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FDTR V14_120712

Food Detective™

Technical Report

Signed:

Matthew Bennett QA Manager

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CONTENTS PAGE

Page

Section 1 Name and Product Code 3 Section 2 Product Description 3 Section 3 Intended Use 4 Section 4 Device Operation 5 Section 5 Technical Requirements 7 Section 6 Specification of Materials and Manufacturing 8 Section 7 Manufacturing Process Flow Chart 9 Section 8 Methods of Manufacture 10 Section 9 Performance Evaluation Data 11 Analytical Sensitivity 11 Detection of Antibodies to 59 foods 11 Sensitivity, Specificity and Accuracy 12 Antigen Absorption Data 13 Intralot Reproducibility 16 Interlot Reproducibility 18 Matrix Effects 19 Section 10 Stability Studies 21 Stability Under Adverse Shipping Conditions 23 Appendix 1 Certificate of Analysis 24 Appendix 2 Bibliography 26 Appendix 3 Labels 27

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1. NAME AND PRODUCT CODE

Name: Food DetectiveTM

Product Code: CNSFDR (Self Test)

CNSFD (Professional)

2. PRODUCT DESCRIPTION A boxed kit (Figure 1) containing:

Cleansing wipe Sterile lancet x 2 Sodium-heparin 50µl end-to-end pipette Plaster Reaction Tray onto which foods are arrayed Sample diluent Detector solution Developer solution Wash solution Instructions for use

Figure 1 - The Food Detective Kit (59 foods)

The Reaction Tray is sealed in a foil pouch with desiccant to maintain stability. All solutions are supplied in inert PET bottles. Developer Solution is supplied in black PET bottles to prevent photolysis. All bottles are capped with machine tightened caps to prevent oxidation, leakage etc. Secondary packaging comprises of a robust cardboard insert to hold the kit components in position and a strong cardboard box which is sealed in Saran wrap to maintain the integrity of the kit.

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3. INTENDED USE The Food DetectiveTM kit is a rapid, qualitative, ELISA-based method for the detection of IgG antibodies to 59 different foods in human sera or plasma.

IgG reactions to food have been implicated in a number of chronic diseases including arthritis, irritable bowel syndrome, bronchitis and depression. Such reactions involve the formation and deposition of food antigen/antibody complexes in a variety of tissues where they cause inflammation, pain and other symptoms.

The measurement of food-specific antibodies by microplate-based immunoassays is currently used for the assessment of these reactions. Such immunoassays require sophisticated equipment and can only be carried out by trained technicians in a laboratory setting. In acknowledgment of this, we have designed and developed ‘The Food DetectiveTM’, a novel macroarray-based immunoassay that is simple to use, requires no sophisticated equipment and gives results which are easily interpreted.

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4. DEVICE OPERATION The Reaction Tray is spotted with food extracts. A small blood sample is collected from a finger-prick into a fixed volume capillary tube. This is added to Sample Diluent which is then poured onto the Reaction Tray. Any food antibodies present in the blood bind to the food extracts on the tray.

After washing away unbound serum components, goat anti-human IgG conjugated to horseradish peroxidase (Detector Solution) is added to the wells, and this binds to surface-bound human antibodies in the second incubation.

Unbound conjugate is removed by washing, and a solution containing 3,3’,5,tetramethylbenzidine and enzyme substrate (Developer Solution) is added to trace specific antibody binding.

The presence of food antibodies is detected through the appearance of one or more blue spots on the tray (Figure 2). Reference to the food layout plan (Table 1) on the Test Report allows the foods causing antibody production to be identified. Further examples of results obtained are shown in Figures 3 and 5.

Figure 2 Table 1

1White fish mix: haddock, cod & plaice; 2Fresh water fish: salmon & trout; 3Shell fish mix: shrimp, prawn, crab, lobster, & mussel; 4Peppers: red, green and yellow; 5Legume mix: pea, lentil & haricot bean; 6Melon mix: cantaloupe & water melon.

1. Oat

2. Wheat

3. Rice

4. Corn

5. Rye

6. Durum wheat

7.

Gluten

8. Almond

9. Brazil

10. Cashew

11. Tea

12. Walnut

13. Cow’s milk

14. Whole

egg

15. Chicken

16. Lamb

17. Beef

18. Pork

19. White fish

mix1

20. Freshwater fish mix2

21. Tuna

22. Shellfish

Mix3

23. Broccoli

24. Cabbage

25. Carrot

26. Leek

27. Potato

28. Celery

29. Cucumber

30. Peppers4

31. Legume

Mix5

32. Grapefruit

33. Melon Mix6

34. Peanut

35. Soya bean

36 Cocoa bean

37.

Apple

38. Black-currant

39. Olive

40. Orange &

lemon

41. Strawberry

42. Tomato

43. Ginger

44. Garlic

45. Mushroom

46. Yeast

47. Negative Control

48. Positive Control

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4.1. Detection of food IgG antibodies by the Food DetectiveTM (59 Food)

A B A sample from an individual with known gluten sensitivity and intolerance to milk, egg and shellfish.

A sample from an individual with wheat and gluten intolerance showing also IgG antibody responses to cashew nut, tea, cow’s milk, egg, carrot, grapefruit, melon mix and yeast.

1.

Oat 2.

Wheat 3.

Rice

4. Corn

5. Rye

6. Durum wheat

7.

Gluten

8. Almond

9. Brazil

10. Cashew

11. Tea

12. Walnut

13. Cow’s milk

14. Whole

egg

15. Chicken

16. Lamb

17. Beef

18. Pork

19. White fish

mix1

20. Freshwater fish mix2

21. Tuna

22. Shellfish

Mix3

23. Broccoli

24. Cabbage

25. Carrot

26. Leek

27. Potato

28. Celery

29. Cucumber

30. Peppers4

31. Legume

Mix5

32. Grapefruit

33. Melon Mix6

34. Peanut

35. Soya bean

36 Cocoa bean

37.

