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Monoclonal antibody-based sandwich ELISA for the detection of mammalian meats
Food Control 110 (2020) 107045
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Monoclonal antibody-based sandwich ELISA for the detection of mammalian meats
T
Xingyi Jiang, Qinchun Rao∗, Kristen Mittl, Yun-Hwa Peggy Hsieh Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL, USA
ARTICLE INFO
ABSTRACT
Keywords: Sandwich ELISA Mammalian meat Skeletal troponin Matrix effect
In order to (1) reduce the risk of intentional or unintentional contamination of foods, (2) better comply with food regulations, and (3) decrease economic loss to the food industry caused by recall, it is necessary to develop reliable methods for the detection of different food adulterants/contaminants. This study was conducted to characterize two mammalian skeletal troponin (sTn) specific monoclonal antibodies (mAbs 6G1 and 8F10), and use them to develop a mAb-based sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of mammalian meats. From our results, both mAbs were positive to porcine sTnI and sTnC but negative to sTnT. The extractability and antigenicity of target analytes in pork were enhanced by the addition of urea and βmercaptoethanol into the extraction buffer. The optimized sandwich ELISA was specific to heated mammalian meats and was adequate to analyze samples subject to the most severe heat treatment (132 °C/2 bar/120 min). Mammalian fat (10–30%, g/g) did not significantly affect the assay signal. The optimized assay could detect as low as 1% (g/g) of heated mammalian meats adulterated in poultry meats. Overall, this assay has the potential to fight food fraud, comply with food regulations, and decrease food recalls, which may open up new diagnostic methods for the food industry and the food regulatory authorities.
1. Introduction It is estimated that food adulteration costs the world economy around $49 billion annually (Moerman, 2018). Around 10% of the foods produced in the U.S. were adulterated and 7% contain fraudulent ingredients (Layton, 2010). From 1980 to 2013, (1) the leading reported type of fraudulent foods was animal products including meat and meat products (7%); (2) 65% of the total incidents were due to substitution or dilution; and (3) in about 30% of the total incidents, the involved food products were produced in the U.S. (National Center for Food Protection and Defense, 2015). Between 2016 and 2017, about 35% (423/1,213) of the food recalls were due to the presence of misbranding and undeclared food residues, which is the No. 1 cause of food recalls in the U.S. (USDA, 2019b; USFDA, 2019). Also, mammalian meat intentional/unintentional adulteration is a serious concern for religious and cultural reasons as well as for those with individual moral aversions (Regenstein, Chaudry, & Regenstein, 2003). Both Muslims and Jews who follow religious law are forbidden from consuming products derived from pigs and animal blood. It was reported that most of the 6 to 8 million Muslims in North America observe halal laws, particularly the avoidance of pork, however, the food industry has essentially ignored this consumer group (Regenstein et al., 2003), while it ∗
is the major authenticity concerns in meat products for Muslim consumers (Nakyinsige, Man, & Sazili, 2012). In addition, as an emergent allergy, mammalian meat allergy is increasingly prevalent in tick-endemic areas worldwide (van Nunen, 2015). As evidenced by the startling statistics noted above, there exists a pressing need to fight food fraud and reduce the risk of foodborne illness. In the U.S., the prohibitions against adulterated and misbranded food appear in many federal laws, including the Food, Drug and Cosmetic Act (USFDA, 2012), the Meat Inspection Act (USDA, 2011; USFDA, 2012), the Poultry Products Inspection Act (USDA, 2012; USFDA, 1995), and the FDA Food Safety Modernization Act (USFDA, 2011). To prevent intentional adulteration from acts intended to cause wide-scale harm to public health, a final rule entitled “Mitigation Strategies to Protect Food Against Intentional Adulteration” was enacted in 2019 (USFDA, 2016). To better comply with the food regulations and decrease the economic loss to the food industry, many monoclonal antibody (mAb) based immunoassays have been commercialized for the detection of different food adulterants/contaminants. It should be noted that Chen and Hsieh (2002) reported that porcine skeletal muscle protein, troponin I (PsTnI), was thermostable, which could still maintain its solubility and antigenicity after severe heat treatment (126 °C for 120 min).
Corresponding author. E-mail address: [email protected] (Q. Rao).
https://doi.org/10.1016/j.foodcont.2019.107045 Received 9 August 2019; Received in revised form 5 December 2019; Accepted 6 December 2019 0956-7135/ © 2019 Published by Elsevier Ltd.
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Table 1 Summary of skeletal muscle troponins from pig (Sus scrofa)a. Protein name
Muscle fiber type
UniProtKB accession number
Troponin T (PsTnT)
Slow
Q75ZZ6 (reviewed)d
Fast
Q75NG9 (reviewed)
Not available
P02587 (reviewed)
Slow
P63317 (reviewed)
Fast
A1XQV5 (unreviewed)
Slowtwitch
Q7YSF4 (unreviewed)
Fasttwitch
Q4JH15 (unreviewed)
Troponin C (PsTnC)
Troponin I (PsTnI)
a b c d
Amino acid sequenceb
1 61 121 181 241 1 61 121 181 241 1 61 121 1 61 121 1 61 121 1 61 121 181 1 61 121 181
MSDAEEQEYEEEQPEEEEAAEEEEAPEEPEPVAEREEERPKPSRPVVPPLIPPKIPEGER VDFDDIHRKRMEKDLLELQTLIDVHFEQRKKEEEELVALKERIERRRAERAEQQRFRTEK ERERQAKLAEEKMRKEEEEAKKRAEDDAKKKKVLSNMGAHFGGYLVKAEQKRGKRQTGRE MKQRILSERKKPLNIDHMGEDQLREKAQELSDWIHQLESEKFDLMAKLKQQKYEINVLYN RISHAQKFRKGAGKGRVGGRWK MSDEEVEHVEEEYEEEEEAQEEAPPPPAEVHEVHEEVHEVHEPEEVQEEEKPRPKLTAPK IPEGEKVDFDDIQKKRQNKDLMELQALIDSHFEARKKEEEELVALKERIEKRRAERAEQQ RIRAEKERERQNRLAEEKARREEEEAKRRAEDDLKKKKALSSMGANYSSYLAKADQKRGK KQTAREMKKKVLAERRKPLNIDHLSEDKLRDKAKELWDALYQLEIDKFEYGEKLKRQKYD IINLRSRIDQAQKHSKKAGTTPKGKVGGRWK TDQQAEARSYLSEEMIAEFKAAFDMFDADGGGDISVKELGTVMRMLGQTPTKEELDAIIE EVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELAECFRIFDRNMDGYIDAEELAEIFR ASGEHVTDEEIESIMKDGDKNNDGRIDFDEFLKMMEGVQ MDDIYKAAVEQLTEEQKNEFKAAFDIFVLGAEDGCISTKELGKVMRMLGQNPTPEELQEM IDEVDEDGSGTVDFDEFLVMMVRCMKDDSKGKSEEELSDLFRMFDKNADGYIDLEELKIM LQATGETITEDDIEELMKDGDKNNDGRIDYDEFLEFMKGVE MTDQQAEARSYLSEEMIAEFKAAFDMFDADGGGDISVKELGTVMRMLGQTPTKEELDAII EEVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELAECFRIFDRNMDGYIDAEELAEIF RASGEHVTDEELESLMKDGDKNNDGRIDFDEFLKMMEGVQ MPEVERKPKITASRKLLLKGLMLAKAKECWEQEHEEREAEKARYLAERIPTLQTRGLSLS ALQDLCRELHAKVEVVDEERYDIEAKCLHNTREIKDLKLKVLDLRGKFKRPPLRRVRVSA DAMLRALLGSKHKVSMDLRANLKSVKKEDTEKERPVEVGDWRKNVEAMSGMEGRKKMFDA AKSPTSQ MGDEEKRHRAITARRQHLKSVMLQIAATELEKEVGRRESEKQNYLSEHCPPLHLPGSMSE VQELCKQLHAKIDAAEEEKYDMEIKVQKSTKELEDMNQKLFDLRGKFKRPPLRRVRMSAD AMLKALLGSKHKVCMDLRANLKQVKKEDTEKERDLRDVGDWRKNIEEKSGMEGRKKMFET ES
60 120 180 240 262 60 120 180 240 271 60 120 159 60 120 161 60 120 160 60 120 180 187 60 120 180 182
Molecular weight (Da)
% Relative concentrationc
31,243
4.70
32,176
18,025
3.25
18,417 18,156 21,616
4.49
21,334
All information was obtained from The UniProt Consortium (2017). The calcium-binding sites are highlighted in green with a dotted underline. Cysteine residues are highlighted in yellow with a single underline. % Relative concentration in myofibrillar proteins from fresh pork meat samples. Data are from Grujic and Savanovic (2018). Reviewed: the entry belongs to the Swiss-Prot section of UniProtKB; Unreviewed: the entry belongs to the computer-annotated TrEMBL section.
As one of the three skeletal troponin (sTn) subunits to regulate muscle contraction, sTnI inhibits actomyosin ATPase activity (Greaser & Gergely, 1973). The other two sTn subunits are sTnT (tropomyosinbinding protein) and sTnC (Ca2+-binding protein) (Zot & Potter, 1987). The relevant molecular information of porcine sTn subunits is summarized in Table 1. It should be noted that there is a high amino acid similarity (> 90%) in sTn subunits among different mammals. As an ideal immunogen, sTnI from different meat species has been used to develop different mAbs for the authentication of meat and meat products (Chen & Hsieh, 2002; Liu, Chen, Dorsey, & Hsieh, 2006; Park, Oh, Kim, & Shim, 2014; Zvereva et al., 2015). The major objectives of this study were to characterize two mammalian sTn-specific mAbs and use them to develop a mAb-based sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of mammalian meats.
mAb8F10 (mAb8F10-HRP, 1 mg IgG/mL) was provided by Neogen Co. (Lansing, MI, USA). One anti-sTnC polyclonal antibody (anti-sTnC pAb, MBS2032663), developed in rabbit against human slow sTnC (UniProtKB accession number P63316), was purchased from MyBioSource, Inc. (San Diego, CA, USA). One anti-sTnT pAb (NBP1-32748), developed in rabbit against human slow sTnT (UniProtKB accession number P13805), was purchased from Novus Biologicals, LLC (Centennial, CO, USA). Both commercial pAbs were reported to cross-react with PsTn subunits (MyBioSource Inc., 2019; Novus Biologicals LLC, 2019). The multicolor near-infrared (IR) fluorescent Western blotting kit (AzureSpectra IR rb700 and ms800, AC2140), containing goat anti-mouse IgG IR800-labeled (green) and anti-rabbit IgG IR700-labeled (red) secondary antibodies, were purchased from Azure Biosystems Inc. (Dublin, CA, USA). Goat anti-mouse IgG (Fc specific) HRP-conjugated secondary antibody (anti-IgG-HRP, A2554) was purchased from SigmaAldrich (St. Louis, MO, USA). All chemicals and reagents were of analytical grade. All solutions were prepared using deionized water from a NANOpure DIamond ultrapure water system (Barnstead International, Dubuque, IA, USA).
