FINAL REPORT EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA. Authors Fernando Hernandez Aaron Peacock
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1 FINAL REPORT EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Authors Fernando Hernandez Aaron Peacock Performing Laboratory Microbac Laboratories, Inc. 800 Oak Ridge Turnpike Suite A-702 Oak Ridge, TN Laboratory Project Identification Number Z8F0069 Sponsor Nima Labs, Inc 450 Alabama Street San Francisco, CA Page 1 of 20
2 Final Report: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Project No. Z8F0069 TABLE OF CONTENTS FINAL REPORT - COVER PAGE TABLE OF CONTENTS COMPLIANCE STATEMENT STUDY DATES AND FACILITIES RECORDS TO BE MAINTAINED TEST SUMMARY 4 TEST CONDITIONS RESULTS CONCLUSIONS APPENDIX 10 Page 2 of 20
3 Final Report: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Project No. Z8F0069 COMPLIANCE STATEMENT The peanut ELISA testing (r-biopharm AOAC-RI PT030404) was conducted under Scope of Accreditation to ISO-17025:2005, certificate number The analysis of the Nima sensor was conducted per manufacturers guidelines. The following technical personnel participated in this study: Fernando Hernandez, Aaron Peacock and Maraea Clark Study Director: Microbac d- Aaron Peacock, PhD Page 3 of 20
4 Final Report: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Project No. Z8F0069 STUDY DATES AND FACILITIES The laboratory phase of this test was performed at Microbac Laboratories, 800 Oak Ridge Turnpike Suite A-702, Oak Ridge, TN Testing was initiated on 6/21/18 and was completed on 7/26/18. RECORDS TO BE MAINTAINED All testing data, protocol, protocol modifications, test material records, the final report, and correspondence between Microbac Laboratories and the sponsor will be stored in the archives at Microbac Laboratories, 800 Oak Ridge Turnpike Suite A-702, Oak Ridge, TN 37830, or at a controlled facility off site. TEST SUMMARY TITLE: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA BACKGROUND: Nima has developed a portable device intended for use by consumers to detect the presence of peanut in foods. Peanut allergy is one of the most common causes of fatality related to food allergies in the US. In one study it accounted for over 50% of reported fatalities (Bock SA, Muñoz-Furlong A, Sampson HA. Further fatalities caused by anaphylactic reactions to food, Journal of Allergy and Clinical Immunology. 2007, 119(4):1016-8). A strict and life-long peanut-free diet is the only current treatment for these patients, but it can be difficult to adhere to because of cross-contamination during food manufacturing or preparation and inadequate labeling and testing (Remington BC, Baumert JL, Marx DB, Taylor SL. Quantitative risk assessment of foods containing peanut advisory labeling. Food and Chemical Page 4 of 20
5 Final Report: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Project No. Z8F0069 Toxicology. 2013, 62:179-87). One potential solution to this public health challenge would be for consumers, including parents of allergic children, to test their food for peanut prior to consumption. Thus, the portable device developed by Nima could fill this void. Prior to development of the peanut sensor, Nima Labs introduced its gluten sensor for the detection of gluten in foods. The peanut sensor was built on the same technology, with modifications made to the underlying chemistry for applicability to peanut. Unlike gluten, there has been no industry or FDA accepted threshold for peanut in foods. The Nima peanut sensor has a threshold or LOD of 10 ppm, corresponding to the lowest LOAEL reported by the FDA (FDA/Threshold Working Group: Journal of Food Protection Vol. 71, No. 5, 2008). OBJECTIVE: The study was a comparative assessment of the portable Nima peanut sensor against a readily available commercial ELISA testing kit, the r-biopharm RIDASCREEN FAST peanut ELISA, in a range of food products containing known levels of peanut. The Nima peanut sensor has been developed to detect 10 ppm of peanut in food matrices. Additional information on assay performance below and above the target concentration level of 10 ppm was assessed as well. APPROACH: A variety of peanut-free foods were formulated by Nima to cover a range of different components and processing/preparation conditions. Peanut in the form of a NIST traceable peanut butter sample (SRM Peanut Butter) was spiked into these foods at various levels (0, 5, 10, 20, 40, and 100 ppm), some before processing or preparation, some after. To perform spiking the peanut butter was added into Nima s proprietary extraction buffer solution. Sensors and capsules were provided by Nima Labs and commercially available ELISA kits were purchased by Microbac. TEST MATERIALS: The following peanut-free foods were formulated from typical recipes to simulate both packaged and processed foods. - Bread Page 5 of 20
