Monoclonal Antibodies as Probes for the Detection of Porcine Blood

May 2, 2016 - The lack of effective methods to monitor the use of porcine blood-derived food ingredients (PBFIs) is a concern for the billions of indi...
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Monoclonal Antibodies as Probes for the Detection of Porcine BloodDerived Food Ingredients Jack A. Ofori and Yun-Hwa P. Hsieh* Department of Nutrition, Food and Exercise Sciences, 420 Sandels Building, Florida State University, Tallahassee, Florida 32306-1493, United States ABSTRACT: The lack of effective methods to monitor the use of porcine blood-derived food ingredients (PBFIs) is a concern for the billions of individuals who avoid consuming blood. We therefore sought to develop a panel of porcine blood-specific monoclonal antibodies (mAbs) for use as probes in immunoassays for the detection of PBFIs. Ten selected mAbs were identified that react with either a 60 or 90 kDa protein in the plasma fraction or a 12 kDa protein in the red blood cell fraction of porcine blood. Western blot analysis of commercially produced PBFIs revealed that these antigenic proteins are not affected by various manufacturing processes. The utility of these mAbs was demonstrated in a prototype sandwich ELISA developed for this study using mAbs 19C5-E10 and 16F9-C11. The new assay is porcine blood-specific and capable of detecting ≤0.03% (v/v) of PBFIs in cooked (100 °C for 15 min) ground meats or fish. KEYWORDS: monoclonal antibodies, immunoassay, porcine blood, food ingredients

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INTRODUCTION Proteins derived from food animal blood (primarily bovine and porcine) are used widely as ingredients in food products and dietary supplements because of the resulting nutritional, functional, environmental, health, and economic benefits.1 However, the use of these blood proteins as food ingredients creates serious concerns for those individuals who avoid consuming blood for religious (Jews, Muslims, and Hindus), health (allergy to blood proteins or the belief that blood is a haven for pathogens), ethical (vegans), cultural, or taboo reasons or just as a matter of preference. The situation is worsened by the fact that these blood proteins are generally declared on labels by their brand names, leaving the consumer unaware that the product contains blood-derived ingredients. Another major concern is the dishonest use of these proteins for economic gain. A typical example is the use of Fibrimex, an ingredient derived from porcine or bovine blood, referred to colloquially as “meat glue”, to bind low-grade pieces of meat together2 to allow them to be fraudulently sold to consumers as expensive steaks. In a report by The Seattle Times, more than 130 meats and deli products checked over a period of 5 months in Seattle, WA, Milwaukee, WI, Omaha, NE, and Denver, CO, USA, were formulated with Fibrimex or another meat glue known as Activa (transglutaminase obtained from bacterial sources). Of these, only four products (all bolognas) had these ingredients shown on the label3 as required by law. To protect consumers, a number of analytical methods have been developed to detect the inclusion of blood or bloodderived products as undeclared ingredients in foodstuffs. Early methods involved the use of spectrophotometry4,5 or a modified version of the Kjeldahl method6 to estimate the amount of added blood in ground meats, but all of these methods are nonspecific, inaccurate, and laborious and/or involve the use of dangerous chemicals. More specific methods have now been developed, most of which focus on the detection of plasma-derived products because they are more © XXXX American Chemical Society

commonly used in comparison with proteins derived from the cellular fraction of blood, such as hemoglobin. These methods, which employ such techniques as isolectric focusing,7 liquid chromatography, triple-quadrupole mass spectrometry,8,9 and immunoassay,10 are also somewhat problematic, however, as they tend to be laborious and ineffective against heat-treated samples, to suffer from matrix interference, and/or to give false positives in the absence of blood proteins. To overcome the shortcomings of the aforementioned methods, in our laboratory we have developed sandwich11 and competitive12 enzyme-linked immunosorbent assays (ELISA) for detecting the presence of bovine blood in feed and meat products. The competitive ELISA (cELISA) employs monoclonal antibody (mAb) Bb1H9, which recognizes a 12 kDa protein in ruminant blood that has been identified to be a monomer of the tetrameric hemoglobin molecule.13 The sandwich ELISA (sELISA), which utilizes two mAbs (Bb6G12 and Bb3D6) that recognize a 60 kDa antigenic protein in bovine plasma, has been shown to be effective in detecting diversely processed plasma-derived proteins in both laboratory-adulterated ground meats and commercially available dietary supplements.14 However, effective methods to monitor the presence of porcine sourced blood proteins are still lacking. The objective of this study was, therefore, to develop porcine-specific mAbs capable of recognizing thermal-stable proteins in porcine blood and then utilize these antibodies either individually or in combination in various formats of immunoassays to detect various kinds of porcine blood-derived proteins in raw and processed foods and dietary supplements. Received: December 30, 2015 Revised: April 1, 2016 Accepted: April 24, 2016

