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DEVELOPMENT and Application of an Enzyme-linked Immunosorbent Assay (ELISA) for the Quantification of Amygdalin, a Cyanogenic Glycoside, in Food Islamiyat F. Bolarinwa, Caroline Orfila, and Michael R. A. Morgan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf501978d • Publication Date (Web): 06 Jun 2014 Downloaded from http://pubs.acs.org on June 10, 2014

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DEVELOPMENT and Application of an Enzyme-linked Immunosorbent Assay (ELISA) for the Quantification of Amygdalin, a Cyanogenic Glycoside, in Food

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Islamiyat F. Bolarinwaa,b,c, Caroline Orfilaa and Michael R.A. Morgana a

4 5 6 7 8 9 10 11 12 13

School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK. Department of Food Science & Engineering,Ladoke Akintola University of Technology (LAUTECH), PMB 4000, Ogbomoso, Oyo state, Nigeria. c Corresponding author, email: [email protected]

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Amygdalin is a member of the cyanogenic glycoside group of plant secondary

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metabolites capable of generating hydrogen cyanide under certain conditions. As a

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consequence, the cyanogenic glycosides have been associated with incidents of acute

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and sub-acute food poisoning. Specific antibodies were raised against an amygdalin-

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bovine serum albumin immunogen synthesised using a novel approach. The antibodies

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were used in a microtitration plate enzyme-linked immunosorbent assay (ELISA) for the

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quantification, for the first time, of amygdalin in commercially-available foods.

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Correlation of results with high-performance liquid chromatography was very high (r =

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0.983). The limit of detection of the immunoassay was 200 ± 0.05 pg mL-1, and the 50%

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inhibitory concentration of amygdalin was 50 ± 0.02ng mL-1, making the ELISA

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particularly sensitive.

b

ABSTRACT

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Keywords: ELISA, Amygdalin, Cyanogenic Glycoside, Food Safety, Analysis

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INTRODUCTION

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Cyanogenic glycosides are plant natural toxicants that are widely distributed in nature,

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being found in more than 2500 plant species.1 Cyanogenic glycosides serve as

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important chemical weapons in the defence of the plant against herbivores because of

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the potential to generate toxic hydrogen cyanide.2 Hydrogen cyanide is released from

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the plant following tissue disruption as a result of animal chewing, bruising or

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mechanical damage. When plant tissues are disrupted in this way, the previously

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inaccessible cyanogenic glycosides located in plant vacuoles are able to come into

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contact with enzymes in the cell wall (β-glucosidases and α-hydroxynitrile lyases) which

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catalyse the degradation of cyanogenic glycosides to benzaldehyde and hydrogen

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cyanide. When cyanogenic plants are ingested by humans, enzymes produced by the

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intestinal microflora are also able to hydrolyse intact cyanogenic glycoside to produce

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hydrogen cyanide in vivo.3 Cyanide toxicity can occur in humans following the

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consumption of cyanide (from foods) at doses between 0.5-3.5 mg kg-1 body weight.4

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Cases of cyanide toxicity with symptoms such as vomiting, diarrhoea, abdominal

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cramps, headache, drowsiness, confusion, nausea, hypotension, paralysis and coma

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have been reported following the consumption of apricot kernels, tapioca cake,

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cassava-based meal and almond.5,6,7,8,9

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Several important plant crops, such as cassava, sorghum, millet, barley, almond,

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cherry, macadamia nut, peach, lima beans, and kidney beans, have been reported to

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biosynthesize and accumulate cyanogenic glycosides.1,10,11,12 Traditional and modern

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food processing techniques such as chopping, grinding, soaking, fermentation, drying,

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roasting, boiling, and steaming have been used to reduce or eliminate the potential

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toxicity of dietary cyanogenic plants.13 These methods can be effective because

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cyanogenic glycosides are soluble in water and hydrogen cyanide is volatile; bringing

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together enzymes and substrate can reduce glycoside concentrations substantially. The

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cyanogenic glycosides, however, are heat stable. Inefficient or inappropriate processing

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may mean that consumers may be exposed to acute and sub-acute doses of the

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glycosides.