6 Final Report: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Project No. Z8F Brownie - Coconut milk - Cookie - Dark chocolate - Fried chicken - Marzipan - Muffin - Taffy - Soy sauce TEST CONDITIONS: Challenge material: Peanut allergen. Various dilutions of peanut were added to each food in quantities needed to provide formulations with 5 ppm, 10 ppm, 20 ppm, 40 ppm and 100 ppm peanut in the finished food product (based on theoretical 100% recovery). Bread, cookie, and muffin doughs as well as brownie batter were spiked prior to baking to simulate incurred samples. The remaining samples, coconut milk, dark chocolate, fried chicken, marzipan, taffy and soy sauce were spiked post processing. Estimates of the amount of diluted peanut added to the formulations were made based on formulation weight loss or gain from processing noted after the production of the peanut-free version of each food product (e.g. bread, brownie, muffin). The peanut-free version of the food served as the unspiked/negative control. TEST METHODS: r-biopharm RIDASCREEN FAST peanut ELISA tests (R6202) AOAC-RI PT were run according to manufacturer s instructions. See Appendix. The ELISA was run with duplicate extractions according to kit instructions, and duplicate measurements were made for each extraction. Nima peanut sensors were run according to manufacturer s instructions. Each sample provided had a target weight of 300 mg. Each food matrix was run in replicates of 6, for each ppm level. Laboratory personnel conducting testing with the Nima sensors were instructed to familiarize themselves with the testing instructions on the Nima website and watch the instructional videos. Nima protocol (in short); - Obtain a supplied food sample. Page 6 of 20
7 Final Report: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Project No. Z8F Add food sample to the capsule and close the cap (to grind the food). - Both chocolate and taffy samples should be broken into small pieces before addition to the capsule. - Close the cap shut until the purple ring disappears. - Immediately insert the capsule into the sensor (ensure capsule is inserted all the way). - Press the button on the sensor and wait for result. - Record Nima result. RESULTS Results are compiled in Exhibit 1. The following key is used for Nima tests. Key for Nima sensor results: - Smile: No peanut allergen detected (Smiley face on sensor) - Found: Peanut allergen detected (Peanut symbol on sensor) - Error: No result, sensor peanut allergen test was not successful (exclamation point symbol on sensor). - NA: Not analyzed. Nima sensor performance was consistent and accurate across most food matrices and ppm levels. Dark chocolate, however, did not follow this trend and will be discussed subsequently. For all samples except for dark chocolate the Nima sensor had a 0% false negative rate (FNR) at or above the target LOD of 10 ppm. The one false negative reported by the sensor in this study occurred in 1 of 6 cookies tested at 5 ppm. Brownie, marzipan, muffin, taffy, soy sauce, fried chicken, and bread were detected by the sensor in all instances at 5 ppm and above. With the exception of the 100 ppm coconut milk sample, this matrix also had a 0% FNR; it is thought that a spiking error occurred in the preparation of this sample as the ELISA results also reported peanut below the LOQ of the assay. The Nima sensor reported two False Positives: 1 of 6 brownies and 1 of 6 soy sauce tests. Combined with the one false negative at 5 ppm for the cookie matrix, the false Page 7 of 20