2. Materials and methods 2.1. Materials Beef chuck, lamb thigh, pork shoulder, turkey thigh, chicken thigh, duck meat, and unrendered fat from pork and beef were purchased from local supermarkets (Tallahassee, FL, USA). Deer steaks were provided by the Fats and Proteins Research Foundation (Bloomington, IL, USA). Horse meat was obtained from the College of Veterinary Medicine, Auburn University (Auburn, AL, USA). All lean meats were ground twice using a meat grinder upon received (Proctor-Silex, Inc., Washington, NC, USA) and stored at −80 °C until use. Two mAbs, 6G1 and 8F10, were previously produced in mouse against purified equine sTnI (Hsieh & Chen, 2002). mAb6G1 (1.37 mg IgG/mL) was purified using a Bio-Scale Mini Affi-Prep Protein A Resin Cartridge (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the manufacturer's instruction. Horseradish peroxidase (HRP) conjugated
2.2. Porcine skeletal troponin complex isolation Porcine skeletal troponin complex (PsTn) was isolated (1) to study the immunoreactivity between each subunit and mAbs; and (2) to study the thermostability of its subunit. The isolation was performed according to (Potter, 1982) with modifications. Briefly, after eight washes (1:5, g/mL) using the ice-cold washing solution (1% (mL/mL) Triton X100, 50 mM KCl, 5 mM Tris, pH 8), proteins soluble in low ionic strength buffer were removed from 8 g of ground pork. Homogenization (11,000 rpm for 1 min using an ULTRA-TURRAX T-25 basic homogenizer, IKA Works, Inc., Wilmington, NC, USA) and centrifugation (11,000 g for 10 min at 4 °C) were performed in each wash step. The 2
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washed pellet was then 1:3 (g/mL) washed with 95% (mL/mL) ethanol for three times. This meshed pellet was further washed with an equal amount (g/g) of acetone three times and dried overnight in a fume hood at room temperature (RT). The acetone-dried powder was mixed with 15 parts (g/mL) of the extraction buffer (1 M KCl, 25 mM Tris, 0.1 mM CaCl2, 1 mM DTT, pH 8). After overnight extraction at 4 °C, the supernatant was collected by centrifugation. The precipitates were resuspended in 7.5 parts (mL/g of the starting tissue) of 1 M KCl and centrifuged. Two supernatants were combined, whose pH was adjusted to 4.6 to precipitate tropomyosin. After centrifugation, the pH of the supernatant was adjusted to 8. Ammonium sulfate (AMS) was added to produce 40% saturation with stirring for 2 h at RT. After centrifugation, the supernatant was brought to 60% AMS saturation and stirred overnight at 4 °C. After centrifugation, the pellet was dissolved in 0.5 M KCl and then replaced with NaCl-PBS (0.5 M NaCl in 10 mM phosphatebuffered saline buffer (PBS), pH 7.0) using Amicon Ultra centrifugal filter units (MWCO 10 kDa, EMD Millipore Co., Billerica, MA, USA). Its 1 mg / mL = 1) protein concentration was determined using UV280 nm (E280 (Thermo Scientific, 2019).
end-over-end rotated for 1 h at 4 °C. After centrifugation (50,000 g for 15 min at 4 °C), the supernatant was collected, and its protein concentration was determined. 2.4. Reducing SDS-PAGE and Western blot Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 4% stacking gel and 15% separating gel) was performed to (1) study protein profile of isolated PsTn and heated meat extracts; (2) compare protein solubility in isolated PsTn before and after heat treatment; and (3) compare porcine meat protein extractability using two extraction buffers (i.e., NaCl-PBS and NaCl-PBS-urea-βME). Briefly, meat protein extracts and PsTn were separated by reducing SDSPAGE according to the method of Laemmli (1970) using a Mini-PROTEAN Tetra Cell (Bio-Rad). The Precision Plus Protein Kaleidoscope Protein Standards (Bio-Rad) was used for band sizing. The protein bands were visualized by the Coomassie blue staining. Double fluorescent Western blot was used to study the immunoreactivity between PsTn and mAb8F10. Briefly, electrophoretically separated PsTn on the unstained gel was transferred onto a 0.2-μm lowfluorescent polyvinylidene fluoride (PVDF) membrane (Azure Biosystems) using a Trans-Blot Turbo Transfer System (Bio-Rad). After blocked with BSA-PBS (PBS containing 1% (g/mL) BSA), each membrane was probed with a cocktail of primary antibodies, either mAb8F10+anti-sTnT pAb or mAb8F10+anti-sTnC pAb. The IgG concentration of each pooled antibody was 1 ppm. Each membrane was then incubated for 2 h in the dark with the mixture of two fluorescently labeled secondary antibodies (anti-mouse and anti-rabbit IgGs, 1:5,000 (μL/μL) diluted in BSA-PBST (PBS containing 0.05% (mL/mL) Tween20 and 1% (g/mL) BSA) before imaging. Chemiluminescent Western blot was performed to study (1) the species selectivity of both mAbs (8F10 and 6G1); and (2) the matrix effect on the antigenicity of PsTn. Briefly, electrophoretically separated protein bands on the unstained gel were transferred onto a 0.45-μm nitrocellulose membrane (Fisher Scientific). The transferred proteins were visualized using the Ponceau S staining. The membrane was blocked with BSA-PBS. After incubation with the primary antibody (mAb6G1 or mAb8F10, 1 ppm in BSA-PBST), the membrane was probed with anti-IgG-HRP (170 ppm in BSA-PBST). The blotted antigens were detected using the luminol chemiluminescence method. For both Western blots, the incubation time for each step was at least 1 h at RT. Between each step, the membrane was washed with PBST several times. The images were captured by Azure c600 Imaging System and analyzed using the AzureSpot software (version 14.2, Azure Biosystems).
2.3. Sample preparation Unless otherwise specified, (1) each nonspiked or fat-spiked or mammalian meat-spiked sample was treated with two heat conditions, 100 °C for 30 min in a water bath and 121 °C/1.2 bar for 30 min in a NAPCO 8000-DSE Benchtop Autoclave (Jouan Inc., Winchester, VA, USA), respectively; (2) all heated samples were cooled immediately in ice-cold water; (3) all samples were two-fold (g/mL) extracted with NaCl-PBS after heat treatment; (4) all samples were homogenized at 11,000 rpm for 2 min; (5) all homogenates were held at 4 °C for 2 h before centrifuging at 3,220 g for 30 min; and (6) all supernatants were filtered through a Whatman No. 4 filter paper (Fisher Scientific, Fair Lawn, NJ, USA), and the filtrates were stored at −20 °C before use. For nonspiked meat samples, ground meat (20 g) from each species (beef, deer, horse, lamb, pork, chicken, duck, and turkey) was prepared with the two abovementioned heat conditions, respectively. In addition, ground beef and pork were autoclaved under the following five conditions, respectively: (1) 128 °C/1.6 bar for 30 min; (2) 132 °C/2 bar for 30 min; (3) 132 °C/2 bar for 60 min; (4) 132 °C/2 bar for 90 min; and (5) 132 °C/2 bar for 120 min. The Protein Assay Kit II (Bio-Rad, Hercules, CA, USA) was used to determine protein concentration, in which bovine serum albumin (BSA) was the protein standard. For spiked meat samples, both fat- and mammalian meat-adulterated samples were prepared with the two abovementioned heating conditions, respectively. For fat-adulterated meat samples, different amounts of unrendered pork and beef fat (10, 20, and 30%, g/g) were mixed with ground pork and beef, respectively. For mammalian meatspiked poultry samples, ground beef and pork were mixed with turkey and chicken (4%, g/g), respectively. The samples spiked with lower levels of mammalian meats were prepared by diluting the 4% adulterated sample extracts with nonspiked poultry extracts at levels of 0.05, 0.1, 0.5, 1, and 2% on a volume basis. To study species selectivity of the two mAbs (6G1 and 8F10), two poultry (chicken and turkey) and two mammalian (beef and pork) meat samples were prepared using NaCl-PBS. To study the matrix effect on the antigenicity of PsTn, two heated samples were prepared: isolated PsTn (1 mg/mL) dissolved in NaCl-PBS and pork meat sample extracted using NaCl-PBS-urea-βME (NaCl-PBS containing 3 M urea and 1 mM βmercaptoethanol). It is noted that no precipitates were visible to the naked eye in heated (100 °C for 15 min) PsTn after centrifugation (20,000 g for 15 min at 4 °C). Poultry and mammalian meat samples for selectivity and thermostability study were two-fold (g/mL) extracted with the extraction buffer after heat treatment (100 °C for 15 min at 600 rpm using a thermomixer (Eppendorf, Hamburg, Germany)). The mixture was homogenized, sonicated (10% amplitude for 10 s three times using a Q125 Sonicator, Qsonica, LLC., Newtown, CT, USA), and
2.5. Sandwich ELISA The performance of the optimized sandwich ELISA was evaluated by studying (1) the species selectivity using nonspiked meat samples; (2) the effect of fat-adulteration and heat treatment on the target analyte detection; and (3) the detection of mammalian meat-spiked poultry meat samples. Unless otherwise specified, during each ELISA step, (1) the added reagent volume was 100 μL/well; (2) the incubation time was 1 h at 37 °C; and (3) three PBST washes were performed using a microplate washer (Bio-Rad). Briefly, a 96-well clear polystyrene microplate (Corning, Inc., Corning, NY, USA) was coated with the capture antibody (1,374 ppm of mAb6G1 in PBS). After blocking with BSA-PBS (200 μL/well), heated meat extracts (nonspiked and fat- and mammalian meat-spiked meat samples) prepared in section 2.3 were added. After incubation for 2 h at 37 °C, the detection antibody (1,000 ppm of mAb8F10-HRP diluted in BSA-PBST), was added. After five PBST washes, the color development was performed by adding the ABTS substrate solution (22 mg of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 15 μL of 30% hydrogen peroxide in 100 mL of 0.1 M phosphate-citrate buffer, pH 4.0), and incubated for 25 min at RT in the 3
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dark, followed by adding 0.2 M citric acid to stop the reaction. The absorbance at 415 nm (A415) was measured by a microplate reader (BioRad).
