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Quality Control System for Beer Developed with Monoclonal Antibodies Specific to Barley Lipid Transfer Protein

Quality Control System for Beer Developed with Monoclonal Antibodies Specific to Barley Lipid... Antibodies 2012, 1, 259-272; doi:10.3390/antib1030259 1 OPEN ACCESS antibodies 3 ISSN 2073-4468 4 www.mdpi.com/journal/antibodies 5 Article 6 Quality Control System for Beer Developed with Monoclonal 7 Antibodies Specific to Barley Lipid Transfer Protein 8 1 2 3 4 Yukie Murakami-Yamaguchi , Junko Hirose , Kumiko Kizu , Fumiko Okazaki , 9 5 4, Wataru Fujii and Hiroshi Narita * 10 Kyoto College of Nutritional & Medical, Setogawa-cho 18, Saga Tenryuji, Ukyo-ku, 11 Kyoto 616-8376, Japan; E-Mail: yukie.yamaguchi@taiwa.ac.jp 12 Department of Food and Nutrition, School of Human Cultures, The University of Shiga Prefecture, 13 Hassaka-cho 2500, Hikone, Shiga 522-8533, Japan; E-Mail: jhirose@shc.usp.ac.jp 14 Department of Life and Living, Osaka Seikei College, Aikawa 3-10-62, Higashiyodogawa, 15 Osaka 533-0007, Japan; E-Mail: kizu@osaka-seikei.ac.jp 16 Department of Food and Nutrition, Kyoto Women’s University, Kitahiyoshi-cho 35, Imakumano, 17 Higashiyama-ku, Kyoto 605-8501, Japan; E-Mail: k1134201@kyoto-wu.ac.jp 18 Suntory Business Expert Limited, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, 19 Japan; E-Mail: Wataru_Fujii@suntory.co.jp 20 * Author to whom correspondence should be addressed; E-Mail: narita@ kyoto-wu.ac.jp; 21 Tel./Fax: +81-75-531-7154. 22 Received: 17 September 2012; in revised form: 28 September 2012 / Accepted: 9 October 2012 / 23 Published: 16 October 2012 24 Abstract: Non-specific lipid transfer protein (LTP) in barley grain reacted with the IgE in 26 sera drawn from food allergy patients. A sandwich-type of enzyme-linked immunosorbent 27 assay (ELISA) was developed with mouse monoclonal antibodies raised against LTP 28 purified with barley flour. This ELISA showed a practical working range of 0.3–3 ng/mL 29 and no cross-reactivity with wheat, adlay and rye. Using this ELISA, LTP was determined 30 in several types of barley-foods, including fermented foods such as malt vinegar, barley- 31 malt miso and beer. LTP content in beer of the same kind was approximately constant, 32 even if manufacturing factory and production days were different. Not only as a factor of 33 foam formation and stability but also as an allergen, controlling and monitoring of LTP in 34 beer should be considered. Taken together, our LTP-detecting ELISA can be proposed as 35 an appropriate system for the quality control of beer. 36 37 Antibodies 2012, 1 260 Keywords: lipid transfer protein; beer; ELISA; food allergy; monoclonal antibody 1 1. Introduction 3 Non-specific lipid transfer protein (LTP) is an ubiquitous 9-kDa protein widely present in the plant 4 kingdom. Its lipid binding property primary postulates the role of LTP in the intracellular transport of 5 lipids [1,2]. However, it is now known that LTP is synthesized as a precursor with a typical N-terminal 6 signal peptide and the mature LTP is secreted outside the cell [1,3,4]. Therefore, the former idea about 7 lipid transportation by LTP has been abandoned. While LTP 2 with a size around 7-kDa is also present 8 as a minor component, we will treat LTP 1 as LTP in this study, hereafter [2]. The highest expression of 9 LTP has been found in peripheral cell layers surrounding aerial organs, associated with the cell wall 10 and cuticle of epidermal tissues. Stress factors, such as drought, cold and high salinity have been described 11 to upregulate the expression of LTP in plants [1,5–7]. Many reports have mentioned the physiological 12 role of LTP in the protective mechanism against the attack of bacteria, fungi and viruses [8–11]. 13 Accordingly, LTP has been classified as pathogenesis-related protein 14 (PR-14) [12]. 14 LTP has been confirmed as to its involvement in the human allergic reaction to fruits, vegetables, 15 nuts, and even pollens [13–17]. LTP is composed of eight cysteine residues forming a network of four 16 disulfide bridges. These disulfide bonds provide stability in the structure of LTP to heat treatment and 17 proteolytic digestion, which is the characteristic feature of food allergen. In addition, the lipid binding 18 domain of LTP also plays an important role in its allergenic property [2,18,19]. Furthermore, LTP is 19 known to be a panallergen according to its highly homologous nature in amino acid sequence and 3D 20 structure among various plants [2,20,21]. Since barley LTP (bLTP) is also resistant to heat treatment 21 and proteolytic digestion, bLTP, as well as protein Z, remains in beer and contributes to foam 22 formation and stability of beer [22,23]. It is noteworthy that LTP performs higher allergenicity than 23 protein Z according to the result of prick test [15,24,25]. 