Apple

38. Black-currant

39. Olive

40. Orange &

lemon

41. Strawberry

42. Tomato

43. Ginger

44. Garlic

45. Mushroom

46. Yeast

47. Negative Control

48. Positive Control

1. Oat

2. Wheat

3. Rice

4. Corn

5. Rye

6. Durum wheat

7.

Gluten

8. Almond

9. Brazil

10. Cashew

11. Tea

12. Walnut

13. Cow’s milk

14. Whole

egg

15. Chicken

16. Lamb

17. Beef

18. Pork

19. White fish

mix1

20. Freshwater fish mix2

21. Tuna

22. Shellfish

Mix3

23. Broccoli

24. Cabbage

25. Carrot

26. Leek

27. Potato

28. Celery

29. Cucumber

30. Peppers4

31. Legume

Mix5

32. Grapefruit

33. Melon Mix6

34. Peanut

35. Soya bean

36 Cocoa bean

37.

Apple

38. Black-currant

39. Olive

40. Orange &

Lemon

41. Strawberry

42. Tomato

43. Ginger

44. Garlic

45. Mushroom

46. Yeast

47. Negative Control

48. Positive Control

Figure 3

IW GD001

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5. TECHNICAL REQUIREMENTS Applicable Directives: In Vitro Medical Devices Directive 98/79EC Medical Devices Directive 94/32EC Solutions adopted to fulfil essential requirements: Compliance with Harmonised Standards BS EN ISO 14971:2009(Medical Devices- Application of Risk management); BS EN 13641:2002 (Elimination or reduction of risk of infection related to in vitro diagnostic reagents); BS EN 13640:2002 (Stability testing of in vitro diagnostic reagents). Also: BS EN ISO 9001:2008 Quality Systems – Model for quality assurance in design / development, production, installation and servicing BS EN 13485:2003 Quality Systems – Medical Devices – Particular requirements for the application of ISO 9001

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6. SPECIFICATION OF MATERIALS AND MANUFACTURING 6.1. Materials included in the kit 6.1.1. Blood Collection

To permit the collection of an accurately measured volume of blood, the following items are supplied in the kit:

Alco-Prep Swab For skin cleansing prior to injection. Manufactured and CE marked by H&W cv-B-3384 Glabbeek.

Sterile Safety Lancet Single use guaranteed. Manufactured and CE marked by Medipurpose Pte. Ltd, 896 Dunearn Road, Sime Darby Centre, Singapore 589472 [CE 0197].

Glass Capillary Tube Manufactured and CE marked by Vitrex Medical A/S, Vasekaer 6-8, 2730 Herlev, Denmark [CE marked]; 50�l volume; lithium-heparinised; colour-code:orange, ID = 1.482+/- 0.02; OD = 1.90 +/- 0.05; L = 29+/- .

Plaster -

6.2. Immunoassay Components

The components selected for the manufacture of the Food DetectiveTM kit are used world-wide for the manufacture of immunoassays for the human healthcare market. None of the solutions contained in The Food DetectiveTM are toxic according to European Directives.

Reaction Tray (59 Food)

Pure white high impact polystyrene Reaction Tray in a sealed foil pouch with desiccant. 48 numbered circular wells arranged in rows of 6 are present on the surface of the tray (Figure 4a, b). 40 wells contain a single food extract (e.g. wheat) and 6 contain a mixture of extracts (e.g. shellfish mix). Wells 47 and 48 provide for negative and positive assay control.

Figure 4a - Reaction Tray Figure 4b - Number wells on surface

Solution A Sample Diluent: Red capped PET bottle containing 10mM Tris buffered saline, a commercially available biomolecular stabiliser, 0.09% sodium azide as preservative, red dye (5ml); ready to use.

Solution B Antibody Detector Solution: Blue capped PET bottle containing horseradish peroxidase (HRP) conjugated goat anti-human IgG in a commercially available HRP enzyme stabiliser; 0.05% Proclin 300 as preservative; blue dye (5ml); ready to use.

Solution C Developer Solution: Black capped, light-proof PET bottle containing a solution of tetramethylbenzidine with a mild oxidising agent (5ml); ready to use.

Solution D Wash Solution: White capped bottle (x2) containing 10mM Tris buffered saline, 0.01% detergent; 100ml; ready to use.

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7. MANUFACTURE PROCESS FLOW CHART

Food DetectiveTM Plate

Control In Process

Preparation of buffers and food extract

solutions according to SOPs - pH determination

- Protein determinations

Manufacture of food extract-coated Reaction Trays according to SOPs

- QC testing against in-house quality control sera

- Reproducibility studies

Plate packaging process according to standard operating procedures - Packaging control

Sample Diluent, Wash Buffer, Antibody Detector Solution, Antibody Developer Solution

Control In Process

Buffers and solutions preparation according to SOPs

- pH measurements - Qc testing using a panel of in-house

quality control sera - Confirmation of assay sensitivity

Filling and Packaging process according to SOPs - Filling control

- Testing bottle check

Final Kit Assembly - Assembly control

FINAL CONTROLS: - QC testing with all the kit

components - Confirmation of acceptance criteria for

batch release

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8. METHODS OF MANUFACTURE

8.1. Food extracts

Where possible, food extracts are prepared from commercially sourced and certified lyophilised foods. Where this is not possible, foods supplied by supermarkets are used. Food extracts are prepared according to published procedures in filtered cabinets under positive pressure. Extracts are filtered through 0.2 micron filters to remove particulate matter. Extracts are treated with 0.09% sodium azide as an anti-microbial agent. Protein determinations are performed for each extract. Extracts are aliquoted and stored at + 4 °C. 8.2. Food extracts