detected by mAb8F10 (green, lane 3) but also simultaneously identified by anti-sTnC pAb (red, lane 2). These overlapping bands were confirmed because their yellow color was generated from the overlapping red and green bands (lane 1, Fig. 2). It should be noted that this antisTnC pAb could react with PsTnC and cross-react with PsTnI according to its product instruction (MyBioSource Inc., 2019). The antigenic band below PsTnC (lanes 3–4, Fig. 2) might be the degraded peptide(s) from sTnI and/or sTnC during PsTn isolation, and/or through post-mortem changes in pork. It was reported that enzymes such as cathepsin and calpain could hydrolyze myofibrillar proteins such as sTn during post mortem (Kemp, Sensky, Bardsley, Buttery, & Parr, 2010). From Western blot II (lanes 4–6, Fig. 2), three antigenic bands (red, lane 5) were identified by anti-sTnT pAb, which is specific to sTnT and its oligomers (Novus Biologicals LLC, 2019). None of these bands overlapped with any of those three green bands detected by mAb8F10 (green, lanes 4 and 6, Fig. 2). Therefore, based on the relative antigenic protein band position (Fig. 2), the images from double fluorescent Western blot verified that (1) isolated PsTn contained three sTn subunits; and (2) mAb8F10 was positive to PsTnI and PsTnC but negative to PsTnT. The same antigenic band pattern of mAb8F10 was confirmed using chemiluminescent Western blot (lane 7, Fig. 1B). It is noted that mAb6G1 shared a similar band pattern with mAb8F10 (lane 7, Fig. 1), indicating that mAb6G1 was also immunoreactive to PsTnI and PsTnC. The protein profiles of four heated meat samples extracted using NaCl-PBS (lanes 1–4) are shown in Fig. 1A. As for antibody selectivity, both mAbs 8F10 and 6G1 were positive to two heated mammalian meat extracts (beef and pork, lanes 3–4, Fig. 1B) but negative to two heated poultry meat samples (turkey and chicken, lanes 1–2). The species
2.6. Statistical analysis Two-way ANOVA with Dunnett post hoc test was performed to study the effect of fat-adulteration and heat treatment on the target analyte detection, and to determine the detection limits of mammalian meat-adulterated poultry meat samples. A p-value of 0.05 or lower was considered statistically significant. Data analysis was performed using GraphPad Prism 8 for Windows (version 8.2.0, GraphPad Software Inc., La Jolla, CA, USA). 3. Results and discussion 3.1. Antibody characterization Through the assistance of two commercial pAbs (anti-sTnC and antisTnT), two fluorescent Western blots were performed to verify the immunoreactivity between three PsTn subunits and mAb8F10, i.e., Western blot I (mAb8F10 and anti-sTnC pAb to study sTnI and sTnC) and Western blot II (mAb8F10 and anti-sTnT pAb to study sTnI and sTnT). From the protein profile of isolated PsTn (lane 7, Fig. 1A), two major bands at 22 kDa and 18 kDa were similar to the molecular weight (MW) of PsTnI and PsTnC (Table 1), respectively. In addition, two weak bands around 37 kDa closed to the MW of PsTnT (Table 1) were observed. From Western blot I (lanes 1–3, Fig. 2), three antigenic bands were not only
Fig. 1. (A) Protein profile of isolated porcine skeletal troponin complex (PsTn) and heated meat extracts using reducing SDSPAGE. (B) Antigenicity of mAbs 6G1 and 8F10 revealed by chemiluminescent Western blot using meat protein extracts and PsTn. The protein loading mass was 5 μg/lane for four meat extracts (lanes 1–4) and 2.5 μg/lane for isolated PsTn (lane 7). The pork meat sample extracted using NaClPBS-urea-βME (lane 5) and heated PsTn (lane 6) were prepared with the same dilution ratio as their counterparts (lane 4 and 7), respectively. The Precision Plus Protein Kaleidoscope Protein Standards (Bio-Rad) was used for band sizing. The IgG concentration of mAbs 6G1 and 8F10 was 1 ppm.
4
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Fig. 2. Immunoreactivity between porcine skeletal troponin subunits (PsTnT, PsTnI, and PsTnC) and mAb8F10 using double fluorescent Western blot. The protein loading mass was 2.5 μg/lane. Western blot I was performed using a mixture of mAb8F10 (green) and anti-sTnC pAb (red), while Western blot II was performed using a mixture of mAb8F10 and anti-sTnT pAb (red). The IgG concentration of each pooled antibody was 1 ppm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
selectivity of both mAbs matched with our previous findings using indirect non-competitive ELISA (Liu et al., 2006; Rao, 2004), indicating that both mAbs were specific to mammalian sTn. As to isolated PsTn, both reducing SDS-PAGE (Fig. 1A) and Western blot (Fig. 1B) results showed that there was no significant difference in the band pattern and color intensity before (lane 7) and after (lane 6) heat treatment. Both PsTnI and PsTnC subunits in isolated PsTn were thermostable and could retain their solubility, molecular integrity, and immunoreactivity after heat treatment. However, in heated mammalian meat extracts, it is noted that sTnI but not sTnC was identified by both mAbs using chemiluminescent Western blot (lanes 3–4, Fig. 1B). It has been verified that sTnI from different meat species was thermostable (Chen & Hsieh, 2001; Chen & Hsieh, 2002; Chen, Hsieh, & Bridgman, 2004; Zvereva et al., 2015). However, few publications reported sTnC as a thermostable marker, which is probably because its thermostability is dependent on the amount of calcium (Brzeska, Venyaminov, Grabarek, & Drabikowski, 1983). It is noted that the addition of urea and βME into the extraction buffer (NaCl-PBS, lane 5, Fig. 1A) enhanced the extractability of pork meat proteins by around 120% compared to its counterpart extracted using NaCl-PBS (lane 4, Fig. 1A) according to the total lane color intensity analysis. Also, the pork protein extracts (lanes 4–5, Fig. 1A) have a greater matrix complexity than the isolated PsTn (lane 7, Fig. 1A). The extractability of target proteins is affected by both internal factors (such as protein isoelectric point and polar/apolar nature) and external factors (such as processing condition and extraction method) (EFSA Panel on Dietetic Products Nutrition and Allergies, 2014; Yu, Morton, Clerens, & Dyer, 2017). The addition of solubilizing (i.e., urea) and reducing (i.e., βME) agents can enhance protein extractability. This is because urea can effectively break non-covalent bonds, such as hydrogen bonds (della Malva et al., 2018), whereas βME can reduce the covalent disulfide bonds between cysteine residues in the protein mass (He et al., 2018). It should be noted that PsTnT has no cysteine residues, while both PsTnI and PsTnC contain more than one (Table 1), indicating that disulfide-induced protein aggregation can occur during heat treatment and lead to PsTnC is not extractable. della Malva et al. (2018) reported that the use of denaturing solutions could improve the extractability of myofibrillar proteins with low MW (< 45 kDa). It is noted that PsTnC was detected by mAb8F10 but not by mAb6G1 (lane 5, Fig. 1B), which might be caused by the destruction of mAb6G1 epitopes on PsTnC during the extraction using urea and βME.
3.2. Sandwich ELISA evaluation Through the epitope comparison study (data not shown), mAbs 6G1 and 8F10-HRP were paired as the capture and detection antibody, respectively, to establish a sandwich ELISA. The optimized sandwich ELISA was specific to heated mammalian (beef, deer, horse, lamb, and pork) meats while had no cross-reaction with heated poultry (chicken, duck, and turkey) meats (Table 2). According to USDA Livestock and Meat Domestic Data, pork and beef are the two mammalian species that are mostly produced and consumed in the U.S. (USDA, 2019a) These two mammalian meats were chosen to evaluate the sandwich ELISA performance. To assess the effect of heat treatment on the performance of the established sandwich ELISA, beef and pork under different heat treatment were studied. For pork protein extracts, only when the heating condition elevated to 132 °C/2 bar/120 min, its relative absorbance decreased about 35% compared with that from the 100 °C/30 min treatment (p < 0.05, Fig. 3). For beef protein extracts, after heating at 132 °C/2 bar for 60 min or longer, it produced a significantly smaller relative A415 compared to that from the 100 °C/30 min treatment (p < 0.05, Fig. 3). It is noted that the A415 of the most severe treatment (132 °C/2 bar/ 120 min) in beef samples remained as high as 0.9, indicating that the developed assay was adequate to analyze mammalian meat samples regardless of the extent of heat processing. Table 2 Species selectivity of sandwich ELISA. Species Mammalian Beef Deer Horse Lamb Pork Poultry Chicken Duck Turkey
100 °C/30 min
121 °C/1.2 bar/30 min
+++* +++ +++ +++ +++
+++ +++ +++ +++ +++
– – –
– – –
* +++: very strong reaction (A415 ≥ 1.2); ++: strong reaction (1.2 > A415 ≥ 0.7); +: weak reaction (0.7 > A415 ≥ 0.2); −: no reaction (A415 < 0.2) (Rao & Hsieh, 2014). 5
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Fig. 3. Effect of heat treatment on the relative immunoreactivity of sandwich ELISA. Different letters within the same meat species indicate the significant difference in % relative absorbance at 415 nm between each heat treatment and 100 °C/30 min (p < 0.05). (A) Pork; (B) Beef. Data are represented as the average % relative absorbance at 415 nm ± SEM (n ≥ 3).
could be predicted using the four-parameter logistic (4 PL) model, a commonly used nonlinear regression analysis in bioassays (Fig. 4). Its goodness of fit (R2) of both heat treatments (100 °C/30 min and 121 °C/ 1.2 bar/30 min) was greater than 96%. The optimized assay could detect as low as 1% (g/g) of mammalian meats adulterated in poultry meats under each heating condition (p ≤ 0.05, black dash-line rectangle, Fig. 4). The EC50 (half-maximal effective concentration) was 1.5% and 2.3% for 100 °C/30 min- and 121 °C/1.2 bar/30 min-heated adulterants, respectively, indicating that different heat treatments could affect the assay sensitivity. Detection of mammalian meat adulteration is important for the enforcement of food-labeling laws. Different methods such as polymerase chain reaction (PCR) (Karabasanavar, Girish, Kumar, & Singh, 2017), mass spectrometry (Ruiz Orduna, Husby, Yang, Ghosh, & Beaudry, 2017) and infrared spectroscopy (Yang et al., 2018) have been utilized for meat species identification. However, it should be noted that no single method can be applied to all cases. For example, PCR was adopted to amplify small DNA segments to determine animal species (Nešić, Stojanović, & Baltić, 2017), while it has the limitation such as prone to contamination (Hsieh, 2004). In addition, due to the variability of DNA at the species and target tissue levels, this type of method is unsuitable for the quantification of the exact percentages of different species in meat or meat products (Nešić et al., 2017). The majority of the published PCR methods are qualitative, and quantitative analysis should be based on real-time PCR (Mayada Ragab Farag, Mahmoud Alagawany, Mohamed Ezzat Abd El-Hack, Ruchi Tiwari, & Dhama, 2018). It is well known that the immunoassay is a rapid, highly sensitive and quantitative method which is suitable for handling numerous samples at a time (Singh & Sachan, 2011). Many studies have developed ELISA to detect mammalian meat adulteration. For example, Liu et al. (2006) reported a sandwich ELISA that was able to detect 0.05% and 0.1% (g/g) of heated pork adulterated in chicken and beef, respectively. Chen and Hsieh (2000) used an indirect non-competitive ELISA to detect 0.5% (g/g) heated pork in meat mixtures. Jones and Patterson (1985) and Martín, Azcona, García, Hernández, and Sanz (1988) applied a sandwich ELISA to detect 1–50% (g/g) of pork in raw beef and horse meat in the raw beef and pork mixtures, respectively. Hsieh, Sheu, and Bridgman (1998) used an indirect non-competitive ELISA to screen as low as 0.5% (g/g) of mammalian meat (cattle, hogs, sheep, horse, and deer) adulterated in heated (100 °C, 15 min) poultry
Table 3 Effect of mammalian fat content on the relative immunoreactivity of sandwich ELISA. Heat treatment % pork fat in pork (g/g) 0 10 20 30 % beef fat in beef (g/g) 0 10 20 30
100 °C/30 min
121 °C/1.2 bar/30 min
100 ± 3.0a * 98.2 ± 0.9a 97.3 ± 1.9a 100.9 ± 0.1a
100 ± 1.2a 98.2 ± 2.4a 98.6 ± 1.3a 97.4 ± 0.9a
100 ± 4.9a 104 ± 1.6a 94.8 ± 0.8a 95.6 ± 1.4a
100 ± 3.4a 89.1 ± 0.5a 90.1 ± 1.3a 95.2 ± 0.1a
* For the same sample model, values in the same column with different letters are significantly different (p < 0.05). Data are represented as the average % relative absorbance at 415 nm ± SEM (standard error of the mean, n ≥ 2).