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DOI: 10.1021/acs.jafc.5b06136 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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prepared. Lower levels of spiking (0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, and 3% v/v) were obtained by diluting 5% v/w spiked sample extracts with the appropriate amount of cooked chicken meat extract (0%) to ensure homogeneity. Another set of samples consisting of cooked ground meats (beef, pork, lamb, or chicken) or fish (cod) spiked with 5% w/w of commercially produced porcine plasma-derived food ingredients (PPDFIs) (PPP, PFP, and APP) were similarly prepared using the procedure described above and tested with the developed sELISA to assess its effectiveness against commercial samples. All spiked sample extracts were tested immediately after preparation. Production of mAbs. Five milliliters of whole porcine blood in a beaker was covered with aluminum foil and heated in a boiling water bath for 15 min to obtain cooked porcine blood. The cooked porcine blood was then broken down into fine particles and 5 mL of extraction buffer (10 mM PBS) added. The mixture was homogenized, centrifuged, and passed through Whatman no. 1 filter paper, as described above. The filtrate, consisting of thermally stable soluble blood proteins, was dialyzed for 24 h in 10 mM PBS with frequent changes, and the dialyzed extract of crude proteins was used as the immunogen. The protein concentration of the immunogen was determined using the Bio-Rad Protein Assay Kit, with BSA as the standard, in accordance with the manufacturer’s instructions. The immunization and ensuing hybridoma procedures were performed as previously described,15 only positive hybridomas that secrete immunoglobulin G (IgG) class of antibodies were selected, screened, cloned, and subcloned. mAbs were obtained from the supernatants of the propagated cell cultures. The isotype of each selected mAb was determined using a mouse mAb isotyping kit (ISO-2 1 Kit, SigmaAldrich) in accordance with the manufacturer’s instructions. Noncompetitive Indirect Enzyme-Linked Immunosorbent Assay (iELISA). The selectivity of the raised mAbs was characterized using antigen-coated noncompetitive iELISA as previously described15 with the following modifications. Antigen and antibody incubation times were 1 h; 0.2% fish gelatin in PBS was used as the blocking buffer; and the antibody buffer was 0.2% fish gelatin in PBST (PBS containing 0.05% (v/v) Tween-20). Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot. SDS-PAGE followed by Western blot was performed to determine the antigenic protein in the blood samples or soluble extracts recognized by each of the selected mAbs. Briefly, soluble proteins (10 μg of protein in 10 μL of sample buffer per lane) from the samples were loaded onto 5% stacking gels and separated on either 12 or 15% (for blots involving mAb 19C4E11, which binds to the 12 kDa antigenic protein) polyacrylamide separating gels at 200 V using the Mini-Protein 3 Electrophoresis Cell (Bio-Rad Laboratories Inc.) in accordance with the method of Laemmli.16 The separated proteins were transferred electrophoretically (1 h at 100 V) using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories Inc.), as described by Towbin and others,17 and the membrane was blocked with 0.2% fish gelatin in TBS containing 0.05% (v/v) Tween-20 (TBST). The blotted membrane was then incubated with the selected mAbs and secondary (goat anti-mouse IgG (H + L)-AP conjugate) antibodies in succession, and color was developed as previously described.15 Precision Plus Protein Kaleidoscope standards were used for the molecular weight estimations on gels and blot. Epitope Comparison. Pairs of mAbs (mAb1 and mAb2) that bind to the same antigenic protein in porcine blood were tested using the additivity test described by Friguet and others18 to determine whether they bind to the same or different epitopes on the common antigenic protein. First, saturation curves were generated using noncompetitive iELISA to ascertain the dilution of each antibody needed to saturate the coated antigen (0.3 μg of cooked porcine blood protein per 100 μL of buffer per well). To the first of these wells containing 0.3 μg/100 μL of bound cooked porcine blood was added 100 μL of mAb1 that had been diluted to saturate the antigen as determined from the saturation curves; 100 μL of the second antibody (mAb2), similarly diluted to saturate the antigen, was added to the second well; and 50 μL each of mAb1 and mAb2 was added to the third. Secondary antibody, color substrate, and stop solution were then added in the

