Bos d 4, Bos d 5, Bos d 6, Bos D 7, Bos D 8, Bos D 9, Bos d 10, Bos d 11, and Bos d 12.

11. The method of claim 2, wherein the food product is egg, the antigen is from an egg white protein, and the second antigen that is degraded during food processing is selected from the group consisting of Gal d 1, Gal d 2, Gal d 3, Gal d 4, Gal d 5, and Gal d 6.

12. The method of claim 2, wherein the food product is wheat, the antigen is from a glutenin protein, and the second antigen that is degraded during food processing is selected from the group consisting of Tri a 14, Tri a 18, Tri a 19, Tri a 20, Tri a 25, Tri a 26, Tri a 36, tri a 37, Tri a 40, Tri a 41, Tri a 42, Tri a 43 Tri a 44, and Tri a 45.

13. The method of claim 1, wherein the second antigen that is degraded during food processing is known to present a high risk of an anaphylactic response in allergic individuals.

14. The method of claim 1, wherein the antigen is suspected to present a lower risk of anaphylactic response in allergic individuals and has greater thermal stability than the second antigen that is degraded during food processing.

15. The method of claim 1, wherein the test device comprises antibodies that bind to the antigen.

Description

DETAILED DESCRIPTION

[0016] The invention provides assays for food allergens that that do not substantially degrade during food processing and thus serve as reliable markers of trace allergenic ingredients. The invention employs the insight that during, for example, roasting, allergens of clinical interest may be destroyed. For example, Ara h 1 is substantially denatured when peanuts are roasted. Ara h 1 has been clinically implicated in anaphylaxis with the consequence that some commercially available, even consumer-facing, food tests are designed to test for Ara h 1 as a marker of the presence of peanut residue in a food product. However, those tests may give false negatives because Ara h 1 is often denatured during roasting and many peanuts used in commercial/industrial food production are roasted. Processes that test only for Ara h 1 thus may result in false negatives. To remedy this problem, the invention provides assays that detect antigens that do not get denatured or destroyed in the food processing and preparation process.

[0017] Assays of the invention may be embodied in test devices that test for antigens from any food of interest including, for example, milk, egg, fish, crustaceans, shellfish, peanut, tree nuts, wheat, or soy. Methods of the invention may include introducing a sample from a food product into a test device.

[0018] The test device may be, for example, a handheld device that includes mechanisms for food processing, antigen detection, result interpretation and data transmission. The device may have a form-factor allowing it to be carried in a purse or bag. Preferably, the device performs all sample processing steps internally, and may be designed to give a quantitative result or, alternatively, a binary positive or negative result.

[0019] In some embodiments, the assay inside the device uses the principle of the enzyme-linked immunosorbent assay (ELISA). Monoclonal antibodies may be raised using standard hybridoma techniques. See Council, 1999, Monoclonal Antibody production, National Academic Press, (Washington, D.C.), incorporated by reference. Alternatively, monoclonal antibodies may be discovered from allergic individuals. See Croote, 2018, High-affinity allergen-specific human antibodies cloned from single IgE B cell transcriptomes, Science 362:1306-1309, incorporated by reference. The device may include vials or wells that include capture antibodies and reporter antibodies. Embodiments provide a test device that comes with a vial or reservoir that contains a bead suspension comprising magnetic beads having capture antibodies conjugated to the magnetic beads. A user introduces a food sample into the vial or reservoir. The bead suspension containing the immobilized capture antibodies is then incubated with the extracted food according to provided instructions (e.g., for around 3 min) before re-suspending with horseradish peroxidase (HRP)-conjugated reporter antibodies. The HRP-bead complex can then be mixed with substrate (TMB) and added into a reading device. The device may be provided as a kit with, or with integrated modules for, an extraction device and the extraction buffers and wash solutions in pre-measured volumes. Optionally, a lid of the extraction vial has a magnetic sheathed bar attached to allow for capture of allergen-magnetic beads. This bar allows easy transfer of the antibody-bead complex to the washing/labelling stages and then for loading onto the magnetic electrode, making sample handling easier for the consumer. A reader/sensor includes a microcontroller unit linked with digital-to-analog converters and a controller that controls the potential difference between reference and working electrodes. Alternatively, a reader/sensor includes a microcontroller unit linked with a light source, optional filters, and a detector. Optionally, the reader may be operated via a Bluetooth connection to a smartphone app. The device may include features as described in Lin, 2017, Integrated magneto-chemical sensor for on-site food allergen detection, ACS Nano 11(10):10062-9, incorporated by reference.