Fat is an important component of animal meat products and varies in different products. For example, regular ground beef contains 30% fat, while in extra beef, the fat content is limited to 5% or less (USDA, 2014). It is important to study the potential interference of fat in meat on the immunoreactivity. To illustrate the effect of mammalian fat content on the performance of the established sandwich ELISA, different fat-adulterated pork and beef were tested. For the same species under the same heat treatment, there was no significant difference in relative A415 among different fat adulteration levels (10%, 20%, and 30%) compared with the lean meat extract (p ≥ 0.05, Table 3). Our results showed that the extraction method was able to extract similar amounts of target analytes from high-fat mammalian meats. The effect of mammalian fat content on the optimized sandwich ELISA was insignificant. As for the preparation of the heated mammalian meat-spiked poultry meat samples, since there was no significant difference in the immunosignal from pork adulterated in chicken samples prepared by either the weight-basis or volume-basis methods (Supplementary Document), the volume-basis method was selected in this study. For the same heat treatment, there was no significant absorbance difference at the same adulteration level (p > 0.05) between pork spiked in chicken and beef spiked in turkey. At each heating condition, two curves were combined, and the ELISA signals of heated adulterated poultry samples 6
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Fig. 4. Immunodetection of heated mammalian meat adulterated in poultry meat samples using sandwich ELISA. The four-parameter logistic model was used for the nonlinear regression analysis. The EC50 of each heat treatment is indicated using a blue and red dotted line, respectively. The detection limit of each heat treatment is indicated using a black dash-line rectangle. The value R2 quantifies the goodness of fit. Data are represented as the average % maximum absorbance at 415 nm ± SEM (n ≥ 4). 100 °C/30 min: blue solid line; 121 °C/ 1.2 bar/30 min: red dash-dotted line. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
products. The limitations in these assays include (1) was selective to only one mammalian meat; (2) was against only adulterated raw meats; (3) using the indirect non-competitive format that is difficult to be commercialized. To the best of our knowledge, the established ELISA is the first sandwich format assay that can be used for the detection of undeclared mammalian meats in poultry meats. Overall, this assay has the potential to fight food fraud, comply with food regulations, and decrease food recalls, which may open up new diagnostic methods for the food industry and the food regulatory authorities.
Chen, F. C., & Hsieh, Y.-H. P. (2002). Porcine troponin I: A thermostable species marker protein. Meat Science, 61(1), 55–60. Chen, F. C., Hsieh, Y.-H. P., & Bridgman, R. C. (2004). Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of prohibited ruminant proteins in feedstuffs. Journal of Food Protection, 67(3), 544–549. EFSA Panel on Dietetic Products Nutrition and Allergies (2014). Scientific opinion on the evaluation of allergenic foods and food ingredients for labelling purposes. EFSA Journal, 12(11), 3894–4180. Greaser, M. L., & Gergely, J. (1973). Purification and properties of the components from troponin. Journal of Biological Chemistry, 248(6), 2125–2133. Grujic, R., & Savanovic, D. (2018). Analysis of myofibrillar and sarcoplasmic proteins in pork meat by capillary gel electrophoresis. Foods and Raw Materials, 6(2), 421–428. He, J., Zhou, G. H., Bai, Y., Wang, C., Zhu, S. R., Xu, X. L., et al. (2018). The effect of meat processing methods on changes in disulfide bonding and alteration of protein structures: Impact on protein digestion products. RSC Advances, 8(31), 17595–17605. Hsieh, Y.-H. P. (2004). Meat species identification. In Y. H. Hui (Vol. Ed.), Handbook of food science, technology, and engineering: Vol. 1, (pp. 1–19). CRC Press. Hsieh, Y.-H. P., & Chen, F. C.. Use of troponin I as a species marker protein for meat speciation in both raw and heat-processed product. (2002). https://patentimages.storage. googleapis.com/d8/28/53/fe927a9dd0d830/US7297500.pdf Accessed December 4, 2019. Hsieh, Y.-H. P., Sheu, S.-C., & Bridgman, R. C. (1998). Development of a monoclonal antibody specific to cooked mammalian meats. Journal of Food Protection, 61(4), 476–481. Jones, S. J., & Patterson, R. L. S. (1985). Double-antibody ELISA for detection of trace amounts of pig meat in raw meat mixtures. Meat Science, 15(1), 1–13. Karabasanavar, N., Girish, P. S., Kumar, D., & Singh, S. P. (2017). Detection of beef adulteration by mitochondrial D-loop based species-specific polymerase chain reaction. International Journal of Food Properties, 20(S2), 2264–2271. Kemp, C. M., Sensky, P. L., Bardsley, R. G., Buttery, P. J., & Parr, T. (2010). Tenderness – an enzymatic view. Meat Science, 84(2), 248–256. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. Layton, L.. FDA pressured to combat rising 'food fraud. (2010). http://www.washingtonpost. com/wp-dyn/content/article/2010/03/29/AR2010032903824_pf.html Accessed December 4, 2019. Liu, L. H., Chen, F. C., Dorsey, J. L., & Hsieh, Y.-H. P. (2006). Sensitive monoclonal antibody-based sandwich ELISA for the detection of porcine skeletal muscle in meat and feed products. Journal of Food Science, 71(1), M1–M6. della Malva, A., Albenzio, M., Santillo, A., Russo, D., Figliola, L., Caroprese, M., et al. (2018). Methods for extraction of muscle proteins from meat and fish using denaturing and nondenaturing solutions. Journal of Food Quality. https://doi.org/10. 1155/2018/8478471. Martín, R., Azcona, J. I., García, T., Hernández, P. E., & Sanz, B. (1988). Sandwich ELISA for detection of horse meat in raw meat mixtures using antisera to muscle soluble proteins. Meat Science, 22(2), 143–153. Moerman, F. (2018). Food defense. In A. M. Holban, & A. M. Grumezescu (Eds.). Food control and biosecurity (pp. 135–223). Academic Press. MyBioSource Inc. Polyclonal antibody to troponin C type 1, slow (TNNC1) instruction manual. (2019). https://cdn.mybiosource.com/tds/protocol_manuals/800000-9999999/ MBS2032663_TDS.pdf Accessed December 4, 2019. Nakyinsige, K., Man, Y. B., & Sazili, A. Q. (2012). Halal authenticity issues in meat and meat products. Meat Science, 91(3), 207–214.
Author contributions Xingyi Jiang. Collected the data, Contributed data or analysis tools, Performed the analysis, Wrote the paper. Qinchun Rao: Conceived and designed the analysis, Collected the data, Contributed data or analysis tools, Performed the analysis, Wrote the paper. Kristen Mittl: Collected the data. Yun-Hwa Peggy Hsieh: Conceived and designed the analysis. Declaration of competing interest The authors declare no competing financial interest. Acknowledgements A part of this study was supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture (2017-70001-25984). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2019.107045. References Brzeska, H., Venyaminov, S. V., Grabarek, Z., & Drabikowski, W. (1983). Comparative studies on thermostability of calmodulin, skeletal muscle troponin C and their tryptic fragments. FEBS Letters, 153(1), 169–173. Chen, F. C., & Hsieh, Y.-H. P. (2000). Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA. Journal of AOAC International, 83, 79–85. Chen, F. C., & Hsieh, Y.-H. P. (2001). Separation and characterization of a porcine‐specific thermostable muscle protein from cooked pork. Journal of Food Science, 66(6), 799–803.