MATERIALS AND METHODS

Hydrogen peroxide, β-mercaptoethanol, isotyping kit (ISO-2 1 Kit), and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Alkaline phosphates (AP) conjugate substrate kit, Protein Assay Kit, goat antimouse IgG (H + L) AP (anti-Ig-AP) conjugate, Tris-buffered saline (TBS), 0.5 M Tris-HCl buffer (pH 6.8), 1.5 M Tris-HCl (pH 8.8), N,N,N′,N′-tetra- methylethylenediamine (TEMED), Precision Plus Protein Kaleidoscope Standards, 30% acrylamide/bis solution, Tris/ glycine buffer, Tris/glycine/SDS buffer, supported nitrocellulose membrane (0.2 μm), and thick blot paper were purchased from BioRad Laboratories Inc. (Hercules, CA, USA). Egg albumin, bovine serum albumin (BSA), and all other chemicals were purchased from Fisher Scientific (Fair Lawn, NJ, USA). All chemicals and reagents were of analytical grade, and solutions were prepared using distilled deionized pure water (DD water) from a NANOpure DIamond ultrapure water system (Barnstead International, Dubuque, IA, USA). Whole blood from pigs, cattle, donkey, horse, goat, sheep, rabbit, turkey, and chicken, porcine plasma, porcine serum, and porcine red blood cells were purchased from Lampire Biological Laboratories (Pipersville, PA, USA). Porcine and bovine gelatins were obtained from GELITA USA Inc. (Sioux City, IA, USA) and soy powder from SoyLink (Oskaloosa, IA, USA). Commercial blood-derived edible ingredients, including porcine plasma powder (PPP), porcine Fibrimex powder (PFP), porcine hemoglobin powder (PHP), porcine hydrolyzed globin (PHG), bovine plasma powder (BPP), bovine Fibrimex powder (BFP), bovine hemoglobin powder (BHP), and bovine fibrinogen powder (BFGP), were obtained from Sonac BV (Suameer, The Netherlands); Aprosan (APS), Aprothem (APT) Apropork (APP), and Aprored (APR) were obtained from Proliant Inc. (Barcelona, Spain); and Immunolin was obtained from Proliant Inc. (Ankeny, IA, USA). Nonfat dry milk was purchased from a local grocery store. Meat samples, including beef eye of round roast, pork loin, lamb shoulder, whole chicken, whole duck, whole goose, turkey breast, bison, and frozen dressed rabbit, were purchased from a local supermarket. Horse meat was obtained from the College of Veterinary Medicine, Auburn University (Auburn, AL, USA). Deer, elk, and African buffalo steak meats were provided by the Fats and Proteins Research Foundation (Bloomington, IL, USA). Sample Preparation. Extraction of Soluble Proteins from Animal Blood and Nonblood Materials. Soluble proteins were extracted from cooked (100 °C, 15 min) blood samples (whole blood, plasma, serum, and RBCs) and nonblood proteins including common food protein ingredients (soy powder, bovine gelatin, porcine gelatin, egg albumin, and BSA) and meat samples (pork, beef, horse, elk, donkey, lamb, bison, African buffalo, rabbit, turkey, chicken, goose, and duck) as previously described.15 Extraction of soluble proteins from commercially produced porcine and bovine blood ingredients was performed as previously reported for commercial feedstuffs.15 Raw blood samples were used as is. The protein extracts were stored at −20 °C until use. Spiked Sample Extracts. One set of extracts of cooked chicken spiked with liquid porcine blood or plasma was prepared as described below to examine the potential of a new sELISA developed for this study as a tool for monitoring porcine blood material in foods. To 9.5 g of ground chicken meat was added 0.5 mL of porcine blood or porcine plasma, and the mixture was stirred thoroughly with a glass rod to obtain 5% v/w porcine blood or porcine plasma in ground chicken meat. The spiked samples were then cooked by immersing the beakers (covered with aluminum foil) in boiling water for 15 min. The cooked samples were broken down into finer particles, 20 mL of 10 mM phosphate-buffered saline (PBS) was added, and the mixture was homogenized for 2 min at 11000 rpm using the ULTRA-TURRAX T25 basic homogenizer (IKA Works Inc., Wilmington, NC, USA). The homogenized samples were centrifuged at 3220g for 1 h at 4 °C (Eppendorf 5810R centrifuge, Brinkman Instruments Inc., Westbury, NY, USA) and then passed through Whatman no. 1 filter paper to obtain 5% v/w spiked sample extracts. Nonspiked cooked chicken meat (0%) containing no added porcine blood or plasma was similarly B

DOI: 10.1021/acs.jafc.5b06136 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry same order. The additivity index (AI) was calculated according to eq 1, where A1 is the absorbance for well 1 (mAb1 alone), A2 is the absorbance for well 2 (mAb2 alone), and A1+2 is the absorbance for well 3 (mAb1 and mAb2 together); AI values between 50 and 100% indicate that the pair of mAbs (mAb1 and mAb2) bind to different epitopes on the common antigenic protein. AI = [(2A1 + 2 )/(A1 + A 2 ) − 1] × 100

screening using positive hybridomas against protein extracts from both cooked and raw blood from different species. Only those hybridomas that secreted IgG class mAbs that recognized both raw and cooked porcine blood extracts were chosen to ensure that all of the selected mAbs would bind to a thermally stable antigenic protein. From two consecutive fusions, a total of 44 hybridomas showing positive reactions with the porcine blood immunogen without cross-reactivity with bovine blood extract were thus selected from the initial screening. Of these, 10 clones were selected after cloning and subcloning on the basis of their cell line stability and their positive reaction to both raw and cooked blood (from porcine or few other species) from the secondary screening. Although the aim was to find mAbs that bind to porcine blood, those that bound to blood from few other uncommon food animal species were also selected as they could be used as candidates to pair with a porcine-specific mAb in a sandwich-type immunoassay if two porcine-specific mAbs cannot be found. Species Selectivity of mAbs. The species selectivity of the 10 selected mAbs was examined against raw and cooked porcine, bovine, horse, donkey, rabbit, goat, sheep, chicken, and turkey blood using noncompetitive iELISA. Table 1 summarizes the reaction patterns of the 10 selected mAbs. All of the mAbs belonged to subclass IgG 1 and showed weak to very strong reactions with both raw and cooked blood from a single species or from multiple species. On the basis of their reaction patterns, these mAbs were categorized into two groups,

(1)