Reaction Trays are coated using automated machinery with 10µl of each food extract at concentrations that have been determined empirically to provide for antibody detection. Manufacture, labelling and packaging are according to ISO9001:2000 quality management system standard operating procedures. 8.3. Solutions Manufacture

Sample diluent, Wash buffer and Antibody Detector are all manufactured according to established and proven standard operating procedures. Developer (Membrane TMB Substrate) is used as supplied by its manufacturer. Manufacture, labelling and bottling are according to ISO9001:2000 quality management system standard operating procedures. 8.4. Quality Control (QC)

For all components, each new manufacture is QC tested against a previous passed batch according to standard operating procedures. Only batches passing the associated acceptance criteria are included in kits. Acceptance criteria include the appearance of the component and its performance in the assay. The results obtained using a newly manufactured batch should be qualitatively the same as the previously passed batch in order for release. QC is according to ISO9001:2000 quality management system standard operating procedures. 8.5. Kit Assembly Kits are assembled, packaged and labelled according to ISO9001:2000 quality management system standard operating procedures.

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9. PERFORMANCE EVALUATION DATA 9.1. Analytical Sensitivity

9.1.1. Limit of Detection

This is defined as the lowest concentration of human IgG detectable by the Food Detective.

Method: Serial dilutions of human IgG from 1mg/ml to 0.1µg/ml were bound to the wells on the Reaction Tray (10µl/well). The tray was then blocked using a commercially available biomolecular stabiliser and dried. Antibody Detector Solution was then added to the Reaction Tray and incubated for 10 minutes as per the Instructions for Use. After washing with Wash Buffer, Developer Solution was added for 2 minutes. The Reaction Tray was then washed with Wash Buffer and dried. The lowest concentration of IgG giving a definite blue spot was then identified. Results: Clear blue spots were detected in wells coated from 1 mg/ml to 1.6µg/ml IgG. Conclusions: The limit of IgG detection was estimated to be 1.6µg/ml or the equivalent of 16ng protein. 9.1.2. Detection of Antibodies to 59 Foods

Method: Samples from coeliac patients that gave positive IgG reactions to all 101 foods in a Mediterranean Food IgG microplate-based ELISA manufactured by Genesis Diagnostics Ltd, UK were used to confirm the detection by the Food Detective of IgG antibodies to 59 foods common to both assays. Assays were conducted according to the Instructions for Use. Results: Figure 5 shows the binding of IgG in the coeliac patient sample. It can be seen that all wells except the negative control (well 47) contain a blue spot. The intensities of the blue spots vary depending on the food in the wells.

Figure 5

Conclusion: The results from this study demonstrate that The Food Detective is able to detect IgG antibodies to all foods arrayed on the Food Detective Reaction Tray where these are present in concentrations that give positive results in the microplate-based ELISA.

Coeliac sample

Negative control

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9.2. Sensitivity, Specific and Accuracy Relative to Conventional Micro-plate Based

ELISA

Method: Relative sensitivity and specificity of the Food Detective was assessed in relation to the detection of positive IgG reactions (>12 U/ml) and negative reactions (<12 U/ml) by the microplate-based 93 Food IgG and Mediterranean Food IgG ELISAs both manufactured by Genesis Diagnostics Ltd. Since the Food Detective is a qualitative assay, borderline results obtained in the semi-quantitative microplate-based assays are considered negative in this study. Results and Conclusions: Table 2 shows the % sensitivity, specificity and accuracy for the detection of IgG's to each food/mixture included in the Food Detective in relation to the target response derived from the microplate-based assay. These data confirm that the Food Detective is suitable for its intended use.

Table 2: Food Detective sensitivity, specificity and accuracy

Food %

Sensitivity %

Specificity %

Accuracy Oat 100 100 100 Wheat 88 100 92 Rice 100 100 100 Corn 100 100 100 Rye 88 100 92 Durum Wheat 100 100 100 Gluten 88 100 92 Almond 100 100 100 Brazil 100 100 100 Cashew 100 100 100 Tea 100 100 100 Walnut 75 100 92 Cow Milk 100 100 100 Whole Egg 100 100 100 Chicken 88 100 92 Lamb 100 100 100 Beef 100 100 100 Pork 100 100 100 White Fish 100 100 100 Freshwater Fish 100 100 100 Tuna 100 100 100 Shellfish 100 100 100 Broccoli 100 100 100 Cabbage 100 100 100

Food %

Sensitivity %

Specificity %

Accuracy Carrot 100 100 100 Leek 100 100 100 Potato 100 100 100 Celery 100 100 100 Cucumber 100 100 100 Peppers 100 100 100 Legume Mix 83 100 92 Grapefruit 100 100 100 Melon Mix 100 100 100 Peanut 100 100 100 Soya Bean 100 100 100 Cocoa Bean 100 100 100 Apple 100 100 100 Blackcurrant 100 100 100 Olive 100 100 100 Orange/Lemon 100 100 100 Strawberry 100 100 100 Tomato 100 100 100 Ginger 100 100 100 Garlic 100 100 100 Mushroom 100 100 100 Yeast 86 100 92