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X. Jiang, et al. National Center for Food Protection and Defense. NCFPD EMA database. (2015). https:// foodprotection.umn.edu/research/ema-database-and-portal Accessed December 4, 2019. Nešić, K., Stojanović, D., & Baltić, Ž. M. (2017). Authentication of meat and meat products vs. detection of animal species in feed – what is the difference? IOP Conference Series: Earth and Environmental Science, 85. https://doi.org/10.1088/1755-1315/1085/ 1081/012043. Novus Biologicals LLC. Troponin T type 1 (slow skeletal) antibody. (2019). https://www. novusbio.com/products/troponin-t-type-1-slow-skeletal-antibody_nbp1-32748# datasheet Accessed December 4, 2019. van Nunen, S. (2015). Tick-induced allergies: Mammalian meat allergy, tick anaphylaxis and their significance. Asia Pacific Allergy, 5(1), 3–16. Park, B. S., Oh, Y. K., Kim, M. J., & Shim, W. B. (2014). Skeletal muscle troponin I (TnI) in animal fat tissues to be used as biomarker for the identification of fat adulteration. Korean Journal for Food Science of Animal Resources, 34(6), 822–828. Potter, J. D. (1982). Preparation of troponin and its subunits. Methods in Enzymology, 85, 241–263. Ragab Farag, M., Alagawany, M., Ezzat Abd El-Hack, Mohamed, Tiwari, R., & Dhama, K. (2018). Identification of different animal species in meat and meat products: Trends and advances. Advances in Animal and Veterinary Sciences, 3(6), 334–346. Rao, Q. C. (2004). Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for the detection of mammalian meat in meat and feed products. Tallahassee, FL: Thesis, Florida State University. Rao, Q. C., & Hsieh, Y.-H. P. (2014). Enhanced immunodetection of bovine central nervous tissue using an improved extraction method. Food Control, 46, 282–290. Regenstein, J. M., Chaudry, M. M., & Regenstein, C. E. (2003). The kosher and halal food laws. Comprehensive Reviews in Food Science and Food Safety, 2(3), 111–127. Ruiz Orduna, A., Husby, E., Yang, C. T., Ghosh, D., & Beaudry, F. (2017). Detection of meat species adulteration using high-resolution mass spectrometry and a proteogenomics strategy. Food Additives & Contaminants: Part A, 34(7), 1110–1120. Singh, V. P., & Sachan, N. (2011). Meat species specifications to ensure the quality of meat-a review. International Journal of Meat Science, 1(1), 15–26. The UniProt Consortium (2017). UniProt: The universal protein knowledgebase. Nucleic Acids Research, 45(D1), D158–D169. Thermo Scientific. Tech tip #6 extinction coefficients. (2019). https://assets.thermofisher. com/TFS-Assets/LSG/Application-Notes/TR0006-Extinction-coefficients.pdf Accessed December 4, 2019. USDA (2011). 9 CFR 301.2 - definitions. http://www.gpo.gov/fdsys/granule/CFR-2011-
title9-vol2/CFR-2011-title9-vol2-sec301-2 Accessed December 4, 2019. USDA (2012). 9 CFR 381.1 - definitions. http://www.gpo.gov/fdsys/granule/CFR-2012title9-vol2/CFR-2012-title9-vol2-sec381-1 Accessed December 4, 2019. USDA. Beef from farm to table. (2014). http://www.fsis.usda.gov/wps/wcm/connect/ c33b69fe-7041-4f50-9dd0-d098f11d1f13/Beef_from_Farm_to_Table.pdf?MOD= AJPERES Accessed December 4, 2019. USDA. Livestock & meat domestic data. (2019). https://www.ers.usda.gov/data-products/ livestock-meat-domestic-data/livestock-meat-domestic-data/#Red%20meat%20and %20poultry%20production Accessed December 4, 2019. USDA (2019b). Recall case archive. http://www.fsis.usda.gov/wps/portal/fsis/topics/ recalls-and-public-health-alerts/recall-case-archive Accessed December 4, 2019. USFDA (1995). 21 U.S.C. 458 - prohibited acts. http://www.gpo.gov/fdsys/granule/ USCODE-1994-title21/USCODE-1994-title21-chap10-sec458 Accessed December 4, 2019. USFDA. FDA food safety modernization act (FSMA). (2011). http://www.fda.gov/Food/ GuidanceRegulation/FSMA/default.htm Accessed December 4, 2019. USFDA (2012). U.S.C. 331 - prohibited acts. http://www.gpo.gov/fdsys/granule/USCODE2011-title21/USCODE-2011-title21-chap9-subchapIII-sec331 Accessed December 4, 2019. USFDA (2016). 21 CFR Parts 11 and 121 Mitigation strategies to protect food against intentional adulteration. https://www.govinfo.gov/content/pkg/FR-2016-05-27/pdf/ 2016-12373.pdf Accessed December 4, 2019. USFDA. Archive for recalls, market withdrawals and safety alerts. (2019). http://www.fda. gov/Safety/Recalls/ArchiveRecalls/default.htm Accessed December 4, 2019. Yang, L., Wu, T., Liu, Y., Zou, J., Huang, Y., Babu, V. S., et al. (2018). Rapid identification of pork adulterated in the beef and mutton by infrared spectroscopy. Journal of Spectroscopy. https://doi.org/10.1155/2018/2413874. Yu, T.-Y., Morton, J. D., Clerens, S., & Dyer, J. M. (2017). Cooking-induced protein modifications in meat. Comprehensive Reviews in Food Science and Food Safety, 16(1), 141–159. Zot, A. S., & Potter, J. D. (1987). Structural aspects of troponin-tropomyosin regulation of skeletal-muscle contraction. Annual Review of Biophysics and Biophysical Chemistry, 16, 535–559. Zvereva, E. A., Kovalev, L. I., Ivanov, A. V., Kovaleva, M. A., Zherdev, A. V., Shishkin, S. S., et al. (2015). Enzyme immunoassay and proteomic characterization of troponin I as a marker of mammalian muscle compounds in raw meat and some meat products. Meat Science, 105, 46–52.
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Monoclonal antibody-based sandwich ELISA for the detection of mammalian meats
T
Xingyi Jiang, Qinchun Rao∗, Kristen Mittl, Yun-Hwa Peggy Hsieh Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL, USA
ARTICLE INFO
ABSTRACT
Keywords: Sandwich ELISA Mammalian meat Skeletal troponin Matrix effect
In order to (1) reduce the risk of intentional or unintentional contamination of foods, (2) better comply with food regulations, and (3) decrease economic loss to the food industry caused by recall, it is necessary to develop reliable methods for the detection of different food adulterants/contaminants. This study was conducted to characterize two mammalian skeletal troponin (sTn) specific monoclonal antibodies (mAbs 6G1 and 8F10), and use them to develop a mAb-based sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of mammalian meats. From our results, both mAbs were positive to porcine sTnI and sTnC but negative to sTnT. The extractability and antigenicity of target analytes in pork were enhanced by the addition of urea and βmercaptoethanol into the extraction buffer. The optimized sandwich ELISA was specific to heated mammalian meats and was adequate to analyze samples subject to the most severe heat treatment (132 °C/2 bar/120 min). Mammalian fat (10–30%, g/g) did not significantly affect the assay signal. The optimized assay could detect as low as 1% (g/g) of heated mammalian meats adulterated in poultry meats. Overall, this assay has the potential to fight food fraud, comply with food regulations, and decrease food recalls, which may open up new diagnostic methods for the food industry and the food regulatory authorities.
1. Introduction It is estimated that food adulteration costs the world economy around $49 billion annually (Moerman, 2018). Around 10% of the foods produced in the U.S. were adulterated and 7% contain fraudulent ingredients (Layton, 2010). From 1980 to 2013, (1) the leading reported type of fraudulent foods was animal products including meat and meat products (7%); (2) 65% of the total incidents were due to substitution or dilution; and (3) in about 30% of the total incidents, the involved food products were produced in the U.S. (National Center for Food Protection and Defense, 2015). Between 2016 and 2017, about 35% (423/1,213) of the food recalls were due to the presence of misbranding and undeclared food residues, which is the No. 1 cause of food recalls in the U.S. (USDA, 2019b; USFDA, 2019). Also, mammalian meat intentional/unintentional adulteration is a serious concern for religious and cultural reasons as well as for those with individual moral aversions (Regenstein, Chaudry, & Regenstein, 2003). Both Muslims and Jews who follow religious law are forbidden from consuming products derived from pigs and animal blood. It was reported that most of the 6 to 8 million Muslims in North America observe halal laws, particularly the avoidance of pork, however, the food industry has essentially ignored this consumer group (Regenstein et al., 2003), while it ∗
is the major authenticity concerns in meat products for Muslim consumers (Nakyinsige, Man, & Sazili, 2012). In addition, as an emergent allergy, mammalian meat allergy is increasingly prevalent in tick-endemic areas worldwide (van Nunen, 2015). As evidenced by the startling statistics noted above, there exists a pressing need to fight food fraud and reduce the risk of foodborne illness. In the U.S., the prohibitions against adulterated and misbranded food appear in many federal laws, including the Food, Drug and Cosmetic Act (USFDA, 2012), the Meat Inspection Act (USDA, 2011; USFDA, 2012), the Poultry Products Inspection Act (USDA, 2012; USFDA, 1995), and the FDA Food Safety Modernization Act (USFDA, 2011). To prevent intentional adulteration from acts intended to cause wide-scale harm to public health, a final rule entitled “Mitigation Strategies to Protect Food Against Intentional Adulteration” was enacted in 2019 (USFDA, 2016). To better comply with the food regulations and decrease the economic loss to the food industry, many monoclonal antibody (mAb) based immunoassays have been commercialized for the detection of different food adulterants/contaminants. It should be noted that Chen and Hsieh (2002) reported that porcine skeletal muscle protein, troponin I (PsTnI), was thermostable, which could still maintain its solubility and antigenicity after severe heat treatment (126 °C for 120 min).
Corresponding author. E-mail address: [email protected] (Q. Rao).
https://doi.org/10.1016/j.foodcont.2019.107045 Received 9 August 2019; Received in revised form 5 December 2019; Accepted 6 December 2019 0956-7135/ © 2019 Published by Elsevier Ltd.
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Table 1 Summary of skeletal muscle troponins from pig (Sus scrofa)a. Protein name
Muscle fiber type
UniProtKB accession number
Troponin T (PsTnT)
Slow
Q75ZZ6 (reviewed)d
Fast
Q75NG9 (reviewed)
Not available
P02587 (reviewed)
Slow
P63317 (reviewed)
Fast
A1XQV5 (unreviewed)
Slowtwitch
Q7YSF4 (unreviewed)
Fasttwitch
Q4JH15 (unreviewed)
Troponin C (PsTnC)
Troponin I (PsTnI)
a b c d
Amino acid sequenceb
1 61 121 181 241 1 61 121 181 241 1 61 121 1 61 121 1 61 121 1 61 121 181 1 61 121 181
MSDAEEQEYEEEQPEEEEAAEEEEAPEEPEPVAEREEERPKPSRPVVPPLIPPKIPEGER VDFDDIHRKRMEKDLLELQTLIDVHFEQRKKEEEELVALKERIERRRAERAEQQRFRTEK ERERQAKLAEEKMRKEEEEAKKRAEDDAKKKKVLSNMGAHFGGYLVKAEQKRGKRQTGRE MKQRILSERKKPLNIDHMGEDQLREKAQELSDWIHQLESEKFDLMAKLKQQKYEINVLYN RISHAQKFRKGAGKGRVGGRWK MSDEEVEHVEEEYEEEEEAQEEAPPPPAEVHEVHEEVHEVHEPEEVQEEEKPRPKLTAPK IPEGEKVDFDDIQKKRQNKDLMELQALIDSHFEARKKEEEELVALKERIEKRRAERAEQQ RIRAEKERERQNRLAEEKARREEEEAKRRAEDDLKKKKALSSMGANYSSYLAKADQKRGK KQTAREMKKKVLAERRKPLNIDHLSEDKLRDKAKELWDALYQLEIDKFEYGEKLKRQKYD IINLRSRIDQAQKHSKKAGTTPKGKVGGRWK TDQQAEARSYLSEEMIAEFKAAFDMFDADGGGDISVKELGTVMRMLGQTPTKEELDAIIE EVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELAECFRIFDRNMDGYIDAEELAEIFR ASGEHVTDEEIESIMKDGDKNNDGRIDFDEFLKMMEGVQ MDDIYKAAVEQLTEEQKNEFKAAFDIFVLGAEDGCISTKELGKVMRMLGQNPTPEELQEM IDEVDEDGSGTVDFDEFLVMMVRCMKDDSKGKSEEELSDLFRMFDKNADGYIDLEELKIM LQATGETITEDDIEELMKDGDKNNDGRIDYDEFLEFMKGVE MTDQQAEARSYLSEEMIAEFKAAFDMFDADGGGDISVKELGTVMRMLGQTPTKEELDAII EEVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELAECFRIFDRNMDGYIDAEELAEIF RASGEHVTDEELESLMKDGDKNNDGRIDFDEFLKMMEGVQ MPEVERKPKITASRKLLLKGLMLAKAKECWEQEHEEREAEKARYLAERIPTLQTRGLSLS ALQDLCRELHAKVEVVDEERYDIEAKCLHNTREIKDLKLKVLDLRGKFKRPPLRRVRVSA DAMLRALLGSKHKVSMDLRANLKSVKKEDTEKERPVEVGDWRKNVEAMSGMEGRKKMFDA AKSPTSQ MGDEEKRHRAITARRQHLKSVMLQIAATELEKEVGRRESEKQNYLSEHCPPLHLPGSMSE VQELCKQLHAKIDAAEEEKYDMEIKVQKSTKELEDMNQKLFDLRGKFKRPPLRRVRMSAD AMLKALLGSKHKVCMDLRANLKQVKKEDTEKERDLRDVGDWRKNIEEKSGMEGRKKMFET ES
60 120 180 240 262 60 120 180 240 271 60 120 159 60 120 161 60 120 160 60 120 180 187 60 120 180 182
Molecular weight (Da)
% Relative concentrationc
31,243
4.70
32,176
18,025
3.25
18,417 18,156 21,616
4.49
21,334
All information was obtained from The UniProt Consortium (2017). The calcium-binding sites are highlighted in green with a dotted underline. Cysteine residues are highlighted in yellow with a single underline. % Relative concentration in myofibrillar proteins from fresh pork meat samples. Data are from Grujic and Savanovic (2018). Reviewed: the entry belongs to the Swiss-Prot section of UniProtKB; Unreviewed: the entry belongs to the computer-annotated TrEMBL section.