Monoclonal Antibody Purification and Biotinylation. Both mAbs 19C5-E10 and 16F9-C11 were purified from the supernatant using a Protein A affinity column on an Econo low-pressure chromatography system (Bio-Rad Laboratories Inc.) per the manufacturer’s instructions. mAb 16F9-C11 was then conjugated with biotin using NHS-CA-biotin in accordance with the standard procedure.19 Concentrations of purified mAb 19C5-E10 and 16F9C11 IgGs and biotin-conjugated mAb 16F9-C11 were determined by UV spectrophotometer (SmartSpec 3000, Bio-Rad Laboratories Inc.) at 280 nm. Sandwich ELISA (sELISA). After the epitope comparison, mAb 19C5-E10 and biotin-conjugated mAb 16F9-C11 were selected to construct a sELISA. Optimization studies were first performed to determine which antibodies to use as the capture and detection antibodies, the optimum dilution for each, and the optimum incubation periods. On the basis of the optimization results, mAb 19C5-E10 was selected as the capture antibody and biotin-conjugated mAb 16F9-C11 as the detection antibody for the sELISA. The detailed sELISA procedure based on the optimized conditions was as follows. The microplate was coated with 100 μL of mAb 19C5-E10 supernatant diluted 500-fold in PBS to contain 0.21 μg of protein and the plate incubated for 1 h at 37 °C. The plate was then blocked for 1 h at 37 °C with 200 μL of blocking buffer. Next, 100 μL of undiluted sample extracts was added to the plate, and the plate was incubated for a further 1 h at 37 °C. One hundred microliters of biotin-conjugated mAb 16F9-C11 diluted 500-fold in antibody buffer to contain 0.1 μg of protein was then added and the plate incubated for 1 h at 37 °C, after which 100 μL of the enzyme, streptavidin peroxidase diluted 3000-fold in antibody buffer, was added to the plate and incubated at 37 °C for 1 h. Finally, 100 μL of the enzyme substrate (22 mg of ABTS and 15 μL of 30% H2O2 in 100 mL of 0.1 M phosphate citrate buffer, pH 4.0) was added and the color developed for 30 min at 37 °C. The enzyme reaction was stopped by the addition of 100 μL of 0.2 M citric acid and the absorbance read at 415 nm using the PowerWave XS microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Between steps, the plate was washed with PBST. Statistical Analysis. Each sample was tested in triplicate, each experiment was repeated at least once, and the data were analyzed using Microsoft Excel 2010. One-way analysis of variance (ANOVA) coupled with Tukey’s pairwise comparisons was performed; P ≤ 0.05 was considered statistically significant.

Table 1. Species Specificity and Antigenic Proteins of the 10 Selected mAbs species specificitya

mAb (subclass) 19C4/C11 (IgG 1) 19C4/E11 (IgG 1) 24C12/E7 (IgG 1) 23F7/E5 (IgG 1) 25E12/D9 (IgG 1) 16F9/C11 (IgG 1) 19C5/D12 (IgG 1) 19C5/E10 (IgG 1) 21F11/E5 (IgG 1) 22E10/A5 (IgG 1)

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RESULTS AND DISCUSSION Production of mAbs. Blood-derived food ingredients typically undergo spray-drying (heating to a minimum internal temperature of 70 or 80 °C) as part of the production process, so to enhance the chances of selecting mAbs capable of recognizing their cognate antigen after heat treatment, cooked porcine blood containing soluble thermally stable peptides was used as the immunogen. As commercial blood protein ingredients are also diverse by nature and may be obtained from either the plasma or cellular fraction of blood, a crude thermally stable protein extract of porcine blood rather than a purified protein was used to immunize animals to increase the likelihood of generating mAbs with diversified antigenic proteins; each protein with a molecular weight >10 kDa in the extract is capable of inducing an immune response for antibody production. mAbs were selected on the basis of their ability to recognize heat-stable epitopes through a secondary

estimated MW of antigenic protein (kDa)

location

raw blood

cooked blood

porcine ++++ horse ++++ donkey ++++ porcine ++++ horse ++++ donkey ++++ porcine ++++

porcine ++++ horse ++++ donkey ++++ porcine ++++ horse ++++ donkey ++++ porcine +++

12

RBC

12

RBC

12

RBC

porcine ++

porcine +++

60

plasma

porcine ++

porcine +++

60

plasma

porcine +

porcine ++

90

plasma

porcine +

porcine ++

90

plasma

porcine +

porcine ++

90

plasma

porcine ++

porcine +

90

plasma

porcine + horse + donkey + goat + chicken +

porcine ++

90

plasma

a +, weak reaction (0.2 ≤ OD < 0.5); ++, moderate reaction (0.5 ≤ OD < 1.0); +++, strong reaction (1 ≤ OD < 2.0); ++++, very strong reaction (OD ≥ 2.0).