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9.3. Antigen Absorption Studies Specificity has been confirmed using antigen absorption studies. Method: Food IgG positive sera were pre-incubated with the reactive food antigen prior to assay. This allows any IgG antibodies to the foods to complex to the food antigens rendering the antibodies unavailable for binding to the food in the subsequent immunoassay. The results for absorbed serum are compared with those for unabsorbed serum. In the example in Table 3, samples positive for IgG's to tea, lentil, oat, almond, egg, beef, shellfish, cabbage, tomato, mustard, yeast and potato were absorbed with the corresponding antigen at room temperature for 1 hour. Unabsorbed sera were treated with the buffer in which the extracts are dissolved. Absorbed and unabsorbed sera were then diluted in Sample Diluent and the assay was performed as per the Instructions for Use. The intensity of blue spots was scored with 6+ indicating a very intense spot and 1 + a weak positive spot; (-) indicates a negative result. Where no effect of absorption was observed following a one hour incubation at room temperature, absorptions were carried out at 37oC for 2 hours (Table 4). Cross-over absorptions were also performed to confirm food IgG specificity (Table 5 and Table 6). Results: It can be seen from Table 3 that absorption of food IgG positive sera with the cognate antigen significantly decreased antibody binding to the corresponding food on the Reaction Tray for all foods except potato.

Table 3: Comparison of unabsorbed sera and sera absorbed with various antigens

for 1 hour at room temperature.

Tea Lentil Oat Almond Unabsorbed 2+ 3+ 6+ 1+ Absorbed +/- 1+ 3+ +/- Egg Beef Shellfish Cabbage Unabsorbed 5+ 2+ 3+ 5+ Absorbed 1+ 1+ 1+ +/- Tomato Mustard Yeast Potato Unabsorbed 4+ 4+ 5+ 1+ Absorbed 2+ 1+ +/- 1+

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Longer absorptions at 37oC reduced binding further as shown in Table 4.

Table 4: Comparison of unabsorbed sera and sera absorbed with various antigens for

2 hours at 37oC.

Plate 1 Tea Lentil Oat Almond Unabsorbed 2+ 3+ 6+ 1+ Absorbed - - 1+ - Egg Beef Shellfish Cabbage 5+ 2+ 3+ 5+ - - - - Plate 3 Tomato Mustard Yeast Potato Unabsorbed 4+ 4+ 6+ 1+ Absorbed 1+ 2+ 1+ -

Table 5 shows the effect of absorption with yeast extract on the binding of IgG to non-yeast extracts and to yeast. It can be seen that only IgG binding to yeast is affected by absorbing serum with yeast.

Table 5: The effect of absorption with yeast on IgG binding to various antigens.

Plate 1 Tea Lentil Oat Almond Unabsorbed 3+ 4+ 4+ 2+ Absorbed with yeast 3+ 4+ 4+ 2+ Plate 2 Egg Beef Shellfish Cabbage Unabsorbed 3+ 2+ 2+ 5+ Absorbed with yeast 3+ 3+ 2+ 5+ Plate 3 Tomato Mustard Yeast Potato Unabsorbed 4+ 4+ 5+ 3+ Absorbed with yeast 3+ 3+ 1+ 3+

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Table 6: The effect of absorption with mustard, oat and cabbage on IgG binding to

various antigens.

Tea Lentil Oat Almond Unabsorbed 2+ 4+ 4+ 2+ Absorbed with mustard 2+ 4+ 4+ 2+ Egg Beef Shellfish Cabbage Unabsorbed 3+ 2+ 2+ 5+ Absorbed with oat 3+ 3+ 2+ 5+ Tomato Mustard Yeast Potato Unabsorbed 4+ 4+ 5+ 3+ Absorbed with cabbage Neg 3+ 5+ 1+

It can be seen from Table 6 that absorption of serum with mustard had no effect on IgG binding to tea, lentil, oat and almond. Absorption with oat had no effect on IgG binding to egg, beef, shellfish and cabbage. Absorption of serum with cabbage significantly reduced binding of IgG to tomato and to a lesser extent to mustard and potato, but had no effect on IgG binding to yeast. Conclusions: Absorption studies, examples of which are shown in Tables 3-6 have confirmed the specificity of food antibody binding for almost all foods. The finding that absorption with cabbage reduced IgG binding to mustard probably reflects antibody cross-reactivity, since cabbage and mustard are both members of the Mustard family. Antibody cross-reactivity may also explain the effect of cabbage absorption on IgG reactivity with tomato and potato. The latter are both members of the Nightshade family. Food antibody cross-reactivity is well documented; further examples are given in Table 7.

Table 7: Food antibody cross-reactivity

Reactive Foods Cross-reactive Foods

Cows’ milk Other animal milk Hens’ eggs Eggs of other birds Peanuts Various tree nuts; rarely other legumes Various tree nuts Cross react with one another and with peanut Soya bean Seldom cross-reacts significantly with other legumes Fish Other fish

Wheat Other cereals

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9.4. Intralot Reproducibility

Method: 5 randomly selected plates were assayed using corresponding kit components and sera with characterised food IgG immunoreactivity. Patterns of immunoreactivity shown by the kits are compared by visual inspection and recorded to confirm reproducibility.

Results: See Table 8 and Table 9

Table 8: Positive Sample

C10271 Sample 18029 Plate C10271

Sample 18029 Plate

Well Food 1 2 3 4 5 Well Food 1 2 3 4 5 1 Oat + + + + + 25 Carrot + + + + + 2 Wheat + + + + + 26 Leek + + + + + 3 Rice + + + + + 27 Potato + + + + + 4 Corn + + + + + 28 Celery + + + + + 5 Rye + + + + + 29 Cucumber + + + + + 6 Durum Wheat + + + + + 30 Peppers + + + + + 7 Gluten + + + + + 31 Legume Mix + + + + + 8 Almond + + + + + 32 Grapefruit + + + + + 9 Brazil + + + + + 33 Melon Mix + + + + + 10 Cashew + + + + + 34 Peanut + + + + + 11 Tea + + + + + 35 Soya Bean + + + + + 12 Walnut + + + + + 36 Cocoa Bean + + + + + 13 Cow Milk + + + + + 37 Apple + + + + + 14 Whole Egg + + + + + 38 Blackcurrant + + + + + 15 Chicken + + + + + 39 Olive + + + + + 16 Lamb + + + + + 40 Orange/Lemon + + + + + 17 Beef + + + + + 41 Strawberry + + + + + 18 Pork + + + + + 42 Tomato + + + + + 19 White Fish + + + + + 43 Ginger + + + + + 20 Freshwater Fish + + + + + 44 Garlic + + + + + 21 Tuna + + + + + 45 Mushroom + + + + + 22 Shellfish + + + + + 46 Yeast + + + + + 23 Broccoli + + + + + 47 Negative - - - - - 24 Cabbage + + + + + 48 Positive + + + + +