As one of the three skeletal troponin (sTn) subunits to regulate muscle contraction, sTnI inhibits actomyosin ATPase activity (Greaser & Gergely, 1973). The other two sTn subunits are sTnT (tropomyosinbinding protein) and sTnC (Ca2+-binding protein) (Zot & Potter, 1987). The relevant molecular information of porcine sTn subunits is summarized in Table 1. It should be noted that there is a high amino acid similarity (> 90%) in sTn subunits among different mammals. As an ideal immunogen, sTnI from different meat species has been used to develop different mAbs for the authentication of meat and meat products (Chen & Hsieh, 2002; Liu, Chen, Dorsey, & Hsieh, 2006; Park, Oh, Kim, & Shim, 2014; Zvereva et al., 2015). The major objectives of this study were to characterize two mammalian sTn-specific mAbs and use them to develop a mAb-based sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of mammalian meats.
mAb8F10 (mAb8F10-HRP, 1 mg IgG/mL) was provided by Neogen Co. (Lansing, MI, USA). One anti-sTnC polyclonal antibody (anti-sTnC pAb, MBS2032663), developed in rabbit against human slow sTnC (UniProtKB accession number P63316), was purchased from MyBioSource, Inc. (San Diego, CA, USA). One anti-sTnT pAb (NBP1-32748), developed in rabbit against human slow sTnT (UniProtKB accession number P13805), was purchased from Novus Biologicals, LLC (Centennial, CO, USA). Both commercial pAbs were reported to cross-react with PsTn subunits (MyBioSource Inc., 2019; Novus Biologicals LLC, 2019). The multicolor near-infrared (IR) fluorescent Western blotting kit (AzureSpectra IR rb700 and ms800, AC2140), containing goat anti-mouse IgG IR800-labeled (green) and anti-rabbit IgG IR700-labeled (red) secondary antibodies, were purchased from Azure Biosystems Inc. (Dublin, CA, USA). Goat anti-mouse IgG (Fc specific) HRP-conjugated secondary antibody (anti-IgG-HRP, A2554) was purchased from SigmaAldrich (St. Louis, MO, USA). All chemicals and reagents were of analytical grade. All solutions were prepared using deionized water from a NANOpure DIamond ultrapure water system (Barnstead International, Dubuque, IA, USA).
2. Materials and methods 2.1. Materials Beef chuck, lamb thigh, pork shoulder, turkey thigh, chicken thigh, duck meat, and unrendered fat from pork and beef were purchased from local supermarkets (Tallahassee, FL, USA). Deer steaks were provided by the Fats and Proteins Research Foundation (Bloomington, IL, USA). Horse meat was obtained from the College of Veterinary Medicine, Auburn University (Auburn, AL, USA). All lean meats were ground twice using a meat grinder upon received (Proctor-Silex, Inc., Washington, NC, USA) and stored at −80 °C until use. Two mAbs, 6G1 and 8F10, were previously produced in mouse against purified equine sTnI (Hsieh & Chen, 2002). mAb6G1 (1.37 mg IgG/mL) was purified using a Bio-Scale Mini Affi-Prep Protein A Resin Cartridge (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the manufacturer's instruction. Horseradish peroxidase (HRP) conjugated
2.2. Porcine skeletal troponin complex isolation Porcine skeletal troponin complex (PsTn) was isolated (1) to study the immunoreactivity between each subunit and mAbs; and (2) to study the thermostability of its subunit. The isolation was performed according to (Potter, 1982) with modifications. Briefly, after eight washes (1:5, g/mL) using the ice-cold washing solution (1% (mL/mL) Triton X100, 50 mM KCl, 5 mM Tris, pH 8), proteins soluble in low ionic strength buffer were removed from 8 g of ground pork. Homogenization (11,000 rpm for 1 min using an ULTRA-TURRAX T-25 basic homogenizer, IKA Works, Inc., Wilmington, NC, USA) and centrifugation (11,000 g for 10 min at 4 °C) were performed in each wash step. The 2
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washed pellet was then 1:3 (g/mL) washed with 95% (mL/mL) ethanol for three times. This meshed pellet was further washed with an equal amount (g/g) of acetone three times and dried overnight in a fume hood at room temperature (RT). The acetone-dried powder was mixed with 15 parts (g/mL) of the extraction buffer (1 M KCl, 25 mM Tris, 0.1 mM CaCl2, 1 mM DTT, pH 8). After overnight extraction at 4 °C, the supernatant was collected by centrifugation. The precipitates were resuspended in 7.5 parts (mL/g of the starting tissue) of 1 M KCl and centrifuged. Two supernatants were combined, whose pH was adjusted to 4.6 to precipitate tropomyosin. After centrifugation, the pH of the supernatant was adjusted to 8. Ammonium sulfate (AMS) was added to produce 40% saturation with stirring for 2 h at RT. After centrifugation, the supernatant was brought to 60% AMS saturation and stirred overnight at 4 °C. After centrifugation, the pellet was dissolved in 0.5 M KCl and then replaced with NaCl-PBS (0.5 M NaCl in 10 mM phosphatebuffered saline buffer (PBS), pH 7.0) using Amicon Ultra centrifugal filter units (MWCO 10 kDa, EMD Millipore Co., Billerica, MA, USA). Its 1 mg / mL = 1) protein concentration was determined using UV280 nm (E280 (Thermo Scientific, 2019).
end-over-end rotated for 1 h at 4 °C. After centrifugation (50,000 g for 15 min at 4 °C), the supernatant was collected, and its protein concentration was determined. 2.4. Reducing SDS-PAGE and Western blot Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 4% stacking gel and 15% separating gel) was performed to (1) study protein profile of isolated PsTn and heated meat extracts; (2) compare protein solubility in isolated PsTn before and after heat treatment; and (3) compare porcine meat protein extractability using two extraction buffers (i.e., NaCl-PBS and NaCl-PBS-urea-βME). Briefly, meat protein extracts and PsTn were separated by reducing SDSPAGE according to the method of Laemmli (1970) using a Mini-PROTEAN Tetra Cell (Bio-Rad). The Precision Plus Protein Kaleidoscope Protein Standards (Bio-Rad) was used for band sizing. The protein bands were visualized by the Coomassie blue staining. Double fluorescent Western blot was used to study the immunoreactivity between PsTn and mAb8F10. Briefly, electrophoretically separated PsTn on the unstained gel was transferred onto a 0.2-μm lowfluorescent polyvinylidene fluoride (PVDF) membrane (Azure Biosystems) using a Trans-Blot Turbo Transfer System (Bio-Rad). After blocked with BSA-PBS (PBS containing 1% (g/mL) BSA), each membrane was probed with a cocktail of primary antibodies, either mAb8F10+anti-sTnT pAb or mAb8F10+anti-sTnC pAb. The IgG concentration of each pooled antibody was 1 ppm. Each membrane was then incubated for 2 h in the dark with the mixture of two fluorescently labeled secondary antibodies (anti-mouse and anti-rabbit IgGs, 1:5,000 (μL/μL) diluted in BSA-PBST (PBS containing 0.05% (mL/mL) Tween20 and 1% (g/mL) BSA) before imaging. Chemiluminescent Western blot was performed to study (1) the species selectivity of both mAbs (8F10 and 6G1); and (2) the matrix effect on the antigenicity of PsTn. Briefly, electrophoretically separated protein bands on the unstained gel were transferred onto a 0.45-μm nitrocellulose membrane (Fisher Scientific). The transferred proteins were visualized using the Ponceau S staining. The membrane was blocked with BSA-PBS. After incubation with the primary antibody (mAb6G1 or mAb8F10, 1 ppm in BSA-PBST), the membrane was probed with anti-IgG-HRP (170 ppm in BSA-PBST). The blotted antigens were detected using the luminol chemiluminescence method. For both Western blots, the incubation time for each step was at least 1 h at RT. Between each step, the membrane was washed with PBST several times. The images were captured by Azure c600 Imaging System and analyzed using the AzureSpot software (version 14.2, Azure Biosystems).
2.3. Sample preparation Unless otherwise specified, (1) each nonspiked or fat-spiked or mammalian meat-spiked sample was treated with two heat conditions, 100 °C for 30 min in a water bath and 121 °C/1.2 bar for 30 min in a NAPCO 8000-DSE Benchtop Autoclave (Jouan Inc., Winchester, VA, USA), respectively; (2) all heated samples were cooled immediately in ice-cold water; (3) all samples were two-fold (g/mL) extracted with NaCl-PBS after heat treatment; (4) all samples were homogenized at 11,000 rpm for 2 min; (5) all homogenates were held at 4 °C for 2 h before centrifuging at 3,220 g for 30 min; and (6) all supernatants were filtered through a Whatman No. 4 filter paper (Fisher Scientific, Fair Lawn, NJ, USA), and the filtrates were stored at −20 °C before use. For nonspiked meat samples, ground meat (20 g) from each species (beef, deer, horse, lamb, pork, chicken, duck, and turkey) was prepared with the two abovementioned heat conditions, respectively. In addition, ground beef and pork were autoclaved under the following five conditions, respectively: (1) 128 °C/1.6 bar for 30 min; (2) 132 °C/2 bar for 30 min; (3) 132 °C/2 bar for 60 min; (4) 132 °C/2 bar for 90 min; and (5) 132 °C/2 bar for 120 min. The Protein Assay Kit II (Bio-Rad, Hercules, CA, USA) was used to determine protein concentration, in which bovine serum albumin (BSA) was the protein standard. For spiked meat samples, both fat- and mammalian meat-adulterated samples were prepared with the two abovementioned heating conditions, respectively. For fat-adulterated meat samples, different amounts of unrendered pork and beef fat (10, 20, and 30%, g/g) were mixed with ground pork and beef, respectively. For mammalian meatspiked poultry samples, ground beef and pork were mixed with turkey and chicken (4%, g/g), respectively. The samples spiked with lower levels of mammalian meats were prepared by diluting the 4% adulterated sample extracts with nonspiked poultry extracts at levels of 0.05, 0.1, 0.5, 1, and 2% on a volume basis. To study species selectivity of the two mAbs (6G1 and 8F10), two poultry (chicken and turkey) and two mammalian (beef and pork) meat samples were prepared using NaCl-PBS. To study the matrix effect on the antigenicity of PsTn, two heated samples were prepared: isolated PsTn (1 mg/mL) dissolved in NaCl-PBS and pork meat sample extracted using NaCl-PBS-urea-βME (NaCl-PBS containing 3 M urea and 1 mM βmercaptoethanol). It is noted that no precipitates were visible to the naked eye in heated (100 °C for 15 min) PsTn after centrifugation (20,000 g for 15 min at 4 °C). Poultry and mammalian meat samples for selectivity and thermostability study were two-fold (g/mL) extracted with the extraction buffer after heat treatment (100 °C for 15 min at 600 rpm using a thermomixer (Eppendorf, Hamburg, Germany)). The mixture was homogenized, sonicated (10% amplitude for 10 s three times using a Q125 Sonicator, Qsonica, LLC., Newtown, CT, USA), and
2.5. Sandwich ELISA The performance of the optimized sandwich ELISA was evaluated by studying (1) the species selectivity using nonspiked meat samples; (2) the effect of fat-adulteration and heat treatment on the target analyte detection; and (3) the detection of mammalian meat-spiked poultry meat samples. Unless otherwise specified, during each ELISA step, (1) the added reagent volume was 100 μL/well; (2) the incubation time was 1 h at 37 °C; and (3) three PBST washes were performed using a microplate washer (Bio-Rad). Briefly, a 96-well clear polystyrene microplate (Corning, Inc., Corning, NY, USA) was coated with the capture antibody (1,374 ppm of mAb6G1 in PBS). After blocking with BSA-PBS (200 μL/well), heated meat extracts (nonspiked and fat- and mammalian meat-spiked meat samples) prepared in section 2.3 were added. After incubation for 2 h at 37 °C, the detection antibody (1,000 ppm of mAb8F10-HRP diluted in BSA-PBST), was added. After five PBST washes, the color development was performed by adding the ABTS substrate solution (22 mg of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 15 μL of 30% hydrogen peroxide in 100 mL of 0.1 M phosphate-citrate buffer, pH 4.0), and incubated for 25 min at RT in the 3
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dark, followed by adding 0.2 M citric acid to stop the reaction. The absorbance at 415 nm (A415) was measured by a microplate reader (BioRad).