C

DOI: 10.1021/acs.jafc.5b06136 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry monospecies-specific and multispecies-specific. The seven monospecies-specific mAbs (16F9-C11, 19C5-D12, 19C5E11, 21F11-E5, 23F7-E5, 24C12-E7, and 25E12-D9) reacted only with porcine blood, whereas the three multispecies-specific mAbs (19C4-C11, 19C4-E11, and 22E10-A5) also reacted with blood from two or more other species. Blood-derived proteins are often used as ingredients in food products and dietary supplements. These dietary products usually also contain nonblood proteins from other sources such as meat, milk, soy, and eggs. To ensure these antibodies do not cross-react with these nonblood edible proteins to give false positives, all of the antibodies were tested against both raw and cooked meat extracts from several species as well as several proteins that are commonly used as food ingredients, including soy, nonfat dry milk, gelatin, egg albumin, and BSA. iELISA results showed that none of the mAbs reacted with either of these common food proteins (OD < 0.2) or with the meat extracts (OD < 0.2) (data not shown). Thermally Stable Antigenic Proteins Recognized by the mAbs. SDS-PAGE was used to resolve the proteins in raw and cooked porcine blood extracts, followed by Western blot using the mAbs as probes, to disclose the molecular weight (MW) of the antigenic proteins recognized by each mAb. Three mAbs (19C4-C11, 19C4-E11, and 24C12-E7) reacted with a 12 kDa peptide, two (23F7-E5 and 25E12-D9) with a 60 kDa protein, and five (16F9-C11, 19C5-D12, 19C5-E10, 21F11-E5, and 22E10-A5) with a 90 kDa antigenic protein in porcine blood (Table 1). Further analysis of porcine whole blood, plasma, serum, and red blood cells using noncompetitive iELISA indicates that the 12 kDa antigenic protein is present in the RBC fraction of porcine blood, whereas the 60 and 90 kDa antigenic proteins are present in the plasma fraction of porcine blood (Table 1). Further studies will be needed to investigate the identity of these antigenic proteins. Effect of Commercial Processing on Thermally Stable Antigenic Proteins. To examine the potential utility of these mAbs as probes in an immunoassay for the detection of commercially produced blood proteins, a variety of these blood protein ingredients (Table 2) were analyzed with Western blot using one mAb selected from each group recognizing a 12 kDa (19C4-E11), 60 kDa (23F7-E5), or 90 kDa (19C5-D12) antigenic protein in porcine blood to study the effect of industrial processing on these antigenic proteins. As the data shown in Figure 1a demonstrate, mAb 19C5D12, which recognizes a 90 kDa antigenic protein in the plasma fraction of blood, reacted with a 90 kDa protein in those commercial products obtained from porcine plasma, namely, porcine plasma powder (PPP, lane a), porcine Fibrimex powder (PFP, lane b), and Apropork (APP, lane g), as well as the whole porcine blood protein ingredient, APS (Aprosan, lane g), as anticipated. For all of these positive porcine plasma ingredients (PPP, PFP, and APP), there was an additional lighter band at around 50 kDa that could be a peptide containing the epitope released from the 90 kDa protein, either by the manufacturing process involved in its production or by the combined action of heat and SDS treatment involved in the sample preparation. As expected, mAb 19C5-D12 did not react with the 90 kDa antigenic protein in any of the porcine RBC-derived proteins [porcine hemoglobin powder (PHP, lane c), porcine hydrolyzed globin (PHG, lane d), Aprothem (APT, lane f), and Aprored (APR, lane h)] or with the bovine blood proteins [(bovine plasma powder (BPP, lane (i), bovine Fibrimex powder (BFP, lane j), Immunolin (ILN, lane k), bovine

Table 2. Commercially Produced Bovine and Porcine Blood Protein Ingredients Examined with Porcine-Selective mAbs in Western Blot blood protein ingredient

abbreviation

source

porcine plasma powder porcine Fibrimex powder porcine hemoglobin powder porcine hydrolyzed globin Aprosan

PPP

porcine plasma

PFP

porcine plasma

PHP

Aprothem

APT

Apropork

APP

porcine red blood cells porcine red blood cells whole porcine blood porcine red blood cells porcine plasma

Aprored

APR

bovine plasma powder bovine Fibrimex powder ImmunoLin bovine hemoglobin powder bovine fibrinogen powder

BPP

porcine red blood cells bovine plasma

BFP

bovine plasma

ILN BHP

bovine plasma bovine red blood cells bovine plasma

PHG APS

BFGP

produced by Sonac BV, Netherlands Sonac BV, Netherlands Sonac BV, Netherlands Sonac BV, Netherlands Proliant Inc., Spain Proliant Inc., Spain Proliant Inc., Spain Proliant Inc., Spain Sonac BV, Netherlands Sonac BV, Netherlands Proliant Inc., USA Sonac BV, Netherlands Sonac BV, Netherlands