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Table 9: Negative Sample

C10271 Sample MIR Plate C10271

Sample MIR Plate

Well Food 1 2 3 4 5 Well Food 1 2 3 4 5 1 Oat - - - - - 25 Carrot - - - - - 2 Wheat - - - - - 26 Leek - - - - - 3 Rice - - - - - 27 Potato - - - - - 4 Corn - - - - - 28 Celery - - - - - 5 Rye - - - - - 29 Cucumber - - - - - 6 Durum Wheat - - - - - 30 Peppers - - - - - 7 Gluten - - - - - 31 Legume Mix - - - - - 8 Almond - - - - - 32 Grapefruit - - - - - 9 Brazil - - - - - 33 Melon Mix - - - - - 10 Cashew - - - - - 34 Peanut - - - - - 11 Tea - - - - - 35 Soya Bean - - - - - 12 Walnut - - - - - 36 Cocoa Bean - - - - - 13 Cow Milk - - - - - 37 Apple - - - - - 14 Whole Egg - - - - - 38 Blackcurrant - - - - - 15 Chicken - - - - - 39 Olive - - - - - 16 Lamb - - - - - 40 Orange/Lemon - - - - - 17 Beef - - - - - 41 Strawberry - - - - - 18 Pork - - - - - 42 Tomato - - - - - 19 White Fish - - - - - 43 Ginger - - - - - 20 Freshwater Fish - - - - - 44 Garlic - - - - - 21 Tuna - - - - - 45 Mushroom - - - - - 22 Shellfish - - - - - 46 Yeast - - - - - 23 Broccoli - - - - - 47 Negative - - - - - 24 Cabbage - - - - - 48 Positive + + + + +

Conclusions: Manufacturing procedures give rise to highly reproducible intralot assay performance.

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9.5. Interlot Reproducibility

Method: Each kit lot is assayed at QC using sera with characterised food IgG immunoreactivity and the results are compared with those obtained with a previously passed kit lot to confirm reproducibility. Results: See Table 10 Table 10

Sample IW Kit Lot Sample IW Kit Lot

Well Food 10276 10289 10303 Well Food 10276 10289 10303 1 Oat - - - 25 Carrot - - - 2 Wheat + + + 26 Leek - - - 3 Rice - - - 27 Potato - - - 4 Corn - - - 28 Celery - - - 5 Rye + + + 29 Cucumber - - - 6 Durum Wheat - - - 30 Peppers - - - 7 Gluten + + + 31 Legume Mix - - - 8 Almond - - - 32 Grapefruit - - - 9 Brazil - - - 33 Melon Mix - - - 10 Cashew + + + 34 Peanut - - - 11 Tea + + + 35 Soya Bean + + + 12 Walnut - - - 36 Cocoa Bean + + + 13 Cow Milk + + + 37 Apple - - - 14 Whole Egg + + + 38 Blackcurrant - - - 15 Chicken - - - 39 Olive - - - 16 Lamb - - - 40 Orange/Lemon - - - 17 Beef - - - 41 Strawberry - - - 18 Pork - - - 42 Tomato - - - 19 White Fish - - - 43 Ginger - - - 20 Freshwater Fish - - - 44 Garlic - - - 21 Tuna - - - 45 Mushroom - - - 22 Shellfish + + + 46 Yeast - - - 23 Broccoli - - - 47 Negative - - - 24 Cabbage - - - 48 Positive + + +

Conclusions: Manufacturing procedures assure interlot reproducibility.

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9.6. Matrix Effects

Method: The effects of common, potentially interfering substances were assessed by spiking samples with triglycerides to 3g/dL, haemoglobin to 100mg/dl and bilirubin to 40mg/dL. Blood samples with added triglycerides, haemoglobin and bilirubin were then tested in parallel with controls in Food Detective.

Results: See Figure 6 for representative results.

Figure 6

GD blood sample with 3g/dL of triglycerides GD blood sample without triglycerides

GD blood sample with 1000mg/dlL of haemoglobin GD blood sample without haemoglobin

+ Triglycerides - Triglycerides

+ Haemoglobin - Haemoglobin

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Figure 6 cont’d

GD blood sample with added 40mg/dl of bilirubin GD blood sample without bilirubin

Conclusions: No interference was observed with hemolytic (up to 1000 mg/dL), lipemic (up to 3 g/dL triglycerides) or bilirubin (up to 40 mg/dL) containing blood samples.

+ Bilirubin - Bilirubin

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10. STABILITY STUDIES

Method: Stability studies were carried out in accordance with British Standard BS EN 13640. Shelf life is defined as the period until expiry date and stability as the ability of an in vitro diagnostic device, when kept under the specified conditions, to retain throughout the shelf life its properties and or performance within the limits specified by the manufacturer. Stability claims are based on real-time testing. Briefly, to cover the claimed shelf-life, studies were conducted with 3 different lots which had reached the end of the shelf-life of 18 months, and which had been stored at 200oC – 250oC. Results were compared with those of an unexpired lot.