8 Final Report: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Project No. Z8F0069 report rate was 0.9% (3 false reports out of 324 tests). A high no result (error) rate was observed with the dark chocolate matrix. There were several attempts to adjust the sample preparation before adding the sample to the capsule including homogenization, adding water, and melting. Neither homogenization or water yielded the desired results, as evidenced by the 0 ppm and 40 ppm results, respectively. It should be noted that testing at 10 ppm was repeated with more successful results (not shown in Exhibit 1). A second round of testing at 10 ppm yielded 100% detection but was performed with a sample that had not undergone a long storage time at -20C. Melting the sample prior to testing (e.g. 100 ppm) showed promise but may be impractical for an end user. Testing of dark chocolate was not completed. The test error rate with the Nima sensor was 2.8% (9 out of 324 tests). Errors occurred with coconut milk (1), soy sauce (1), fried chicken (4), and bread (3). CONCLUSIONS This study was a comparative assessment of the portable Nima peanut sensor against a readily available commercial ELISA testing kit in a range of food products containing known levels of peanut. At 10 ppm and above, the Sensitivity of the Nima Peanut Sensor was 100.0% and the Accuracy was 99.2% (CI: 98.1% - 100%). The FNR was 0.0% and the False Positive Rate (FPR) was 4.2% (CI: 0% - 9.9%). The Dark Chocolate samples were excluded from the testing due to the issues with matrix preparation. The Nima sensor was found to have a 0.9% false report rate for the foods and peanut concentrations tested (three false reports out of 324 tests). The error rate for the Nima sensor was 2.8% for the foods and concentrations tested (nine errors out of 324 tests). It is possible that the run errors could be minimized by optimizing the preparation of the food sample prior to loading the capsule. Page 8 of 20
9 Final Report: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Project No. Z8F0069 *Only three samples were run at selected concentration. NA: Not Analyzed. Std. Dev.: Standard Deviation. Page 9 of 20
10 Final Report: EVALUATION OF THE NIMA PEANUT SENSOR: VALIDATION WITH MULTIPLE MATRICES AND COMPARISON TO ELISA Project No. Z8F0069 Appendix r-biopharm RIDASCREEN FAST Art.Nr. R6202 Peanut ELISA Kit Instructions Page 10 of 20
11 RIDASCREEN FAST Peanut Art. Nr. R6202 Enzymimmunoassay zur quantitativen Bestimmung von Erdnuss Enzyme immunoassay for the quantitative determination of peanut In vitro Test Lagerung bei 2-8 C Storage at 2-8 C
12 RIDASCREEN FAST Peanut Brief information RIDASCREEN FAST Peanut (Art. No.: R6202) is a sandwich enzyme immunoassay for the quantitative analysis of peanut and parts of peanut in food. Breakfast cereals, cookies, ice cream and milk chocolate have been approved by the AOAC Research Institute according to the Performance Tested Method Program with the license No All reagents required for the enzyme immunoassay - including standards - are contained in the test kit. The test kit is sufficient for 48 determinations (including standards). A microtiter plate spectrophotometer is required for quantification. The ELISA RIDASCREEN FAST Peanut is certified at AOAC-RI (030404). Sample preparation: Time requirement: Limit of detection: homogenization, extraction and centrifugation sample preparation (for 10 samples)... approx. 20 min test implementation (incubation time) min 0.13 mg/kg (ppm) peanut Limit of quantification: 2.5 mg/kg (ppm) peanut Standard material: Specificity: The reference material NIST SRM 2387 peanut butter is used as standard material. The antibodies specifically detect peanut proteins, including the peanut allergen Ara h 1 and Ara h 2. There is a cross reactivity to green pea, lentils and fenugreek. Cross reactivities of the used antibodies have been determined for the pure food (e.g. corn flour). In a composed / processed food (e.g. maize bread) cross 12 RIDASCREEN FAST Peanut