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Amygdalin (D-mandelonitrile-β-D-gentiobioside; Figure 1) is the most common of

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the cyanogenic glycosides. It is present in abundance in the seeds and kernels of fruits

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such as apricot, almond, apple, cherry, plum, peach and nectarine. Although the seeds

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of some of these fruits are not consumed directly, fruits with high contents of

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cyanogenic glycosides in their seeds are likely to contain cyanogenic glycosides in the

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edible portion. Indeed, stewed fruit has been reported by Voldřich & Kyzlink14 to contain

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3-4 mg kg-1 HCN equivalents. The same author also reported levels of up to 4 mg kg-1

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HCN equivalents for canned stone fruits.

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Conventional methods for cyanogenic glycoside detection include high

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performance liquid chromatography (HPLC) with ultra-violet, refractive index or

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amperometric detection15,16 and gas chromatography-mass spectrophotometry (GC-

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MS).17 An improved reversed-phase HPLC-UV method for detection and quantification

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of amygdalin in commercially-available food products has been reported recently,

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utilizing an optimised extraction procedure.18 Although these methods can be sensitive,

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they utilise expensive equipment and require complicated sample pre-treatment. There

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is therefore the need for a method that is simple, rapid, sensitive and cost-effective.

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Enzyme-linked immunosorbent assay (ELISA) would fulfill these requirements in

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addition to having the potential for high sample throughput. Immunoassays are now

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widely utilized in food analysis for determination of food contaminants and in the

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agrochemical industry for detection of chemical residues in plants, water and soil.19,20,21

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Different types of immunoassay techniques have been developed for the detection and

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quantification of hazardous compounds in food. Quan et al.22 developed a sensitive

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enhanced chemiluminescence enzyme linked immunosorbent assay (ECL-ELISA) for

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the detection of fumonisin B1 (FB1) in food and animal feeds. An enzyme-linked

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immunosorbent assay (ELISA) developed by Pastor-Navarro et al.23 was able to detect

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different tetracycline residues in honey. ELISA methods have also been developed for

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monitoring the levels of herbicides in water,24 pesticides and estrogenic compounds in

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agricultural

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previously27,28 but have not been fully characterized or used quantitatively. We describe

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the development, characterization and application (for the first time) of an ELISA for the

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specific quantification of amygdalin in commercially-available food products.

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 MATERIALS AND METHODS

products.25,26

Antibodies

against

amygdalin

have

been

described

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Chemicals. Amygdalin, dhurrin, TMB (ready-to-use ELISA substrate), acetone,

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cyanuric chloride, Tween 20, gelatin, casein and buffer salts were all purchased from

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the Sigma Chemical Company, Poole, Dorset, UK. Prunasin, mandelonitrile and

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linamarin were purchased from Santa Cruz Biotechnology, Dallas, Texas, USA.

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6-trinitrobenzene sulfonic acid (TNBS; 5%, w/v) was obtained from VWR International

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Ltd., Leicestershire, UK. Keyhole Limpet Haemocyanin (KLH) was from Merck

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Chemicals Ltd., Nottingham, UK. Bovine serum albumin (BSA) was purchased from

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Fisher Scientific Ltd., Leicestershire, UK. Goat anti-rabbit IgG (H+L) horseradish

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peroxidase conjugate was obtained from Invitrogen Life Technology, Paisley, UK.

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Microtitration plates (96-well, MaxiSorp) were purchased from Nunc, Rosklide,

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Denmark. Microtitration plate washing was carried out using a Labsystem 4MK2

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Wellwash (Labsystems, Uxbridge, UK) and the optical densities at 405nm were

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recorded using a plate reader (ThermoFisher Scientific, Vantaa, Finland). Phosphate–

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buffered saline (PBS, 0.01 M, pH 5.5 and 7.4), phosphate–buffered saline (pH 7.4)

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containing 0.05% Tween-20 (PBST), sodium carbonate-bicarbonate buffer (0.05 M, pH

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9.4 and 9.6) were used.