Results: See Tables 11 and 12

Table 11: Positive Sample

Sample 18029 In-date Batch

Expired Sample 18029 In-date Batch

Expired

Well Food 1 2 3 Well Food 1 2 3 1 Oat + + + + 25 Carrot + + + + 2 Wheat + + + + 26 Leek + + + + 3 Rice + + + + 27 Potato + + + + 4 Corn + + + + 28 Celery + + + + 5 Rye + + + + 29 Cucumber + + + + 6 Durum Wheat + + + + 30 Peppers + + + + 7 Gluten + + + + 31 Legume Mix + + + + 8 Almond + + + + 32 Grapefruit + + + + 9 Brazil + + + + 33 Melon Mix + + + + 10 Cashew + + + + 34 Peanut + + + + 11 Tea + + + + 35 Soya Bean + + + + 12 Walnut + + + + 36 Cocoa Bean + + + + 13 Cow Milk + + + + 37 Apple + + + + 14 Whole Egg + + + + 38 Blackcurrant + + + + 15 Chicken + + + + 39 Olive + + + + 16 Lamb + + + + 40 Orange/Lemon + + + + 17 Beef + + + + 41 Strawberry + + + + 18 Pork + + + + 42 Tomato + + + + 19 White Fish + + + + 43 Ginger + + + + 20 Freshwater Fish + + + + 44 Garlic + + + + 21 Tuna + + + + 45 Mushroom + + + + 22 Shellfish + + + + 46 Yeast + + + + 23 Broccoli + + + + 47 Negative - - - - 24 Cabbage + + + + 48 Positive + + + +

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Table 12: Negative Sample

Sample MIR In-date Batch

Expired Sample MIR In-date Batch

Expired

Well Food 1 2 3 Well Food 1 2 3 1 Oat - - - - 25 Carrot - - - - 2 Wheat - - - - 26 Leek - - - - 3 Rice - - - - 27 Potato - - - - 4 Corn - - - - 28 Celery - - - - 5 Rye - - - - 29 Cucumber - - - - 6 Durum Wheat - - - - 30 Peppers - - - - 7 Gluten - - - - 31 Legume Mix - - - - 8 Almond - - - - 32 Grapefruit - - - - 9 Brazil - - - - 33 Melon Mix - - - - 10 Cashew - - - - 34 Peanut - - - - 11 Tea - - - - 35 Soya Bean - - - - 12 Walnut - - - - 36 Cocoa Bean - - - - 13 Cow Milk - - - - 37 Apple - - - - 14 Whole Egg - - - - 38 Blackcurrant - - - - 15 Chicken - - - - 39 Olive - - - - 16 Lamb - - - - 40 Orange/Lemon - - - - 17 Beef - - - - 41 Strawberry - - - - 18 Pork - - - - 42 Tomato - - - - 19 White Fish - - - - 43 Ginger - - - - 20 Freshwater Fish - - - - 44 Garlic - - - - 21 Tuna - - - - 45 Mushroom - - - - 22 Shellfish - - - - 46 Yeast - - - - 23 Broccoli - - - - 47 Negative - - - - 24 Cabbage - - - - 48 Positive + + + +

Conclusions: The results obtained using the 3 expired lots were not different from those obtained with a newly manufactured lot. It is concluded that product performance is maintained for a period of 18 months after the date of manufacture when the product is stored at room temperature (200oC – 250oC).

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10.1. Stability Under Adverse Shipping Conditions

Method: Food Detective kits were stored at +37oC and -20oC for 3 days to mimic extreme temperatures during shipping. A full plate negative sample (AS) and a sample giving a high number of positives (GD) were then tested in the kits and the performance of the kits evaluated against kits stored under the recommended storage conditions.

Results: Storage at -20oC and +37oC had no effect on the performance of Food Detective™ as shown in Figure 7A, B).

Figure 7A

Figure 7B

Conclusions: Food Detective™ is stable under the temperatures investigated and is unlikely to be adversely affected by storage at extreme temperatures during shipping.

Recommended +37oC AS -20oC AS

Recommended +37oC GD -20oC GD

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APPENDIX 1 - Certificate of Analysis (Example)

25

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English Deutsch Italiano Español Français Português

I Refer to instructions for use Gebrauchsanweisung lesen Consultare le istruzioni per l'uso Consulte instrucciones para su uso

Consulter le mode d'emploi Consultar instruções de uso

Light Sensitive Nicht direktem Sonnenlicht aussetzen Proteggere dalla Luce Solare Proteger de la luz solar Sensible à la lumière Sensivel á luz

Storage Temperature Lagerungstemperatur Limiti di Temperatura Temperatura de almacenamiento La température de stockage Temperatura de armazenamento

e Use By Verfallsdatum Utilizzare entro Fecha de caducidad Date de péremption Data de validade

Hungarian Polish Norwegian Swedish Danish Slovakian

I Olvassa el a Használati ÚtmutatótPrzed użyciem zapoznaj się z instrukcją

Henviser til bruksanvisning Hänvisar till bruksanvisning Henvis til brugsanvisning Pred použitím si pozorne prečítajte návod na použitie

Fényérzékeny Światłoczułe Sensitiv for lys Ljuskänslig Lyosfølsom Nevystavujte priamemu slnečnému žiareniu

Tárolási hőmérséklet Przechowywać w temperaturze Lagringstemperatur Förvaringstemperatur Opbevaringsteperatur Skladujte pri teplote

e Felhasználható Zużyć przed Brukes innen Bäst före datum Mindst holdbar til Spotrebujte do