13 reactivities might be different. Interfering substances (e.g. polyphenols) can be detected by spike experiments. In order to increase the quality of assessment when performing ELISAprocedures, we refer additionally to our Good ELISA Practice (GEP) Manual in the respective version. Such lists minimum standards concerning the framework conditions when using test-kits of R-Biopharm AG and performing ELISA-analysis. The manual can be retrieved, printed and downloaded under the website Related products Bioavid Lateral Flow Erdnuss / Peanut (Art. Nr. BL / BL606-25) SureFood ALLERGEN ID Peanut (Art. No. S3103) SureFood ALLERGEN QUANT Peanut (Art. No. 3203) 1. Intended use RIDASCREEN FAST Peanut test is a sandwich enzyme immunoassay for the quantitative analysis of peanuts or parts of peanuts in food. Peanut can be present as an ingredient or as a contamination in raw or heated food. 2. General The allergen can be present as an ingredient or as a contamination in raw and cooked products. According to the regulation (EU) No. 1169/2011, peanut and products thereof must be declared on food labels. Similar regulations exist e.g. in the USA, Canada, Australia and New Zealand. RIDASCREEN FAST Peanut was validated with breakfast cereals, cookies, ice cream and milk chocolate in an independent study by three independent US laboratories under the supervision of the AOAC Research Institute. Each laboratory analyzed 20 blank samples and 20 samples spiked at 5 mg/kg peanut of each matrix. In total, two of 240 blank samples were measured positive (> 2.5 mg/kg peanut) corresponding to a specificity of 99.2 % (238/240). All of the spiked samples (240/240) were found positive (sensitivity = 100 %). 3. Test principle RIDASCREEN FAST Peanut
14 The wells of the microtiter strips are coated with specific antibodies to peanut. By adding standards and samples to the wells, peanut present will bind to the specific antibodies. In a washing step components not bound are removed. Then antibody conjugated to peroxidase is added. This antibody conjugate is bound to the Ab- Ag-complex. An antibody-antigen-antibody (sandwich) complex is formed. Any unbound conjugate is then removed in a washing step. The detection of peanut takes place by adding Substrate/Chromogen solution. The enzyme conjugate converts the chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm. The absorbance is proportional to the peanut concentration of the sample. The result is expressed in mg/kg peanut. 4. Reagents provided Each kit contains sufficient materials for 48 measurements (including standard analyses). Component Cap color Format Volume Microtiter plate - Ready to use 48 wells Allergen extraction buffer Green Concentrate 10x 100 ml Standard 1* Transparent Ready to use 0 mg/kg 2.6 ml Standard 2* Transparent Ready to use 2.5 mg/kg 2.6 ml Standard 3* Transparent Ready to use 5.0 mg/kg 1.3 ml Standard 4* Transparent Ready to use 10.0 mg/kg 1.3 ml Standard 5* Transparent Ready to use 20.0 mg/kg 1.3 ml Wash buffer Brown Concentrate 10x 100 ml Conjugate Red Ready to use 6 ml Substrate/Chromogen Red Chromogen Pro Brown Ready to use 10 ml Stop solution Yellow Ready to use 14 ml *) The dilution factor 20 for the sample has already been considered when labeling. Therefore, the peanut concentration of a sample can directly be read from the standard curve. 14 RIDASCREEN FAST Peanut
15 5. Reagents required but not provided 5.1. Equipment: microtiter plate spectrophotometer (450 nm) centrifuge + centrifugal vials shaker water bath laboratory mincer / grinder, pestle and mortar, Ultra-Turrax or homogenisator graduated pipettes variable µl and µl micropipettes 5.2. Reagents: distilled or deionized water skim milk powder (food quality) 6. Warnings and precautions for the users This test should only be carried out by trained laboratory employees. The instruction for use must be strictly followed. This kit may contain hazardous substances. For hazard notes on the contained substances please refer to the appropriate material safety data sheets (MSDS) for this product, available online at 7. Storage instructions Store the kit at 2-8 C (35-46 F). Do not freeze any of the test kit components. Return any unused microwells to their original foil bag, reseal them together with the desiccant provided and further store at 2-8 C (35-46 F). The substrate/chromogen is light sensitive, therefore, avoid exposure to direct light. No quality guarantee is accepted after the expiration date on the kit label. Do not interchange individual reagents between kits of different lot numbers. RIDASCREEN FAST Peanut