[0020] In certain embodiments, the assay devices of the invention use the principle of the lateral flow immunoassay (LFIA). The device may have a test strip, to which colloidal gold nanoparticles (red) are conjugated with antibody against the antigen to be tested for. Antibody may be mixed with the nanoparticles at a ratio of 12 μg antibody/ml gold particle (optical density=2) in a basic solution (pH=8.2-8.6) for 30 min at room temperature, and the complex may be dispensed onto the conjugate pad (red pad at the base of strip). A test line containing a second, other antibody may be printed onto a nitrocellulose membrane. The test may include a control line. Components may be assembled into lateral flow strips. E.g., a 25 mm wide nitrocellulose membrane may be glued onto a paper-back card and a wick pad, and the preprinted conjugate pad assembled on top of the rear card with a 2-3 mm overlap. Cover tape may be applied to keep the assembly intact and air was squeezed out by pressing on the assembly. The resulting immunoassay strip is exposed to food sample that has passed through a receiving and processing well of the device. E.g., the device may include a flip cap that opens a to receiving and processing well (of, e.g., about 5 mL volume) into a which a user introduces a food sample. The device may then run homogenizer such as a burr grinder or blade grinder in the well while washing with an extraction solution, e.g., saline or a mild organic solvent such as acetone or ethanol before washing the processed sample to the test strip. Test strips are preferably exposed to the sample, e.g., for about 2 min. Control, hook, and test lines may be included. Intensities may be e captured using a sensor such as a photodiode, CMOS pixel array, or linear-array camera in the device. The device may include features described in U.S. Pub. 2016/0266083 A1, incorporated by reference.

[0021] A simple processor (e.g., a field programmable gate array or application specific integrated circuit) may read the sensor and report the presence and/or quantity of the analyte(s), e.g., give an output showing that the antigen was present in the input sample.

[0022] In some embodiments of the assay device, the device includes a reusable testing instrument and a disposable testing unit. Users put a food sample into the testing unit, e.g., into the receiving well, and fasten (e.g., screw down) a lid. Once the lid is fastened, the testing unit mechanically grinds the food sample and releases an extraction solution. E.g., the user may place the testing unit into the testing instrument and press a button to have the instrument initiate mixing in the unit. Optionally, fully attaching the unit to the instrument causes the opening of an internal valve or passage allowing liquid from the extraction chamber to flow onto the LFIA strip. Mechanical features may be as described in U.S. Pat. No. 9,939,431 B2, incorporated by reference. The assay device assays the food sample for at least one antigen that occurs naturally in the food product and is more stable during food processing as compared to another antigen that may be present in the food product. Using, e.g., the sensor and the processor, the device reports a risk of the allergen being in the food product when the stable (detected) antigen is detected. In certain embodiments, the unstable allergen is one that is often denatured by a cooking process and the stable antigen is one that is rarely denatured by the cooking process. E.g., the detected antigen may be a thermo-stable antigen of a non-allergenic protein of the food product. In some embodiments, the antigen that is tested for is Ara h 2 and/or Ara h 6 and the unstable antigen is Ara h 1 and or Ara h 3 (which may also be tested for).

[0023] The test device may test for a plurality of antigens that occur naturally in the food product. Table 1 lists antigens that may be tested for.

TABLE-US-00001 TABLE 1 antigens that may be tested for; reproduced from Sharma, 2017, Detection of allergen markers in food: analytical methods, FDA Papers 6:65-121, incorporated by reference. Allergen Biochemical name Allergen Biochemical name MILK Bos d 4 α-lactalbumin Bos d 9 α-S1-casein Bos d 5 β-lactoglobulin Bos d 10 α-S2-casein Bos d 6 Serum albumin Bos d 11 β-casein Bos d 7 Immunoglobulin Bos d 12 K-casein Bos d 8 Casein EGG Allergen Biochemical name Allergen Biochemical name Gal d 1 Ovomucoid Gald 4 Lysozyme C Gal d 2 Ovalbumin Gald 5 Serum albumin Gal d 3 Ovotransferrin Gald 6 YGP4 FISH Allergen Biochemical name Allergen Biochemical name Yellowfin tuna Atlantic cod Thu a 1 β-parvalbumin Gad m 1 β-parvalbumin Thu a 2 β-enolase Gad m 2 β-enolase Thu a 3 Aldolase A Gad m 3 Aldolase A Atlantic salmon Baltic cod Sal s 1 β-parvalbumin 1 Gad c 1 β-parvalbumin Sal s2 β-Enolase Sal s 3 Aldolase A CRUSACEAN SHELLFIS Allergen Biochemical name Allergen Biochemical name Black tiger shrimp American lobster Pen m l Tropomyosin Hom a 1 Tropomyosin Pen m 2 Arginine kinase Hom a 3 Myosin light chain 2 Pen m 3 Myosin light chain 2 Hom a 6 Troponin C Pen m 4 Sarcoplasmic Ca Spiny lobster binding protein Pen m 6 Troponin C Pan s 1 Tropomyosin Crab Cha f 1 Tropomyosin PEANUT Allergen Biochemical name Allergen Biochemical name Ara h 1 7S globulin Ara h 10 16 kDa oleosin Ara h 2 2S albumin Ara h 11 14 kDa oleosin Ara h 3 11S globulin Ara h 12 Defensin Ara h 4 renamed Ara h 3.02 Ara h 13 Defensin Ara h 5 Profilin Ara h 14 Oleosin Ara h 6 2S albumin Ara h 15 Oleosin Ara h 7 2S albumin Ara h 16 nsLTP Ara h 8 PR-10 Ara h 17 nsLTP Ara h 9 nsLTP TREE NUTS Allergen Biochemical name Allergen Biochemical name Almond Brazil nut Pru du 3 nsLTP 1 Ber e 1 2S albumin Pru du 4 Profilin Ber e 2 11S globulin Pru du 5 60s acidic ribosomal Hazelnut protein P2 Pru du 6 Amandin, US Cor a 1 PR-10 globulin Cashew nut Cor a 2 Profilin Ana o 1 7S globulin Cor a 8 nsLTP 1 Ana o 2 11S globulin Cor a 9 11S globulin Ana o 3 2S albumin Cor all 7S globulin Pecan Cor a 12 17 kDa oelosin Cari 1 2S albumin Cor a 13 14-16 kDa oleosin Car i 2 7S globulin Cor a 14 2S albumin Car i 4 11S globulin English walnut Pistachio Jug r 1 2S albumin Pis v 1 2S albumin Jug r 2 7S globulin Pis v 2 11S globulin Jug r 3 nsLTP 1 Pis v 3 7S globulin Jug r 4 11S globulin Pis v 4 Manganese Jug r 5 PR-10 superoxide dismutase Pis v 5 11S globulin WHEAT Allergen Biochemical name Allergen Biochemical name Tri a 14 nsLTPl Tri a 3 purothionin Tri a 18 Agglutinin isolectin 1 Tri a 40 amylase inhibitor Tri a 1 Omega-5 gliadin Tri a 41 Mitochondrial ubiquitin ligase activator of NFKB 1 Tri a 2 Gliadin Tri a 42 Hypothetical protein Tri a 25 Thioredoxin Tri a 43 Hypothetical protein Tri a 26 High molecular Tri a 44 Endosperm transfer weight glutenin cell specific PR60 precursor Tri a 36 Low molecular Tri a 45 Elongation factor 1 weight glutenin (EIF1) GluB3-23 SOY Allergen Biochemical name Allergen Biochemical name Gly m 3 Profilin Gly m 6 11S globulin Gly m 4 PR-10 Gly m 7 Seed biotinylated protein Gly m 5 7S globulin Gly m 8 2S albumin