Bulgarian Česky Hrvatski ภาษาไทย Estonian Icelandic

I Прочетете инструкцията Před použitím pečlivě prostudujte návod k použití

Uputstvo za korištenje คําแนะนําสาํหรับการใช ้ Vaata instruktsiooni Fylgið leiðbeiningunum

Чувствителен на светлина Chraňte před slunečním zářením Osjetljivo na svjetlo ไวต่อแสง Valgustundlik ljósnæmt

Температура за съхранение Skladujte při teplotě Temperatura skladištenja อุณหภูมทิ ี@ใชใ้นการเก็บรักษา Säilitamise temperatuur Geymslu-hitastig

e Употреба: Spotřebujte do Upotrijebiti do ใชโ้ดย Aegumise kuupäev Notist fyrir

Português - Brasil

I Consultar instruções de uso

Sensivel á luz

Temperatura de armazenamento

e Data de validade

Version 20.0

Tabuleiro de reacção[TRAY] Reaction Tray Testplatte Vassoio di reazione Bandeja de reacción Bac de réaction

Solução C - Solução de Revelação

[SOLN|D] Solution D - Wash Solution Lösung D - Waschlösung Soluzione D - Soluzione di lavaggio Solución D - Líquido de lavado Solution D - Solution de lavage Solução D - Solução de Lavagem

[SOLN|C] Solution C - Developer Solution Lösung C - Entwicklerlösung Soluzione C - Soluzione di sviluppo Solución C - Líquido revelador Solution C - Solution de développement

Solução A - Diluente de Amostra

[SOLN|B] Solution B - Detector Solution Lösung B - Nachweislösung Soluzione B - Soluzione di rilevazione Solución B - Líquido detector Solution B - Solution de détection Solução B - Solução de Detecção

[SOLN|A] Solution A - Sample Diluent Lösung A - Probenverdünner Soluzione A - Diluente per campioni Solución A - Líquido diluyente Solution A - Diluant d'échantillon

Fabricante

[LOT] Lot Number Lotnummer Numero del lotto Número de lote Numéro du lot Número de Lote

M Manufacturer Hersteller Fabbricante Fabricante Fabricant

SYMBOLS USED ON LABEL / SYMBOLE DIE AUF DEN ETIKETTEN VERWENDET WERDEN /SIMBOLI UTILIZZATI NELLE ETICHETTESIMBOLOS UTILIZADOS EN LA ETIQUETA/ LES SYMBOLES ONT UTILISE SUR L'ETIQUETTE / SÍMBOLOS USADOS NO RÓTULO

[IVD] In Vitro Diagnostic Medical Device In vitro Diagnostikum Disposit ivo medico diagnostico in vitro Dispositivo médico de diagnóstico in vitro

Appareil médical pour diagnostic in vitro Dispositivo médico para diagnóstico in vitro

Greiningar Bakki[TRAY] Реакционен диск Reakční podložka Reakcijska pločica ถาดที@ใชทํ้าปฏกิริ ิยา Tulemuste alus

Lausn C- mótefna binding

[SOLN|D] Разтвор D – Измиващ разтвор Roztok D - Promývací roztok Otopina D - otopina za ispiranje สารละลาย D - สารละลายที@ใชล้า้ง Lahus D - Pesemise lahus Lausn D- skol lausn

[SOLN|C] Разтвор С – Проявител Roztok C - Barvící roztok Otopina C - otopina za razvijanje สารละลาย C - สารละลายที@ทําให ้เกดิส ี

Lahus C - Ilmutamise lahus

Lausn A- þynnunarvökvi fyrir sýni

[SOLN|B] Разтвор В – Индикатор Roztok B - Detetekční roztok Otopina B - otopina za otkrivanje สารละลาย B - สารละลายที@ใชจั้บสิ@งสง่ตรวจ

Lahus B - Tuvastamise lahus Lausn B- Nemur mótefni

[SOLN|A] Разтвор А – Дилуент за проба Roztok A - Ředicí roztok Otopina A - otopina za razrjeđenje uzorka สารละลาย A - สารที@ใชล้ะลายสิ@งสง่ตรวจ

Lahus A - Proovi lahusti

Framleiðandi

[LOT] Лот номер Výrobní šarže Broj serije ตัวเลขล๊อตสนิคา้ของการผลติ Seeria number Lotu númer

M Производител Výrobce Proizvođač โรงงานผูผ้ลติ Tootja

Reakčná podložka

ИЗПОЛЗВАНИ СИМВОЛИ / SYMBOLY UVEDENÉ NA ETIKETĚ/SIMBOLI KOJI SE KORISTE NA ETIKETI/ สัญลักษณ์ที@แสดงอยูบ่นฉลาก / ETIKETIL KASUTATUD SÜMBOLID/Tákn notuð á miða

[IVD] Тест за Ин-витро диагностика In-vitro diagnostikum In vitro dijagnostika อุปกรณว์เิคราะหท์างการแพทย์ Kehavälise diagnostika meditsiiniseade Greiningarpróf

[TRAY] Reakció tálca Taca reakcyjna Testpanel Reaktionsplatta Reaktionsbakke

Roztok C - Vývojový roztok

[SOLN|D] "D" Oldat - Mosó oldat Roztwór D - Do przemywania Oppløsning D - skylle oppløsning Lösning D Løsning D- Vaskeopløsning Solution D - Čist iaci roztok

[SOLN|C] "C" Oldat - Előhívó oldat Roztwór C - Do wywoływania Oppløsning C - fremkaller væske Lösning C Løsning C-Fremkalderopløsning

Roztok A - Riedidlo na vzorku

[SOLN|B]"B" Oldat - Ellenanyag kimutató

oldat Roztwór B - Do wykrywacz przeciwciał Oppløsning B - detektor væske Lösning B Løsning B-Detektor opløsning Roztok B - Detekčný roztok