hemoglobin powder (BHP, lane l), and bovine fibrinogen powder (BFGP, lane m)]. Similarly, mAb 23F7-E5, which binds to a 60 kDa antigenic protein in the porcine plasma fraction, reacted with a 60 kDa protein in the plasma-derived proteins (PPP, PFP, and APP) and the whole blood product (APS), as expected (Figure 1b). Interestingly, although mAb 23F7-E5 reacted strongly with a 60 kDa protein in the porcine RBC-derived protein PHG, it did not react with the remaining porcine RBC-derived proteins (PHP, APT, and APR). This positive reaction with PHG could be due to contamination with plasma material during the commercial production process, but it is also possible that the hydrolysis action involved in its production may have released a peptide and exposed a new epitope recognized by mAb 23F7E5 that then cross-reacted with the mAb. For the porcine plasma ingredients PPP, PFP, and APP, mAb 23F7-E5 also reacted with a band at around 40 kDa that may again be a peptide released from the 60 kDa antigenic protein by processing. There was no reaction with bovine blood proteins. In contrast, mAb 19C4-E11, which recognizes a 12 kDa protein in the porcine RBC fraction, reacted with the porcine RBC-derived products PHP and APT as well as the porcine whole blood product APS (Figure 1c). A weaker and slightly heavier band (∼13 kDa) was, however, present in APR. APR is a proprietary formulation for which very little information is available save for the disclosure that it is a pigment obtained from porcine red blood cells. Our assumption is that this product consists primarily of the heme component. The observed band is probably due to a peptide containing the epitope recognized by mAb 19C4-E11 that has remained attached to the heme prosthetic group. The lack of binding with the other RBC-derived protein, PHG, is likely due to the destruction of the epitope by the hydrolysis involved in its production. The mAb 19C4-E11 did not react with any of the bovine blood proteins. For the products PHP, APT, and APS, D

DOI: 10.1021/acs.jafc.5b06136 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Western blot of soluble proteins extracted from commercially produced bovine and porcine blood proteins using (a) mAb 19C5-D12, (b) 23F7-E5, and (c) mAb 19C4-E11. Protein extracts were loaded at 10 μg/10 μL per lane alongside Precision Plus protein kaleidoscope prestained standards. Lanes: S, standard; a, porcine plasma powder (PPP); b, porcine Fibrimex powder (PFP); c, porcine hemoglobin powder (PHP); d, porcine hydrolyzed globin (PHG); e, Aprosan (APS); f, Aprothem (APT); g, Apropork (APP); h, Aprored (APR); i, bovine plasma powder (BPP); j, bovine Fibrimex powder (BFP); k, Immunolin (ILN); l, bovine hemoglobin powder (BHP); m, bovine fibrinogen powder (BFGP).

Figure 2. Detection limit of cooked ground chicken (Cm) spiked with porcine blood (Pb) or porcine plasma (Pp) using sELISA with 19C5-E10 as capture antibody and biotin-conjugated 16F9-C11 as detection antibody. Soluble proteins extracted from spiked cooked ground chicken were added undiluted. Results are expressed as A415 ± SD, n = 3. ∗ indicates the detection limit.

bands at around 25, 40, and 50 kDa were also present, which may correspond to the dimers, trimers, and tetramers of the 12 kDa protein, respectively. In summary, these antigenic proteins do indeed remain unaffected by the diverse food preparation processes to which these blood proteins are subjected except the hydrolyzed product, PHG, indicating that they should serve as useful and reliable markers for the immunodetection of these porcine blood-derived proteins using their cognate antibodies as probes. Epitope Comparison. Of all the various formats of ELISA that are available, sELISA is generally preferred because it is both sensitive and user-friendly. A requirement for developing a sELISA is that the two mAbs between which the antigen is sandwiched must bind to different epitopes on the sandwiched

antigenic protein without inhibiting each other’s binding sites. This section of the study therefore sought to determine if pairs of antibodies that bind to the same antigenic protein (at either 12, 60, or 90 kDa) can bind cooperatively (to different epitopes) or inhibitively (to the same epitope or overlapped epitopes) on the common antigenic protein. From the results of the additivity test, only pairs of mAbs from the panel of mAbs that bind to the 90 kDa antigenic protein were found to bind cooperatively, producing AI values >50%; these mAbs could thus be paired to construct a sELISA for the detection of porcine plasma-derived proteins in food products. Although the other mAbs did not show cooperative binding, they could still be employed as probes in other types of immunoassays (noncompetitive or competitive iELISA) to detect target blood E