[0024] In various embodiments, the test device tests for a plurality of antigens from a plurality of respective food items, wherein each antigen is selected because it is relatively more stable than other allergens found in that food source. Optionally, the test device includes a plurality of molecular binders (e.g., antibodies) attached to a surface that bind to the stable antigen when it is introduced into the test device. For example, the test device may be useful for performing an enzyme-linked immunosorbent assays or a lateral flow assay for the stable antigen.

[0025] The test device may include antibodies that bind specifically to the stable antigen and capture it for detection. For example, antibodies may be raised against, or characterized to be specific to, antigens indicative of any of the allergens shown in Table 1 and included in the test device. The antigen that is tested for is selected because it is more robust to processing (e.g., thermostable, and less likely to denature) than a known, clinically-important allergen (e.g., one that is thermolabile). In some cases, the unstable allergen is known to present a high risk of an anaphylactic response in allergic individuals.

[0026] The invention provides methods of testing food for antigens that are more stable to food processing than antigens that, while diagnostically-relevant, are often degraded during processing. When clinically-significant allergens are disproportionately broken down by common preparation methods, the presence of certain food ingredients may be “masked” to some tests. To prevent clinically problematic allergens from being masked by food preparation, the invention provides tests that test for specific food antigens that are selected on the basis of their stability under processing. Antigens are selected for inclusion in the test not because they are the most clinically relevant allergens, but rather because they are robust to processing (e.g., and do not denature during cooking). Tests of the invention may also test for the most clinically relevant allergens, but importantly, by testing for stable protein products/antigens, the tests report the presence of food residues even after commercial processing.

[0027] In fact, embodiments of the invention may address a specific phenomenon by which global allergy incidence is unevenly distributed in a manner that may be linked to predominant geographical cooking practices. It may be found that peanuts are processed in North America and other parts of the Western world primarily by roasting, while peanuts in Asia and other parts of the Eastern world are primarily processed by boiling or frying. It may be found that the times and temperatures of those different cooking practices have different effects on allergen stability with a consequence of early childhood immune sensitization having different patterns across the globe. It may be found that Ara h 1 or Ara h 3 are denatured by roasting in such a way that some populations are not desensitized to it and develop proportionally higher incidence of allergy. However, some commercial tests may employ antibodies specific to Ara h 1 or Ara h 3 with the consequence that the roasting masks the presence of trace peanut amounts to the test due to denaturation. Tests of the invention include assays that test for antigens other than Ara h 1 or Ara h 3 to detect trace amounts of peanut without requiring the specific molecular detection of Ara h 1 or Ara h 3.

[0028] It is important to note that assays of the invention may find particular utility in, and are intended to be used for, detecting trace amounts of ingredients in food products for which the ingredient is not intended to be an ingredient. For example, tests of the invention are useful to detect trace amounts of peanut dust in foods that are not expected to include any peanuts. Tests of the invention may be used, for example, to test for trace amounts of peanut in foods and environments such as in cotton candy at a fair, or in butter cookies at a bakery, or in a gelatin desert at a social gathering, or in a fruit smoothie from a juice bar.