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Food Samples. All fruits and processed products used in this study were

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purchased from local supermarkets in Leeds, UK. The products were Toasted Almond

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Kernels (produced in the USA, packaged in the UK by Sainsbury’s), EcoMil Almond

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Cocoa Dessert (8% almond) produced in Spain, Tesco own-brand Almond Flour

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(produced in the USA, packaged in the UK by Tesco), Morrison’s own-brand White

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Marzipan (25% almond) produced in the UK, Sainsbury’s own-brand Apple Juice

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(produced in the UK), Copella Apple Juice (hand-picked English apples, produced in the

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UK), Del Monte Quality Long Life Apple Juice (100% concentrate), squashes, melons

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and black cherries (from the UK), black plums (from Chile), yellow plums (from South

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Africa), apples (from Brazil), pears (from Holland), courgettes and cucumbers (from

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Spain), peaches (from Argentina), and marrows (from Egypt). Three packs from

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different batches were purchased for each brand or product. All products were stored at

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4 °C for two days after purchase and extracted as soon as possible.

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Synthesis of Hapten-Protein Conjugates. The hapten-protein synthesis route

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is illustrated in Figure 1. Amygdalin was first activated with cyanuric chloride (CC) using

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a modification of the method of Abuknesha et al.29 used for antibody-enzyme

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conjugations. CC (1 mg) was dissolved in acetone (1 ml) and 0.3 ml of the solution was

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measured into a glass vial. The content of the vial was allowed to evaporate. Amygdalin

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was activated by adding a solution of amygdalin (2.3 mg in 0.5 ml sodium carbonate-

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bicarbonate buffer, pH 9.4) to the CC residue. The mixture was stirred for 6hr at room

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temperature (20 ± 2 oC) to produce the activated hapten (amygdalin-CC). BSA (0.1

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µmol, 6.6 mg in 0.5 ml sodium carbonate-bicarbonate buffer, pH 9.4) was added to the

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hapten and the mixture was incubated overnight at 37 oC and dialysed for 6 hr against

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0.01 M phosphate buffer (pH 5.5). The resulting material (amygdalin-BSA conjugate,

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Figure 1) was freeze-dried to obtain a white powder. The conjugate was stored at –20

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o

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Amygdalin-KLH conjugate used for the immobilized phase of the ELISA was

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synthesized using the same method as for immunogen production.

C until use. The synthesized amygdalin-BSA conjugate was used as the immunogen.

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Assessment of Hapten-Protein Conjugates. The ratios of coupled hapten to

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protein in the conjugates were assessed by both spectrophotometric and chemical

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methods.30,31,32,33 Firstly, verification of conjugate synthesis and estimation of the

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number of moles of hapten conjugated per mole of protein was estimated by UV-visible

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spectrometry. Amygdalin (1 mg), BSA (1 mg) or amygdalin-BSA conjugate (1 mg) were

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dissolved in sodium carbonate-bicarbonate buffer (0.05 M, pH 9.6, 1 ml) was added.

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Absorption over 200-400 nm were determined, and the number of moles of hapten in

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the conjugate was calculated using the maximum absorbance for amygdalin at 262 nm

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to calculate the molar extinction coefficient. The number of moles of conjugated hapten

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was calculated using A = εcl.

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Secondly, the trinitrobenzene sulfonic acid assay (TNBS; Habeeb30) was used to

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estimate the molar conjugation ratio of amygdalin-BSA and amygdalin-KLH conjugates.

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The method is based on the determination of available amino acid groups present in

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carrier protein before and after hapten conjugation following its reaction with 2,4,6-

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trinitrobenzenesulfonic acid. The end product of the reaction of TNBS produces

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trinitophenyl derivatives which can be measured spectrophotometrically.

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protocol used was as modified by Kemp & Morgan.31 Carrier protein or conjugate (1 mg)

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were dissolved in 1 ml of 0.05 M sodium carbonate-bicarbonate buffer, pH 9.4.

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Standard curves for the proteins were constructed using a total volume of 200µl in the

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wells of a microtitration plate. To each of the wells, 50 µl of TNBS (0.1%) in carbonate

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buffer (4%) were added. The plate was incubated for 2 hr at 37oC for colour

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development and the absorbance was read at 405 nm on a plate reader. The

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percentage substitution of amino acid in the carrier protein was used to estimate the

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conjugation ratio of hapten to carrier protein.