DOI: 10.1021/acs.jafc.5b06136 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry proteins. For example, mAbs that recognize the 12 kDa antigenic protein could be used individually or pooled together in an immunoassay for the detection of porcine RBC-derived proteins. Likewise, mAbs recognizing the 12, 60, and 90 kDa antigenic proteins could be pooled together variously in an immunoassay for the detection of both porcine plasma and RBC-derived proteins in a single assay. Development of a sELISA for the Detection of Laboratory-Prepared Porcine Blood Material. In this study, a sELISA was constructed from a pair of mAbs, 19C5E10 and 16F9-C11, that bind cooperatively to the 90 kDa antigenic protein, and the potential of this sELISA as an analytical method for porcine blood detection was assessed by testing against cooked porcine blood in chicken meat. Cooked porcine blood was employed because these blood-derived food ingredients are typically available commercially as heat-treated (spray-dried) products. The sELISA based on mAb 19C5-E10 as the capture antibody and biotin-conjugated mAb 16F9-C11 as the detection antibody was found to be particularly promising for the detection of cooked porcine blood. After step-by-step optimization, this sELISA based on mAbs 19C5E10 and 16F9-C11 reacted strongly with cooked porcine blood (OD415 nm = 2.29) but not with cooked blood from any of the other species tested (bovine, horse, donkey, rabbit, goat, sheep, chicken, and turkey) (OD415 nm < 0.2) or with the nonblood proteins (OD415 nm < 0.1). The sELISA results matched the initial screening iELISA results from each of the two mAbs exactly. Consequently, this prototype sELISA was evaluated in terms of its potential as a tool for detecting the presence of porcine blood material in foods using cooked ground chicken spiked with porcine blood or porcine plasma. The assay demonstrated its ability to detect as little as 0.3% v/v of porcine blood or porcine plasma in cooked ground chicken meat (Figure 2), indicating that this assay has excellent potential for monitoring the presence of low levels of porcine blood material in cooked foods. The detection limit of the sELISA used here is taken to be the minimum amount of porcine blood or porcine plasma in the cooked chicken matrix that could be significantly (P < 0.05) distinguished from nonspiked cooked chicken meat (0%). Performance of the sELISA for the Detection of Commercial Porcine Blood Protein Products. Finally, the performance of the newly developed sELISA against commercial blood-derived ingredients was assessed. As anticipated, the sELISA did not react with any of the bovine blood proteins (BPP, BFP, BHP, ILN, and BFGP) tested, but did react with the porcine plasma-derived proteins, namely, PPP (OD415 nm = 1.88), PFP (OD415 nm = 0.80), and APP (OD415 nm = 0.66), and also weakly with the whole porcine blood product, APS (OD415 nm = 0.33) (Figure 3). The assay also failed to react (OD415 nm < 2) with any of the porcine RBC-derived proteins (PHG, APT, and APR), again as expected, except for the product PHP, which produced a strong positive reaction (OD415 nm = 2.14) (Figure 3). As before, this may be due to contamination with plasma material, either from poor separation of the RBC from the plasma fraction or from contamination with other porcine plasmaderived products (e.g., PPP and PFP) that are produced by the same company. Cooked ground meats and fish spiked with these porcine plasma-derived food ingredients were then analyzed using this sELISA, and the results are shown in Figure 4. The sELISA was found to be capable of detecting levels down to 0.01% (v/v)

Figure 3. Reactivity of sandwich ELISA using purified mAb 19C5-E10 as the capture antibody and biotin-conjugated mAb 16F9-C11 as the detection antibody with porcine and bovine blood-derived food ingredients. Soluble proteins extracted from blood-derived ingredients were added undiluted to microplates precoated with 19C5-E10. PPP, porcine plasma powder; PFP, porcine Fibrimex powder (PFP); PHP, porcine hemoglobin powder; PHG, porcine hydrolyzed globin; APS, Aprosan; APT, Aprothem; APP, Apropork; APR, Aprored; BPP, bovine plasma powder; BFP, bovine Fibrimex powder; ILN, ImmunoLin; BHP, bovine hemoglobin powder; BFGP, bovine fibrinogen powder. The line indicates the cutoff point (A415 = 0.2) between positive (A415 nm ≥ 0.2) and negative (A415 nm < 0.2) samples.

Figure 4. Detection limit of cooked meats and fish spiked with (a) porcine plasma powder (PPP), (b) porcine Fibrimex powder (PFP), and (c) Apropork (APP) using sandwich ELISA with purified mAb 19C5-E10 as capture antibody and biotin-conjugated purified mAb 16F9-C11 as the detection antibody. Results are expressed as A415 nm ± SD, n = 3. ∗ denotes a significant difference (P < 0.05) from the background (0%).

PPP in cooked beef, pork, lamb, and cod; and 0.03% (v/v) PPP in cooked chicken. In the case of PFP-spiked samples, the F

DOI: 10.1021/acs.jafc.5b06136 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