detected by mAb8F10 (green, lane 3) but also simultaneously identified by anti-sTnC pAb (red, lane 2). These overlapping bands were confirmed because their yellow color was generated from the overlapping red and green bands (lane 1, Fig. 2). It should be noted that this antisTnC pAb could react with PsTnC and cross-react with PsTnI according to its product instruction (MyBioSource Inc., 2019). The antigenic band below PsTnC (lanes 3–4, Fig. 2) might be the degraded peptide(s) from sTnI and/or sTnC during PsTn isolation, and/or through post-mortem changes in pork. It was reported that enzymes such as cathepsin and calpain could hydrolyze myofibrillar proteins such as sTn during post mortem (Kemp, Sensky, Bardsley, Buttery, & Parr, 2010). From Western blot II (lanes 4–6, Fig. 2), three antigenic bands (red, lane 5) were identified by anti-sTnT pAb, which is specific to sTnT and its oligomers (Novus Biologicals LLC, 2019). None of these bands overlapped with any of those three green bands detected by mAb8F10 (green, lanes 4 and 6, Fig. 2). Therefore, based on the relative antigenic protein band position (Fig. 2), the images from double fluorescent Western blot verified that (1) isolated PsTn contained three sTn subunits; and (2) mAb8F10 was positive to PsTnI and PsTnC but negative to PsTnT. The same antigenic band pattern of mAb8F10 was confirmed using chemiluminescent Western blot (lane 7, Fig. 1B). It is noted that mAb6G1 shared a similar band pattern with mAb8F10 (lane 7, Fig. 1), indicating that mAb6G1 was also immunoreactive to PsTnI and PsTnC. The protein profiles of four heated meat samples extracted using NaCl-PBS (lanes 1–4) are shown in Fig. 1A. As for antibody selectivity, both mAbs 8F10 and 6G1 were positive to two heated mammalian meat extracts (beef and pork, lanes 3–4, Fig. 1B) but negative to two heated poultry meat samples (turkey and chicken, lanes 1–2). The species
2.6. Statistical analysis Two-way ANOVA with Dunnett post hoc test was performed to study the effect of fat-adulteration and heat treatment on the target analyte detection, and to determine the detection limits of mammalian meat-adulterated poultry meat samples. A p-value of 0.05 or lower was considered statistically significant. Data analysis was performed using GraphPad Prism 8 for Windows (version 8.2.0, GraphPad Software Inc., La Jolla, CA, USA). 3. Results and discussion 3.1. Antibody characterization Through the assistance of two commercial pAbs (anti-sTnC and antisTnT), two fluorescent Western blots were performed to verify the immunoreactivity between three PsTn subunits and mAb8F10, i.e., Western blot I (mAb8F10 and anti-sTnC pAb to study sTnI and sTnC) and Western blot II (mAb8F10 and anti-sTnT pAb to study sTnI and sTnT). From the protein profile of isolated PsTn (lane 7, Fig. 1A), two major bands at 22 kDa and 18 kDa were similar to the molecular weight (MW) of PsTnI and PsTnC (Table 1), respectively. In addition, two weak bands around 37 kDa closed to the MW of PsTnT (Table 1) were observed. From Western blot I (lanes 1–3, Fig. 2), three antigenic bands were not only
Fig. 1. (A) Protein profile of isolated porcine skeletal troponin complex (PsTn) and heated meat extracts using reducing SDSPAGE. (B) Antigenicity of mAbs 6G1 and 8F10 revealed by chemiluminescent Western blot using meat protein extracts and PsTn. The protein loading mass was 5 μg/lane for four meat extracts (lanes 1–4) and 2.5 μg/lane for isolated PsTn (lane 7). The pork meat sample extracted using NaClPBS-urea-βME (lane 5) and heated PsTn (lane 6) were prepared with the same dilution ratio as their counterparts (lane 4 and 7), respectively. The Precision Plus Protein Kaleidoscope Protein Standards (Bio-Rad) was used for band sizing. The IgG concentration of mAbs 6G1 and 8F10 was 1 ppm.
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Fig. 2. Immunoreactivity between porcine skeletal troponin subunits (PsTnT, PsTnI, and PsTnC) and mAb8F10 using double fluorescent Western blot. The protein loading mass was 2.5 μg/lane. Western blot I was performed using a mixture of mAb8F10 (green) and anti-sTnC pAb (red), while Western blot II was performed using a mixture of mAb8F10 and anti-sTnT pAb (red). The IgG concentration of each pooled antibody was 1 ppm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
selectivity of both mAbs matched with our previous findings using indirect non-competitive ELISA (Liu et al., 2006; Rao, 2004), indicating that both mAbs were specific to mammalian sTn. As to isolated PsTn, both reducing SDS-PAGE (Fig. 1A) and Western blot (Fig. 1B) results showed that there was no significant difference in the band pattern and color intensity before (lane 7) and after (lane 6) heat treatment. Both PsTnI and PsTnC subunits in isolated PsTn were thermostable and could retain their solubility, molecular integrity, and immunoreactivity after heat treatment. However, in heated mammalian meat extracts, it is noted that sTnI but not sTnC was identified by both mAbs using chemiluminescent Western blot (lanes 3–4, Fig. 1B). It has been verified that sTnI from different meat species was thermostable (Chen & Hsieh, 2001; Chen & Hsieh, 2002; Chen, Hsieh, & Bridgman, 2004; Zvereva et al., 2015). However, few publications reported sTnC as a thermostable marker, which is probably because its thermostability is dependent on the amount of calcium (Brzeska, Venyaminov, Grabarek, & Drabikowski, 1983). It is noted that the addition of urea and βME into the extraction buffer (NaCl-PBS, lane 5, Fig. 1A) enhanced the extractability of pork meat proteins by around 120% compared to its counterpart extracted using NaCl-PBS (lane 4, Fig. 1A) according to the total lane color intensity analysis. Also, the pork protein extracts (lanes 4–5, Fig. 1A) have a greater matrix complexity than the isolated PsTn (lane 7, Fig. 1A). The extractability of target proteins is affected by both internal factors (such as protein isoelectric point and polar/apolar nature) and external factors (such as processing condition and extraction method) (EFSA Panel on Dietetic Products Nutrition and Allergies, 2014; Yu, Morton, Clerens, & Dyer, 2017). The addition of solubilizing (i.e., urea) and reducing (i.e., βME) agents can enhance protein extractability. This is because urea can effectively break non-covalent bonds, such as hydrogen bonds (della Malva et al., 2018), whereas βME can reduce the covalent disulfide bonds between cysteine residues in the protein mass (He et al., 2018). It should be noted that PsTnT has no cysteine residues, while both PsTnI and PsTnC contain more than one (Table 1), indicating that disulfide-induced protein aggregation can occur during heat treatment and lead to PsTnC is not extractable. della Malva et al. (2018) reported that the use of denaturing solutions could improve the extractability of myofibrillar proteins with low MW (< 45 kDa). It is noted that PsTnC was detected by mAb8F10 but not by mAb6G1 (lane 5, Fig. 1B), which might be caused by the destruction of mAb6G1 epitopes on PsTnC during the extraction using urea and βME.
3.2. Sandwich ELISA evaluation Through the epitope comparison study (data not shown), mAbs 6G1 and 8F10-HRP were paired as the capture and detection antibody, respectively, to establish a sandwich ELISA. The optimized sandwich ELISA was specific to heated mammalian (beef, deer, horse, lamb, and pork) meats while had no cross-reaction with heated poultry (chicken, duck, and turkey) meats (Table 2). According to USDA Livestock and Meat Domestic Data, pork and beef are the two mammalian species that are mostly produced and consumed in the U.S. (USDA, 2019a) These two mammalian meats were chosen to evaluate the sandwich ELISA performance. To assess the effect of heat treatment on the performance of the established sandwich ELISA, beef and pork under different heat treatment were studied. For pork protein extracts, only when the heating condition elevated to 132 °C/2 bar/120 min, its relative absorbance decreased about 35% compared with that from the 100 °C/30 min treatment (p < 0.05, Fig. 3). For beef protein extracts, after heating at 132 °C/2 bar for 60 min or longer, it produced a significantly smaller relative A415 compared to that from the 100 °C/30 min treatment (p < 0.05, Fig. 3). It is noted that the A415 of the most severe treatment (132 °C/2 bar/ 120 min) in beef samples remained as high as 0.9, indicating that the developed assay was adequate to analyze mammalian meat samples regardless of the extent of heat processing. Table 2 Species selectivity of sandwich ELISA. Species Mammalian Beef Deer Horse Lamb Pork Poultry Chicken Duck Turkey
100 °C/30 min
121 °C/1.2 bar/30 min
+++* +++ +++ +++ +++
+++ +++ +++ +++ +++
– – –
– – –
* +++: very strong reaction (A415 ≥ 1.2); ++: strong reaction (1.2 > A415 ≥ 0.7); +: weak reaction (0.7 > A415 ≥ 0.2); −: no reaction (A415 < 0.2) (Rao & Hsieh, 2014). 5
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Fig. 3. Effect of heat treatment on the relative immunoreactivity of sandwich ELISA. Different letters within the same meat species indicate the significant difference in % relative absorbance at 415 nm between each heat treatment and 100 °C/30 min (p < 0.05). (A) Pork; (B) Beef. Data are represented as the average % relative absorbance at 415 nm ± SEM (n ≥ 3).