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% Substitution ≡ O.D of carrier protein – O.D of hapten-protein conjugate X 100

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O.D of carrier protein

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The assay

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Immunization Protocol and Polyclonal Antibody Production. Antibodies were

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raised by Covalab Ltd, Cambridge, UK. Two female New Zealand White rabbits (2-2.5

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kg) were immunized with the immunogen (amygdalin-BSA conjugate) to generate

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polyclonal antibodies. The first injection consisted of the immunogen (50 µg in 0.5 ml of

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phosphate-buffered saline) emulsified with 0.5 mL of Freund’s complete adjuvant and

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injected intradermally on multiple sites on the rabbits back. The rabbits were further

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boosted by injecting intradermally with the immunogen (50 µg) dissolved in 0.5 ml of

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Freund’s incomplete adjuvant three times at three weeks intervals, with the final booster

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injection administered subcutaneously. The antisera were stored at –20 oC until use.

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Enzyme-Linked Immunosorbent Assay (ELISA). For determination of antibody

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titer, each well of the 96-well microtitration plate was coated with amygdalin-KLH

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overnight at 4 oC using 300 µl of a solution of conjugate dissolved in 0.05M carbonate-

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bicarbonate buffer, pH9.6. The plate was then washed three times with water and twice

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with 0.01M phosphate-buffered saline containing 0.05% Tween 20 (PBST). Antibody

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titer was determined by incubating serial dilutions of antisera and pre-immune sera in

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PBST (200 µl per well, in triplicate). The plate was incubated overnight at 4 oC, washed

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five times with PBST and dried. A species-specific secondary antibody, goat anti-rabbit

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horseradish peroxidase IgG conjugate (diluted 1/1000 in PBST, v/v; 200 µl per well) was

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added. The plate was incubated at 37 oC for 2 hr, washed and dried as before, and then

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TMB substrate (200 µl) was added to each well. The plate was further incubated for 30

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min at 37 oC and the reaction was stopped with sulfuric acid (2 M, 50 µl per well). The

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absorbance of each well was determined at 405 nm. The development of yellow colour

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was proportional to the amount of antibody present. The mean absorbance values of

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triplicate determinations were plotted against antiserum dilutions.

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For the indirect competitive ELISA (icELISA), standard curves were constructed

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with100 µl of solution containing amygdalin standard (10 pg/well -10 µg/well) dispensed

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into appropriate wells of the coated plates prior to adding antibody (100 µl). The plate

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was then processed for the antibody titer curve determination. The limit of detection of

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the assay was estimated as the concentration that corresponds to the optical density at

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zero binding minus three times the standard deviation of the mean of zero binding.

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To optimise the ELISA, the sensitivity of the immunoassay was improved by

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varying the antiserum dilution, the incubation time, the concentration of the coating

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antigen and by using different protein buffers. The protein buffers that were evaluated

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were PBST containing 1% BSA, 1% gelatin or 1% casein. Evaluation of the optimisation

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process was based on the IC50 value and the coefficient of evaluation of the linear

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equation (R2) as described by Qian et al.32

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Cross-Reactions. The optimised assays were applied to cross-reactivity studies

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using standard solutions of amygdalin and other cyanogenic glycosides. The cross-

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reactivity of the antibody with some closely related cyanogenic glycosides such as

200

prunasin, dhurrin, mandelonitrile and linamarin was determined by icELISA. The

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specificity of the polyclonal antibody was determined using:

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% Cross-reactivity (CR) = (IC50 of amygdalin/ IC50 of analogue) x 100

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Application of the icELISA to Quantification of Amygdalin in Commercially-

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available Food Products. The extraction method described previously18 was used for

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the extraction of amygdalin from food samples. Solid samples (2 g) were ground in a

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blender (20 sec; Moulinex Optiblend 2000, France) and 1 g was weighed into a round-

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bottom flask (500 ml). In the case of liquid samples, ethanol (50 ml) was added to the

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samples (10 ml) in a round-bottom flask, and the mixture was boiled under reflux for 100

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min. The extracts were filtered (Whatman No. 1 filter paper) and transferred into plastic

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polypropylene tubes (50 ml). Ethanol was completely evaporated from the filtrate with a

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rotary evaporator (low BP, 35 oC, 7 mbar). Diethyl ether (10 ml) was added to the dried

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sample and the mixture was vortexed (1 min) at room temperature (20

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precipitate amygdalin. The diethyl ether was allowed to evaporate overnight in a fume

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hood and the extracted amygdalin was dissolved in 1%BSA-PBST (1 ml). The food

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extract (100 µl) was then incubated with antibody on coated plate as previously

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described for setting up the standard curve. Quantification of amygdalin in the sample

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was by reference to standard curves. Samples were also analysed by HPLC as

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described previously in order to show the correlation of the results from the 2 methods.