16 8. Indication of instability or deterioration of reagents any bluish coloration of the red stained substrate/chromogen prior to test implementation a value of less than 0.8 absorbance units (A 450 nm 0.8) for standard 5 9. Preparation of Samples Working devices such as a mill, glass vials or spatulas must be cleaned before and after each sample preparation to remove any remains of peanut and to avoid contamination. The Allergen Extraction buffer is provided as a 10fold concentrate. Before dilution of the buffer concentrate dissolve any crystals in a water bath at 37 C (99 F) completely and mix well. After that dilute the heated buffer concentrate 1:10 (1+9) with distilled water before use (i.e. 100 ml buffer concentrate ml distilled water). The diluted buffer is stable at room temperature for approx. twelve weeks Sample preparation (without skim milk powder) 5 g of the sample should be ground well and thoroughly mixed weigh 1 g of sample and add 20 ml diluted extraction buffer (the extraction buffer should have already been heated to approx. 60 C (140 F)) mix intensively and extract for 10 min at 60 C (140 F) by shaking casually centrifuge: 10 min / 2500 g / if possible at 4 C (39 F) and/or filter the extract (alternatively 2 ml of the extract can be centrifuged for 10 minutes at high speed in reaction caps by using a microcentrifuge) use 100 µl of the supernatant or filtrate per well in the assay 9.2. Sample preparation (with skim milk powder) for tannin or polyphenol containing samples like spices (e.g. pepper, paprika) 5 g of the sample should be ground well and thoroughly mixed weigh 1 g of sample and add 1 g skim milk powder add 20 ml diluted extraction buffer (the extraction buffer should have already been heated to approx. 60 C (140 F)) mix intensively and extract for 10 min at 60 C (140 F) by shaking casually centrifuge: 10 min / 2500 g / if possible at 4 C (39 F) and/or filter the extract (alternatively 2 ml of the extract can be centrifuged for 10 minutes at high speed in reaction caps by using a microcentrifuge) 16 RIDASCREEN FAST Peanut
17 use 100 µl of the supernatant or filtrate per well in the assay Remark: To improve the recovery the addition of skim milk powder is also recommended for chocolate containing samples and ice cream. The sample extracts can be stored at 2-8 C (35-46 F) for 5 days. The extracts can be stored at -20 C (-4 F) for several months. 10. Test implementation Preliminary comments Bring all reagents to room temperature (20-25 C / F) before use. The wash buffer is provided as a 10fold concentrate. Before dilution of the buffer concentrate dissolve any crystals in a water bath at 37 C (99 F) completely and mix well. After that dilute the heated buffer concentrate 1:10 (1+9) with distilled water before use (i.e. 100 ml buffer concentrate ml distilled water). The diluted wash buffer is stable at room temperature for approx. four weeks Test procedure Carefully follow the recommended washing procedure. Do not allow microwells to dry between working steps. Do not use more than 3 strips (24 wells) at a time. In the case of more than three strips, a second uncoated plate (e.g. low binding from Greiner bio-one Cat.-No or Mikrotiter Assembly Breakable Strip 1x8, Thermo Scientific) should be used as a pre-plate to avoid a time shift over the microtiter plate. All standards and samples are pipetted into the uncoated plate (at least 150 μl per well) and then quickly transferred to the coated microtiter plate with an 8-channel pipette. It is recommended to pipette the conjugate, the substrate/chromogen and the stop solution with a multi-channel or stepper pipette to avoid a time shift over the plate. 1. Insert a sufficient number of wells into the microwell holder for all standards and samples to be run. Record standard and sample positions. RIDASCREEN FAST Peanut