(2) Flores, N. C.; Boyle, E. A. E.; Kastner, C. L. Instrumental and consumer evaluation of pork restructured with activa or with fibrimex formulated with and without phosphate. Food Sci. Technol. 2007, 40, 179−185. (3) Schneider, A. First pink slime, now “meat glue”. Seattle Times, May 10, 2012. (4) Karasz, A. B.; Andersen, R.; Pollman, R. Determination of added blood in ground beef. J. Assoc. Off. Anal. Chem. 1976, 59, 1240−1243. (5) Maxstadt, J. J.; Pollman, R. M. Spectrophotometric determination of sulfites, benzoates, sorbates, ascorbates, and added blood in ground beef: collaborative study. J. Assoc. Off. Anal. Chem. 1980, 63, 667−674. (6) Bjarno, O. C. Multicomponent analysis of meat-products. J. Assoc. Off. Anal. Chem. 1981, 64, 1392−1396. (7) Bauer, F.; Stachelberger, H. [Detection of blood plasma in heattreated meat products by ultrathin-layer isoelectric focusing]. Z. Lebensm.-Unters. Forsch. 1984, 178, 86−89. (8) Grundy, H. H.; Reece, P.; Sykes, M. D.; Clough, J. A.; Audsley, N.; Stones, R. Screening method for the addition of bovine bloodbased binding agents to food using liquid chromatography triple quadrupole mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 2919−2925. (9) Grundy, H. H.; Reece, P.; Sykes, M. D.; Clough, J. A.; Audsley, N.; Stones, R. Method to screen for the addition of porcine bloodbased binding products to foods using liquid chromatography/triple quadrupole mass spectrometry. Rapid Commun. Mass Spectrom. 2008, 22, 2006−2008. (10) Otto, W.; Sinell, H. J. [The influence of thermal load on the immunochemical detection of dried blood plasma supplements in meat mixtures]. Berl. Munch. Tierarztl. Wochenschr. 1989, 102, 14−18. (11) Ofori, J. A.; Hsieh, Y. H. Sandwich enzyme-linked immunosorbent assay for the detection of bovine blood in animal feed. J. Agric. Food Chem. 2007, 55, 5919−5924. (12) Rao, Q.; Hsieh, Y. H. Competitive enzyme-linked immunosorbent assay for quantitative detection of bovine blood in heatprocessed meat and feed. J. Food Prot. 2008, 71, 1000−1006. (13) Ofori, J. A.; Hsieh, Y. H. Characterization of a 12 kDa thermalstable antigenic protein in bovine blood. J. Food Sci. 2011, 76, C1250− C1256. (14) Ofori, J. A.; Hsieh, Y.-H. P. Characterization of a 60 kDa thermally stable antigenic protein as a marker for the immunodetection of bovine plasma-derived food ingredients. J. Food Sci. 2015, 80, C1654−C1660. (15) Hsieh, Y. H.; Ofori, J. A.; Rao, Q.; Bridgeman, C. R. Monoclonal antibodies specific to thermostable proteins in animal blood. J. Agric. Food Chem. 2007, 55, 6720−6725. (16) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680− 685. (17) Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4350− 4354. (18) Friguet, B.; Djavadi-Ohaniance, L.; Pages, J.; Bussard, A.; Goldberg, M. A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the beta 2-subunit of Escherichia coli tryptophan synthase. J. Immunol. Methods 1983, 60, 351−358. (19) Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 1988.

detection limit was 0.03% (v/v) PFP in cooked beef, pork, lamb, cod, and chicken. The sELISA could detect 0.01% (v/v) of APP in cooked beef, pork, lamb, chicken, and cod. Not only are the detection limits achieved by this assay lower than any of those reported for other methods,7,9,10 but this new immunoassay does not involve laborious sample preparation, does not suffer from matrix interference, and is not hampered by heat treatment, as is the case with other methods. In particular, the new sELISA reliably detected porcine plasma-derived proteins in ground meats and fish that had been cooked at 100 °C for 15 min, despite the fact that they had already undergone heat treatment during the spray-drying process. In conclusion, 10 mAbs were developed and characterized in terms of their selectivity, cross-reactivity, antigenic proteins, and the thermostability of their cognate antigens for this study using both laboratory-prepared and commercially produced bloodderived food ingredients. All of the mAbs were found to react with porcine blood and to bind either to a 12 kDa protein in the RBC fraction or to a 60 or 90 kDa protein in the plasma fraction of porcine blood. None of these mAbs reacted with other commonly used proteins of nonblood origin tested, including meat, soy, and gelatin that are likely to be present in a food and dietary supplement formulation, thus minimizing the likelihood of false-positive results. Extracts of diversely processed commercial blood protein samples that were analyzed with Western blot using these mAbs revealed that these antigenic proteins in commercially produced porcine blood-derived food ingredients remained unchanged, indicating that these antigenic proteins are unaffected by the various processes that the blood protein products are subjected to and are therefore stable markers for the immunodetection of porcine blood-derived ingredients in foods. Consequently, a prototype sandwich ELISA utilizing two of these mAbs, 19C5E10 (as the capture antibody) and biotin-conjugated 16F9-C11 (as the detection antibody), was constructed to demonstrate the use of these selected mAbs in a sELISA system. The resulting assay was shown to sensitively and effectively detect levels as low as 0.3% of porcine blood or plasma in cooked ground chicken without exhibiting a cross reaction with other proteins tested. Cooked ground meat or fish spiked with various PBFIs could also be detected at very low inclusion levels of ≤0.03%, demonstrating the excellent potential of the newly developed sELISA as an analytical tool for monitoring PBFIs in foods and dietary supplements. Once successfully developed, these mAbs can be used either singly or in combination in various formats of immunoassays (as demonstrated with this developed sELISA) to detect porcine plasma and/or RBC-derived food ingredients, thus protecting the billions of individuals that avoid consuming blood for various reasons and also discouraging the fraudulent use of these blood-derived materials.

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AUTHOR INFORMATION

Corresponding Author

*(Y.-H.P.H.) E-mail: [email protected]. Phone: (850) 644-1744. Fax: (850) 645-5000. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Ofori, J. A.; Hsieh, Y.-H. P. The use of blood and derived products as food additives. In Food Additive; El-Samragy, Y., Ed.; InTech: Rijeka, Croatia, 2012; pp 229−25610.5772/32374. G

DOI: 10.1021/acs.jafc.5b06136 J. Agric. Food Chem. XXXX, XXX, XXX−XXX