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The HPLC extraction method was, however, different from the ELISA method. In the

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HPLC method, extracted amygdalin was further purified by dissolving it in water

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followed by centrifugation (14000 rpm, 22 oC, 10 min) and filtration with 0.45 µm PTFE

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filters prior to HPLC analysis.

223 224 225 226 227

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± 2 oC) to

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 RESULTS AND DISCUSSION

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Immunoassays are now widely used in quantitative analysis for agric-food

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components for their advantages of cost-effectiveness and simplicity which arise directly

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from the affinity of the antibody interaction with its target. Surprisingly, perhaps, given

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that immunoassays can be used away from fully-equipped laboratories to monitor food

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processing, only 2 previous reports have described antibody production against

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cyanogenic glycosides, both against amygdalin.27,28 No antibody characterization was

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carried out, no assay validation reported and nor was any quantitative data provided.

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The present report provides antibody characterization, assay validation and quantitative

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data on commercially-available food products.

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Hapten Synthesis. Methods for production of hapten-protein conjugates need to

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be efficient, be carried out under mild conditions and be effective. In order to produce

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the amygdalin immunogen we used a method previously used by Abuknesha et al.29 to

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synthesise antibody-enzyme conjugates. Cyanuric chloride has reactivity towards

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hydroxyl groups under mild conditions and the coupling method did not affect the

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structure of the target analyte (amygdalin). Figure 1 shows the synthetic scheme

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followed. In order to provide the immobilized phase of the assay, a KLH-amygdalin

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conjugate was produced using the same chemistry.

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Estimation of Hapten-Protein Ratios. Amygdalin exhibited a UV maximum at

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262nm; BSA at 278nm. The BSA-amygdalin conjugate had a peak at 278nm and a

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significant shoulder at 262nm. Similar results were seen with KLH. The magnitude of

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the 262 absorption was used to calculate hapten-protein ratios, which were found to be

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23:1 and 19:1 for the BSA-amygdalin and KLH-amygdalin conjugates, respectively.

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Reaction of the proteins with TNBS generated standard curves at 405nm.

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Theoretically, 59 lysine residues of BSA are available for coupling to hapten.33 The

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percentage substitution of amygdalin calculated from the standard curves was 34.4%

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giving a hapten-protein ratio for the immunogen of 20:1. The same procedure was

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employed for the KLH coating conjugate and the ratio was calculated to be 17:1.

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The hapten:protein ratios obtained for the BSA conjugate (23 and 20:1) and the

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coating conjugate (19 and 17:1) using the spectrophotometric and chemical methods

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were very close. This is an indication that the use of cyanuric chloride as a coupling

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agent for conjugate production was efficient and that the procedure merits wider

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utilization in future. The hapten:protein ratios obtained in this study were in line with

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recommended ratios for good antibody production. Kemp & Morgan31 reported that a

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conjugate with a high hapten: protein ratio is good as immunogen, while the one with a

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low ratio may be desirable as a coating antigen. Hapten densities of 15-30 molecules

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per carrier protein were also reported to be good for the production of high antibody

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titers.33

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Antibody Titer. The BSA-amygdalin conjugate was used in antibody production

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in rabbits. The presence of anti-amygdalin antibodies was observed through production

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of titer curves using KLH-amygdalin as the immobilized phase on microtitration plates.

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Antibody binding as measured by optical density was 2.1 at a dilution of 1:10,000 (v:v)

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compared to that given by pre-immune serum (0.03 at 1:10,000). Even at dilutions of

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1:100,000, the antisera showed significant binding above that of the pre-immune serum.