could be predicted using the four-parameter logistic (4 PL) model, a commonly used nonlinear regression analysis in bioassays (Fig. 4). Its goodness of fit (R2) of both heat treatments (100 °C/30 min and 121 °C/ 1.2 bar/30 min) was greater than 96%. The optimized assay could detect as low as 1% (g/g) of mammalian meats adulterated in poultry meats under each heating condition (p ≤ 0.05, black dash-line rectangle, Fig. 4). The EC50 (half-maximal effective concentration) was 1.5% and 2.3% for 100 °C/30 min- and 121 °C/1.2 bar/30 min-heated adulterants, respectively, indicating that different heat treatments could affect the assay sensitivity. Detection of mammalian meat adulteration is important for the enforcement of food-labeling laws. Different methods such as polymerase chain reaction (PCR) (Karabasanavar, Girish, Kumar, & Singh, 2017), mass spectrometry (Ruiz Orduna, Husby, Yang, Ghosh, & Beaudry, 2017) and infrared spectroscopy (Yang et al., 2018) have been utilized for meat species identification. However, it should be noted that no single method can be applied to all cases. For example, PCR was adopted to amplify small DNA segments to determine animal species (Nešić, Stojanović, & Baltić, 2017), while it has the limitation such as prone to contamination (Hsieh, 2004). In addition, due to the variability of DNA at the species and target tissue levels, this type of method is unsuitable for the quantification of the exact percentages of different species in meat or meat products (Nešić et al., 2017). The majority of the published PCR methods are qualitative, and quantitative analysis should be based on real-time PCR (Mayada Ragab Farag, Mahmoud Alagawany, Mohamed Ezzat Abd El-Hack, Ruchi Tiwari, & Dhama, 2018). It is well known that the immunoassay is a rapid, highly sensitive and quantitative method which is suitable for handling numerous samples at a time (Singh & Sachan, 2011). Many studies have developed ELISA to detect mammalian meat adulteration. For example, Liu et al. (2006) reported a sandwich ELISA that was able to detect 0.05% and 0.1% (g/g) of heated pork adulterated in chicken and beef, respectively. Chen and Hsieh (2000) used an indirect non-competitive ELISA to detect 0.5% (g/g) heated pork in meat mixtures. Jones and Patterson (1985) and Martín, Azcona, García, Hernández, and Sanz (1988) applied a sandwich ELISA to detect 1–50% (g/g) of pork in raw beef and horse meat in the raw beef and pork mixtures, respectively. Hsieh, Sheu, and Bridgman (1998) used an indirect non-competitive ELISA to screen as low as 0.5% (g/g) of mammalian meat (cattle, hogs, sheep, horse, and deer) adulterated in heated (100 °C, 15 min) poultry
Table 3 Effect of mammalian fat content on the relative immunoreactivity of sandwich ELISA. Heat treatment % pork fat in pork (g/g) 0 10 20 30 % beef fat in beef (g/g) 0 10 20 30
100 °C/30 min
121 °C/1.2 bar/30 min
100 ± 3.0a * 98.2 ± 0.9a 97.3 ± 1.9a 100.9 ± 0.1a
100 ± 1.2a 98.2 ± 2.4a 98.6 ± 1.3a 97.4 ± 0.9a
100 ± 4.9a 104 ± 1.6a 94.8 ± 0.8a 95.6 ± 1.4a
100 ± 3.4a 89.1 ± 0.5a 90.1 ± 1.3a 95.2 ± 0.1a
* For the same sample model, values in the same column with different letters are significantly different (p < 0.05). Data are represented as the average % relative absorbance at 415 nm ± SEM (standard error of the mean, n ≥ 2).
Fat is an important component of animal meat products and varies in different products. For example, regular ground beef contains 30% fat, while in extra beef, the fat content is limited to 5% or less (USDA, 2014). It is important to study the potential interference of fat in meat on the immunoreactivity. To illustrate the effect of mammalian fat content on the performance of the established sandwich ELISA, different fat-adulterated pork and beef were tested. For the same species under the same heat treatment, there was no significant difference in relative A415 among different fat adulteration levels (10%, 20%, and 30%) compared with the lean meat extract (p ≥ 0.05, Table 3). Our results showed that the extraction method was able to extract similar amounts of target analytes from high-fat mammalian meats. The effect of mammalian fat content on the optimized sandwich ELISA was insignificant. As for the preparation of the heated mammalian meat-spiked poultry meat samples, since there was no significant difference in the immunosignal from pork adulterated in chicken samples prepared by either the weight-basis or volume-basis methods (Supplementary Document), the volume-basis method was selected in this study. For the same heat treatment, there was no significant absorbance difference at the same adulteration level (p > 0.05) between pork spiked in chicken and beef spiked in turkey. At each heating condition, two curves were combined, and the ELISA signals of heated adulterated poultry samples 6
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Fig. 4. Immunodetection of heated mammalian meat adulterated in poultry meat samples using sandwich ELISA. The four-parameter logistic model was used for the nonlinear regression analysis. The EC50 of each heat treatment is indicated using a blue and red dotted line, respectively. The detection limit of each heat treatment is indicated using a black dash-line rectangle. The value R2 quantifies the goodness of fit. Data are represented as the average % maximum absorbance at 415 nm ± SEM (n ≥ 4). 100 °C/30 min: blue solid line; 121 °C/ 1.2 bar/30 min: red dash-dotted line. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
products. The limitations in these assays include (1) was selective to only one mammalian meat; (2) was against only adulterated raw meats; (3) using the indirect non-competitive format that is difficult to be commercialized. To the best of our knowledge, the established ELISA is the first sandwich format assay that can be used for the detection of undeclared mammalian meats in poultry meats. Overall, this assay has the potential to fight food fraud, comply with food regulations, and decrease food recalls, which may open up new diagnostic methods for the food industry and the food regulatory authorities.
Chen, F. C., & Hsieh, Y.-H. P. (2002). Porcine troponin I: A thermostable species marker protein. Meat Science, 61(1), 55–60. Chen, F. C., Hsieh, Y.-H. P., & Bridgman, R. C. (2004). Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of prohibited ruminant proteins in feedstuffs. Journal of Food Protection, 67(3), 544–549. EFSA Panel on Dietetic Products Nutrition and Allergies (2014). Scientific opinion on the evaluation of allergenic foods and food ingredients for labelling purposes. EFSA Journal, 12(11), 3894–4180. Greaser, M. L., & Gergely, J. (1973). Purification and properties of the components from troponin. Journal of Biological Chemistry, 248(6), 2125–2133. Grujic, R., & Savanovic, D. (2018). Analysis of myofibrillar and sarcoplasmic proteins in pork meat by capillary gel electrophoresis. Foods and Raw Materials, 6(2), 421–428. He, J., Zhou, G. H., Bai, Y., Wang, C., Zhu, S. R., Xu, X. L., et al. (2018). The effect of meat processing methods on changes in disulfide bonding and alteration of protein structures: Impact on protein digestion products. RSC Advances, 8(31), 17595–17605. Hsieh, Y.-H. P. (2004). Meat species identification. In Y. H. Hui (Vol. Ed.), Handbook of food science, technology, and engineering: Vol. 1, (pp. 1–19). CRC Press. Hsieh, Y.-H. P., & Chen, F. C.. Use of troponin I as a species marker protein for meat speciation in both raw and heat-processed product. (2002). https://patentimages.storage. googleapis.com/d8/28/53/fe927a9dd0d830/US7297500.pdf Accessed December 4, 2019. Hsieh, Y.-H. P., Sheu, S.-C., & Bridgman, R. C. (1998). Development of a monoclonal antibody specific to cooked mammalian meats. Journal of Food Protection, 61(4), 476–481. Jones, S. J., & Patterson, R. L. S. (1985). Double-antibody ELISA for detection of trace amounts of pig meat in raw meat mixtures. Meat Science, 15(1), 1–13. Karabasanavar, N., Girish, P. S., Kumar, D., & Singh, S. P. (2017). Detection of beef adulteration by mitochondrial D-loop based species-specific polymerase chain reaction. International Journal of Food Properties, 20(S2), 2264–2271. Kemp, C. M., Sensky, P. L., Bardsley, R. G., Buttery, P. J., & Parr, T. (2010). Tenderness – an enzymatic view. Meat Science, 84(2), 248–256. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. Layton, L.. FDA pressured to combat rising 'food fraud. (2010). http://www.washingtonpost. com/wp-dyn/content/article/2010/03/29/AR2010032903824_pf.html Accessed December 4, 2019. Liu, L. H., Chen, F. C., Dorsey, J. L., & Hsieh, Y.-H. P. (2006). Sensitive monoclonal antibody-based sandwich ELISA for the detection of porcine skeletal muscle in meat and feed products. Journal of Food Science, 71(1), M1–M6. della Malva, A., Albenzio, M., Santillo, A., Russo, D., Figliola, L., Caroprese, M., et al. (2018). Methods for extraction of muscle proteins from meat and fish using denaturing and nondenaturing solutions. Journal of Food Quality. https://doi.org/10. 1155/2018/8478471. Martín, R., Azcona, J. I., García, T., Hernández, P. E., & Sanz, B. (1988). Sandwich ELISA for detection of horse meat in raw meat mixtures using antisera to muscle soluble proteins. Meat Science, 22(2), 143–153. Moerman, F. (2018). Food defense. In A. M. Holban, & A. M. Grumezescu (Eds.). Food control and biosecurity (pp. 135–223). Academic Press. MyBioSource Inc. Polyclonal antibody to troponin C type 1, slow (TNNC1) instruction manual. (2019). https://cdn.mybiosource.com/tds/protocol_manuals/800000-9999999/ MBS2032663_TDS.pdf Accessed December 4, 2019. Nakyinsige, K., Man, Y. B., & Sazili, A. Q. (2012). Halal authenticity issues in meat and meat products. Meat Science, 91(3), 207–214.
Author contributions Xingyi Jiang. Collected the data, Contributed data or analysis tools, Performed the analysis, Wrote the paper. Qinchun Rao: Conceived and designed the analysis, Collected the data, Contributed data or analysis tools, Performed the analysis, Wrote the paper. Kristen Mittl: Collected the data. Yun-Hwa Peggy Hsieh: Conceived and designed the analysis. Declaration of competing interest The authors declare no competing financial interest. Acknowledgements A part of this study was supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture (2017-70001-25984). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2019.107045. References Brzeska, H., Venyaminov, S. V., Grabarek, Z., & Drabikowski, W. (1983). Comparative studies on thermostability of calmodulin, skeletal muscle troponin C and their tryptic fragments. FEBS Letters, 153(1), 169–173. Chen, F. C., & Hsieh, Y.-H. P. (2000). Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA. Journal of AOAC International, 83, 79–85. Chen, F. C., & Hsieh, Y.-H. P. (2001). Separation and characterization of a porcine‐specific thermostable muscle protein from cooked pork. Journal of Food Science, 66(6), 799–803.
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