18 2. Add 100 µl of each standard or prepared sample to separate wells and incubate for 10 min at room temperature (20-25 C / F ). 3. Pour the liquid out of the wells and tap the microwell holder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. Fill all the wells with 250 µl wash buffer (see 10.1.) and pour out the liquid again. Repeat two more times. 4. Add 100 µl of the enzyme conjugate to each well. Mix gently by shaking the plate manually and incubate for 10 min at room temperature (20-25 C / F). 5. Pour the liquid out of the wells and tap the microwell holder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. Fill all the wells with 250 µl wash buffer (see 10.1.) and pour out the liquid again. Repeat two more times. 6. Add 100 µl of the reddish substrate/chromogen to each well. Mix gently by shaking the plate manually and incubate for 10 min at room temperature (20-25 C / F) in the dark. 7. Add 100 µl of the stop solution to each well. Mix gently by shaking the plate manually and measure the absorbance at 450 nm. Read within 10 minutes after addition of stop solution. 11. Results A special software, the RIDA SOFT Win / RIDA SOFT Win.net (Art. Nr. Z9996), is available for evaluation of the RIDASCREEN enzyme immunoassays. The calculation should be done by use of a cubic spline function. The course of the standard curve is shown in the Quality Assurance Certificate enclosed in the test kit. In comparison with the certificate, higher values of the absorbance (A 450 nm ) for the standard curve, especially for the zero standard, may be a result of insufficient washing or peanut contamination. A further dilution and new detection of the samples is recommended for absorbance values (A 450 nm ) > standard 5. Please note: When working according to the test kit instructions, the dilution factor is 20. The allergen concentration can be read directly from the standard curve (see 4. *) - the sample dilution factor of 20 is already taken into account). 18 RIDASCREEN FAST Peanut
19 For sample dilutions of more than 1:20 the further dilution factor must be considered for the calculation of the peanut concentration. In general: Samples tested negative still could contain an allergen contamination below the limit of detection of the assay, or they might contain other allergen components like lipids for example. Due to the multitude of food types, matrix effects cannot be excluded. In processed food (e.g. heat treatment, dehydration, etc.), proteins may be altered or fragmented, this may have an impact on the recovery/cross reactivity. Allergen containing samples that have been heat treated show a reduced recovery because the proteins denature and are no longer recognized by the antibody. The reduction in recovery depends strongly on the temperature and the duration of the heat treatment. If samples are heat treated at high temperature the recovery can be significantly reduced. For evaluation of the cross reactivity only one exemplary sample was analyzed, other samples may show a different result. All cross reactivities and exemplary analyzed matrices are described in the Validation report. Recommendation: In order to ensure a high analytical performance we recommend: to analyze each sample material in duplicates to use also allergen-free and allergen- containing (spiked) samples as test controls in case of extremely acid or alkaline samples, the ph should be adjusted to a neutral ph due to the multitude of food types, matrix effects cannot be excluded. to carry out spiking experiments for an accurate and correct procedure to perform SureFood PCR for confirmation of the result For details using the ChemWell or GEMINI automation please contact sales@rbiopharm.de. The product information folder with further information is available on request from your local distributor or R-Biopharm AG. RIDASCREEN FAST Peanut
20 Further application notes: More sensitive analysis with RIDASCREEN FAST Peanut (dilution of standards down to mg/kg peanut) The data corresponds to our present state of technology and provides information on our products and their uses. R-Biopharm makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. Defective products will be replaced. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. R-Biopharm shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product. R-Biopharm AG Postanschrift / Postal Address: An der neuen Bergstraße Darmstadt, Germany Sitz / Corporate Seat: Pfungstadt Tel.: +49 (0) Fax: +49 (0) info@r-biopharm.de Vorsitzender des Aufsichtsrats / Chairman of Supervisory Board: Dietrich Mollat Vorstand / Board of Management: Dr. Ralf M. Dreher (Vorsitzender / Chairman), Dr. Carsten Bruns, Jochen Hirsch, Dr. Peter Schubert Handelsregister / Commercial Register: Amtsgericht Darmstadt HRB RIDASCREEN FAST Peanut
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