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The anti-amygdalin antiserum was used at a dilution of 1:30,000 for subsequent

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experiments unless otherwise stated. For anti-hapten antisera, such a dilution is at the

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high end of what is achievable.

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Optimisation of the ELISA. The antibody dilution, incubation time, coating

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conjugate concentration and the use of protein buffers were each investigated for ELISA

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optimization (Table 1). Antibody dilution significantly influenced the sensitivity of the

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ELISA. A lower dilution (1:10,000) of antibody resulted in higher IC50 (7 µg/well) than at

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a 1:30,000 dilution which gave an IC50 of 0.80 µg mL-1. When the antibody dilution was

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increased beyond 1:30,000, the IC50 increased significantly. Consequently, an antibody

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dilution of 1:30,000 in PBST was used throughout.

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The incubation time for anti-amygdalin antibody and amygdalin on the

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microtitration plate was varied from 1 to 5 hr (at 37 oC) and overnight at 4 oC. A higher

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IC50 (5.51 µg/ml) and lower R2 (0.97) resulted from a 1 hr incubation at 37 oC. As the

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incubation time increased, the IC50 decreased and R2 values increased.

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incubation at 4 oC resulted in the lowest IC50 with R2 > 0.9. Subsequent assays utilised

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an overnight incubation at 4 oC.

Overnight

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The concentration of the coating conjugate, KLH-amygdalin, strongly influenced

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ELISA performance. As the concentration of the coating conjugate was decreasing, the

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concentration

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Consequently, an increase in the binding of the antibody to the unconjugated amygdalin

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was evidenced for coating conjugate concentrations of 0.01 to 0.05 µg mL-1. The IC50 of

of

the

free

hapten

(unconjugated

amygdalin)

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the standard curve decreased with decreased coating conjugate concentration and

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associated R2 value increased. The assay was most sensitive with a coating conjugate

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concentration of 0.05 µg mL-1. The assay sensitivity was good at this concentration (50

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ng mL-1), possibly because antibody bivalent binding were prevented at this lower

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concentration of conjugate.31 A coating conjugate concentration of 0.05 µg mL-1 was

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used for subsequent assays.

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The ELISA absorbance and sensitivity was greatly enhanced by using 1% (w/v)

300

BSA-PBST as buffer. A significantly lower IC50 value was observed when 1% BSA-

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PBST was used as buffer compared to when PBST only or 1% casein-PBST or

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1%gelatin-PBST was used as buffer. On the basis of the IC50 values, R2 values and the

303

ratios of the maximum and minimum absorbances for the amygdalin standard curves,

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1% BSA-PBST was selected as the buffer for subsequent experiments.

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Indirect Competitive ELISA (icELISA). The optimised conditions were used to

306

obtain an indirect competitive standard curve for amygdalin (Figure 2), represented by:

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Y = A2 + (A1-A2)/(1 + (x/x0)^p), R2 = 0.9995. The curve was fitted using OriginPro 8.6

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software. The IC50 of the assay was estimated to be 50 ± 0.02 ng/ml with a detection

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limit of 200 ± 0.05 pg mL-1 (as determined by the concentration that corresponded to the

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optical density of zero binding reduced by 3 times the standard deviation of the mean

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zero binding). The working concentration range of the icELISA was 10 ng mL-1 (IC20)

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and 1 µg mL-1 (IC80). The detection limit of the ELISA reported here was significantly

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lower than previously reported limits using a polyclonal antibody (100 µg mL-1)27 or a

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recombinant antibody (500 pg mL-1).28

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Cross-Reactivity. The specificity of the assay was investigated by testing the

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cross-reactivity of the antibody with other cyanogenic glycosides that are closely related

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to amygdalin. The results obtained (Table 2) showed that the antibody was very specific

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for amygdalin, with cross-reactions of 1.3% for prunasin, 0.1% for mandelonitrile and

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500

500

Development and application of an enzyme-linked immunosorbent assay (ELISA) for the quantification of amygdalin, a cyanogenic glycoside, in food.

Amygdalin is a member of the cyanogenic glycoside group of plant secondary metabolites capable of generating hydrogen cyanide under certain conditions...
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