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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
3
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] 14
Amygdalin is a member of the cyanogenic glycoside group of plant secondary
15
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
17
and sub-acute food poisoning. Specific antibodies were raised against an amygdalin-
18
bovine serum albumin immunogen synthesised using a novel approach. The antibodies
19
were used in a microtitration plate enzyme-linked immunosorbent assay (ELISA) for the
20
quantification, for the first time, of amygdalin in commercially-available foods.
21
Correlation of results with high-performance liquid chromatography was very high (r =
22
0.983). The limit of detection of the immunoassay was 200 ± 0.05 pg mL-1, and the 50%
23
inhibitory concentration of amygdalin was 50 ± 0.02ng mL-1, making the ELISA
24
particularly sensitive.
b
ABSTRACT
25
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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
31
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
136
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
149
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
182
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
189
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
195
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
198
using standard solutions of amygdalin and other cyanogenic glycosides. The cross-
199
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
213
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
216
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
218
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
220
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
222
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
234
cyanogenic glycosides, both against amygdalin.27,28 No antibody characterization was
235
carried out, no assay validation reported and nor was any quantitative data provided.
236
The present report provides antibody characterization, assay validation and quantitative
237
data on commercially-available food products.
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Hapten Synthesis. Methods for production of hapten-protein conjugates need to
239
be efficient, be carried out under mild conditions and be effective. In order to produce
240
the amygdalin immunogen we used a method previously used by Abuknesha et al.29 to
241
synthesise antibody-enzyme conjugates. Cyanuric chloride has reactivity towards
242
hydroxyl groups under mild conditions and the coupling method did not affect the
243
structure of the target analyte (amygdalin). Figure 1 shows the synthetic scheme
244
followed. In order to provide the immobilized phase of the assay, a KLH-amygdalin
245
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
248
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%
254
giving a hapten-protein ratio for the immunogen of 20:1. The same procedure was
255
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
257
coating conjugate (19 and 17:1) using the spectrophotometric and chemical methods
258
were very close. This is an indication that the use of cyanuric chloride as a coupling
259
agent for conjugate production was efficient and that the procedure merits wider
260
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
262
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
265
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
277
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
279
a 1:30,000 dilution which gave an IC50 of 0.80 µg mL-1. When the antibody dilution was
280
increased beyond 1:30,000, the IC50 increased significantly. Consequently, an antibody
281
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
283
microtitration plate was varied from 1 to 5 hr (at 37 oC) and overnight at 4 oC. A higher
284
IC50 (5.51 µg/ml) and lower R2 (0.97) resulted from a 1 hr incubation at 37 oC. As the
285
incubation time increased, the IC50 decreased and R2 values increased.
286
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
288
The concentration of the coating conjugate, KLH-amygdalin, strongly influenced
289
ELISA performance. As the concentration of the coating conjugate was decreasing, the
290
concentration
291
Consequently, an increase in the binding of the antibody to the unconjugated amygdalin
292
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-
301
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:
307
Y = A2 + (A1-A2)/(1 + (x/x0)^p), R2 = 0.9995. The curve was fitted using OriginPro 8.6
308
software. The IC50 of the assay was estimated to be 50 ± 0.02 ng/ml with a detection
309
limit of 200 ± 0.05 pg mL-1 (as determined by the concentration that corresponded to the
310
optical density of zero binding reduced by 3 times the standard deviation of the mean
311
zero binding). The working concentration range of the icELISA was 10 ng mL-1 (IC20)
312
and 1 µg mL-1 (IC80). The detection limit of the ELISA reported here was significantly
313
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
317
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