[SOLN|A] "A" Oldat - Mintahígító Roztwór A - Do rozcieńczania próbki Oppløsning A - prøvefortynner Lösning A Løsning A- Opløsningsvæske test (prove)

In Vitro diagnostická pomôcka

Výrobca

[LOT] Gyártási szám Numer Partii Lot nummer LOT nummer Varenummer

In vitro diagnostiseringsredskap Invitro diagnostik In vitro diagnostik medicinsk apparat

CIMKÉKEN HASZNÁLT JELÖLÉSEK /OZNACZENIA UŻYTE NA NAKLEJCE/ SYMBOLER PǺ ETIKETTENE /SYMBOLER SOM ANVÄNDS PǺ FÖR PACKNINGEN /SYMBOLER BRUGT PǺ ETIKETTERNE/SYMBOLY UVEDENÉ NA ETIKETE

[IVD] In-vitro diagnosztikum Medyczne urządzenie do diagnostyki in Vitro

Producent Fabrikant Tillverkare Producent (fabrikant)

[SOLN|C] Solução C - Solução Reveladora

Číslo dávky

M Gyártó

[TRAY] Placa de Reação

[SOLN|D]

[SOLN|B]Solução B - Detector de

Anticorpo (Conjugado)

Solução D - Solução de Lavagem

M Fabricante

[SOLN|A] Solução A - Diluente de Amostra

[LOT] Número de Lote

SÍMBOLOS USADOS NO RÓTULO/

[IVD]Dispositivo médico para

diagnóstico in vitro

2oC

25oC

t

2oC

25oC

t

2oC

25oC

t

2oC

25oC

t

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APPENDIX 2 - Bibliography

CODE PUBLICATION DETAILS

W1 Dietary advice based on food specific IgG Results Geoffrey Hardman, Gillian Hart, University of York, Heslington, York, UK Nutrition and food science Vol 37 No 1 2007 pp 16-23

GA1 Food elimination based on IgG antibodies in irritable bowel syndrome: a randomised controlled trial. W Atkinson, T A Sheldon, N Shaath, PJ Whorwell Gut 2004:53 1459-1464 doi: 10.1136

GA2 Food allergy in irritable bowel syndrome: new facts and old fallacies. E Isolauri, S.Rautava, M.Kalliomaki Gut 2004; 53 1391-1393 10.1136

M1 A prospective audit of Food Intolerance among Migraine patients in primary care clinical practise. Trevor Rees, David Watson, Susan Lipscombe, Helen Speight, Peter Cousins, Geoffrey Hardman and Andrew J. Dowson. Headache care Vol.2 No 2 2005 105-110

GA3 Celiac Disease. Peter H.R Green M.D. and Christopher collier, M.D. PhD The New England Journal of Medicine 2007; 357:1731-43

GA4

Alterations of food antigen-specific serum immunoglobulins G and E in patients with irritable bowel syndrome and functional dyspepsia. X.L.Zuo, Y.Q. Li, W.J.Li, Y.T. Guo, X.F. Lu, J.M. Li and P.V. Desmond Clinical and Experimental Allergy, 37, 823-830

W2

IgG Antibodies against Food Antigens are Correlated with Inflammation and Intima Media Thickness in Obese Juveniles M. Wilders-Truschnig, H.Mangge, C.Lieners, H.J.Gruber, C Mayer, W Marz Exp Clin Endocrinol Diabetes 2008; 116:241-245

R1 A Vegan diet free of gluten improves the signs and symptoms of Rheumatoid Arthritis.. I Hafstöm, B.Ringertz, A. Spångberg, L. Von Zweigbergk, S. Brannemark, I. Nylander, J.Rönnelid, L.Laasonen, L.Klareskog. British Society of Rheumatology, 2001 pp 1175-1179

R2 The gut-joint axis: cross reactive food antibodies in rheumatoid arthritis. M Hvatum, L Kanerud, R Hällgren, P Brandtzaeg Gut 2006:55 1240-1247 originally published online 16 feb 2006

GN1 Toward an understanding of Allergy and In-Vitro Testing By Mary James N.D Great Smokies Diagnostic Laboratory

GN2 Reported food Intolerance and respiratory symptoms R.K.Woods, M.Abramson, J.M.Raven, M Bailey, JM Weiner, E.H.Walters Eur Respir J 1998 11:151-155

GN3 A critical review of IgG Immunoglobulins and Food Allergy- Implications in systemic health Raymond M. Suen, MT (ASCP), Shalima Gordon, ND 13500 Linden Ave. N. Seattle, WA 98133 Ph: (206) 365-1256, (877) 318-8728, Fax: (206) 363-8790

GN4 The clinical relevance of IgG food allergy testing through ELISA (Enzyme-Linked Immunosorbent Assay). Townsend Letter for Doctors and Patients | Date: 1/1/2004 Author: Suen, Raymond M.; Gordon, Shalima

GN6

Gastrointestinal Candida colonisation promotes sensitisation against food antigens by affecting the mucosal barrier in mice. N Yamaguchi, R Sugita, A Miki, N Takemura, J Kawabata, J Watanabe, K Sonoyama Gut2006; 55:954-960. Doi: 10.1136/gut.2005.084954

GN7

Ovalbumin-specific immunoglobulin G and subclass responses through the first 5 years of life in relation to duration of egg sensitization and the development of asthma. G.H.S Vance*, C.A. Thornton*, T.N.Brynat, J.A.Warner * and J.O.Warner*Child Health, Infection, Inflammation & Repair Division and Information & Computing Division, University of Southhampton, UK

27

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APPENDIX 3 - Labels