ELISA of acrylamide in foods

Transcription

1 ELISA of acrylamide in foods 1

2 The present situation of acrylamide in foods Acrylamide generation during food manufacturing process Reducing sugar+asparagine Acrylamide Acrylamide (CH 2 =CH-CO-NH 2 ; MW=71) is inevitably formed during the food manufacturing process, where ingredients containing reducing sugar and asparagine are exposed to high temperatures. Acrylamide thus produced in foods is reported potentially hazardous for human health. Administration of acrylamide reduction Codex Alimentarius Commission adopted CODE OF PRACTICE FOR THE REDUCTION OF ACRYLAMIDE IN FOODS (CAC/RCP ) in In Japan, food companies are administered under the guidance of the Ministry of Agriculture, Forestry and Fisheries so as to promote activities of reducing acrylamide contents in foods. Method of acrylamide analysis The current methods of quantifying acrylamide usually employ expensive equipments, including LC/MS, LC/MS/MS, and GC/MS, which require skilful techniques for operation. A major cause that hampers the effective reduction of acrylamide in foods, especially in small and medium-sized enterprises, is the lack of easy and inexpensive quantification methods. In this context, a new method of quantifying acrylamide is required.

3 Antibody preparation and ELISA development Antibody to acrylamide cannot be obtained by the direct immunization of animals with acrylamide, because acrylamide is a low molecular weight compound (F.W. =71). It is not realistic to develop a sandwich ELISA of acryamide. An indirect competitive ELISA was developed with a polyclonal antibody to 3-[(2-carbamoylethyl)thio] benzoic acid (3-CTBA), which is an acrylamide derivative. Principle of indirect competitive ELISA of 3-CTBA Addition1 Addition2 3-CTBA in samples Antibody to 3-CTBA 3-CTBA coated well Antibody amount bound to the well is determined. 3

4 Procedures of acryamide extraction from foods Step 1 Extraction of acrylamide from the food sample The food sample in an amount of 1 g is homogenized in the presence of 19 ml of distilled water, by using a Polytron homogenizer or equivalent (2 min/sample) The homogenate is centrifuged and the supernatant fraction is retained (20 min) Filter the supernate through a membrane of 0.45μm in pore size (1 min/sample) Pass the filtrate through a solid phase extraction column (ISOLUTE Multimode 500 mg/3 ml) (5 min/sample) (To step 2) Total time required for extraction: approx. 2 h/10 samples

5 Derivatization of acrylamide to 3-CTBA and ELISA Step 2 Derivatization (in a test tube) Total time required for derivatization (2 h) COOH 2 SH MBA + CH 2 =CH-CO-NH 2 Acrylamide Incubation for 2 h at 37 under alkaline condition COOH 2 S-CH 2 -CH 2 -CO-NH CTBA Total time required for ELISA (2 h) Addition1 (after neutralization) Step 3 ELISA (on an immunoplate coated with 3-CTBA-Protein) Anti 3-CTBA antibody Addition2 Incubation for 2 h at 25 Protein-NH CO S-CH 2 -CH 2 -CO-NH 2 Acrylamide is quantified by determining the antibody amount bound to the solid phase.

6 A representative calibration curve and LOD of ELISA 110% Average±SD (n=3) 100% 90% 80% 70% 60% LOD = 0.4 ng/ml 50% 40% 30% B/B 0 = 1/(1+(X/c)^b) 20% 10% 0% Concentration of acrylamide (ng/ml) The LOD is determined according to ISO :2008(E) [Capability of detection; Part 5: Methodology in the linear and non-linear calibration cases].

7 RSD(%) The LOQ and quantification range of ELISA 30% 20% 10% 0% Concentration of acrylamide (ng/ml) The range of quantification (RSD 10%) is ng/ml (equivalent to 90-30,000 ppb in food) 7

8 Cross-reactivity to the acrylamide analogues Acrylamide Methacrylamide Acrylonitrile 100% Acrylic acid Maleic acid Maleamic acid CH 2 =CH-CO-NH 2 Acrylamide 90% 80% CH 2 =C(CH 3 )-CO-NH 2 Methacrylamide B/B 0 70% 60% 50% 40% CH 2 =CH-CN CH 2 =CH-COOH Acrylonitrele Acrylic acid 30% 20% HOOC-CH=CH-COOH Maleic acid 10% 0% HOOC-CH=CH-CO-NH 2 Maleamic acid Concentration (nm) Cross-reactivity (%) (Acrylamide = 100%) Analogue IC 50 (nm) 1/IC 50 (nm -1 ) Cross-reactivity (%) Acrylamide % Methacrylamide % Acrylonitrile % Acrylic acid > < <0.04% Maleic acid > < <0.04% Maleamic acid > < <0.04%

9 ELISA (ppb) Correlation of assay results between ELISA and LC/MS for model samples (potato chips) y = x R² = Model sample LC/MS (ppb) ELISA (ppb) Mean±SD ± ± ± ± ± ± Model samples (potato chips of differing acrylamide contents) were specially prepared by Calbee, Inc LC/MS analysis was performed by the Research Institute of Morinaga & Co. Inc,. LC/MS (ppb)

10 ELISA (ppb) Correlation of assay results between ELISA and LC/MS for commercial foods Comparison with 25 commercial foods y = x R² = LC/MS (ppb) Name of foods Main materials LC/MS (ppb) ELISA Mean±SD (ppb) Biscuit A Wheat ± 30 Biscuit B Wheat ± 41 Biscuit for baby A Wheat nd nd Biscuit for baby C Wheat ± 15 Biscuit for baby D Wheat ± 15 Cracker E Wheat ± 7 Butter cracker F Wheat ± 115 Fried sugar coated dough (Karintou) Wheat ± 21 Bread Wheat 49 nd Doughnut Wheat 28 nd Corn snack Corn ± 28 Corn flake Corn 19 nd Deep-frying rice cracker (Age sennbei) Rice ± 25 Mashed potatoes Potato ± 9 Potato snack Potato ± 283 French fly Potato ± 9 Fried sweet potato Sweet potato ± 30 Muscovado Sugarcane ± 89 Cocoa Cacao ± 92 Black chocolate Cacao ± 78 Roasted bean curd Soy bean <6 nd Thick deep fried bean curd Soy bean <6 nd Roasted tea Tea leaf ± 40 Tea Tea leaf <6 nd Green tea Tea leaf <6 nd nd= not detected

11 Advantages of ELISA ELISA Initial investment (a microplate reader) is less than five thousand Euros. Cost of analysis for one sample is less than 10% that of LC/MS (Cheap). Total time required for quantification is approx. 6 h, and no special technique is required (Fast and easy). Up to 24 different samples can be quantified simultaneously (Multi-sample assay). Current methods (LC/MS, LC/MS/MS, GC/MS etc.) Initial investment is very costly (several hundred thousand Euros), and the running cost as well. Skillful techniques are required.

12 SUMMARY 1. ELISA Performance 1) The limit of detection (LOD) is as low as 0.4 ng/ml, and the quantification range (RSD 10%) is ng/ml, corresponding to ,000 ppb in foods. 2) The assay is highly specific to acrylamide. 3) The assay results are well correlated with those of conventional LC/MS method (R 2 = ). 2. Advantages of ELISA Up to 24 samples can be assayed simultaneously, cheaply, easily, and quickly. 3. The ELISA kit will be placed on market very soon.