24 Globally, food manufacturers have faced the growing of consumers’ concerns regarding the 25 presence of allergens in their products. Japanese Ministry of Health, Labor and Welfare made the first 26 decision in the world for the mandatory labeling of several foods containing allergenic ingredients in 27 2002 from the viewpoint of preventing the occurrence of health hazard [26]. Development of the 28 proper analytical method for the determination of allergens in foods is necessary in order to ensure the 29 quality, safety and fair trade of foods for consumers. Enzyme-linked immunosorbent assay (ELISA) is 30 well known, and is widely utilized as a method for determining the presence of specific proteins in 31 processed foods [26]. In this study, we have developed ELISA with monoclonal antibodies (mAbs) 32 specific to bLTP and proposed its application in quality controlling of beer production. 33 2. Results and Discussion 34 2.1. Barley Grain LTP 35 Barley LTP (bLTP) was purified from commercial barley grain flour by using (NH ) SO 36 4 2 4 precipitation, hydrophobic chromatography and cationic exchange chromatography. Corresponding to 37 Antibodies 2012, 1 261 the position on SDS-PAGE, the homogenous protein with a molecular mass about 9-kDa was obtained. 1 To clearly identify whether this protein is bLTP, the N-terminal amino acid sequence of this protein 2 was analyzed. The result showed the eight amino acid sequence LN(X)GQVDS, that was identical to 3 the first eight amino acid sequence of bLTP reported by Douliez et al. [2]. Thus, the purified 9-kDa 4 protein was bLTP, as confirmed by its apparent molecular mass and a part of amino acid sequence 5 (Figure 1). The concentration of purified bLTP was determined by absorbance assay (280 nm) and 6 TM TM DC protein assay [27] using BSA as a standard protein. According to the result of DC protein 7 assay, concentration of bLTP was three times higher than that determined by measuring absorbance at 8 280 nm (E = 10). The difference in concentration determined by these two methods might be due to 9 1% the lack of tryptophan and small amount of tyrosine (three residues) in the amino acid sequence of 10 bLTP. The purified bLTP showed lipid transfer activity measured according to the Paridon’s 11 method [28], suggesting no changing in bLTP structure by purification process (data not shown). This 12 pure bLTP, obtained as described, was used here for further investigation. 13 Figure 1. Deduced amino acid sequences of barley seed and leaf lipid transfer proteins 14 (LTPs) and wheat seed LTP from their nucleotide sequences (NLT1_HORVU, 15 NLT3_HORVU, and NLTA_WHEAT). The blue highlight indicated the homologous 16 amino acid in the alignment of these amino acid sequences. 17 5 10 15 20 barley （seed） L N C G Q V D S K M K P C L T Y V Q G G barley （leaf） A I S C G Q V S S A L S P C I S Y A R G N wheat （seed） I D C G H V D S L V R P C L S Y V Q G G 25 30 35 40 barley （seed） - P G P S G E C C N G V R D L H N Q A Q barley （leaf） G A K P P V A C C S G V K R L A G A A Q wheat （seed） - P G P S G Q C C D G V K N L H N Q A R 45 50 55 60 barley （seed） S S G D R Q T V C N C L K G I A R G I H barley （leaf） S T A D K Q A A C R C L K S L A T S I K wheat （seed） S Q S D R Q S A C N C L K G I A R G I H 65 70 75 80 barley （seed） N L N L N N A A S I P S K C N V N V P Y barley （leaf） G I N M G K V S G V P G K C G V S V P Y wheat （seed） N L N E D N A R S I P P K C G V N L P Y 85 90 barley （seed） T I S P D I D C S R I Y barley （leaf） P I S M S T D C N K V H wheat （seed） T I S L N I D C S R V 2.2. Allergenicity of bLTP 19 Human sera possessing IgE of barley, wheat, or apple obtained from 10 food allergy patients (Table 1) 20 were tested individually to determine IgE reactivity to bLTP by immunoblotting analysis (Figure 2). 21 The sera No. 5 and 10 showed no reactivity to bLTP in the extract. On the other hand, the high 22 Antibodies 2012, 1 262 reactivity to bLTP in the extract was detected in sera No. 1–4 and 6–9. Despite other protein bands at the 1 molecular mass of 14, 28 and 60-kDa also were present in the extract and showed similar band intensity 2 as that of bLTP on protein staining; a high intensity of the IgE-binding band was only observed at the 3 position of bLTP. This result indicates the importance of bLTP as the major allergen in barley food 4 products. Thus, preventing bLTP contamination in food processing system should be seriously concerned. 5 Table 1. Characteristics of sera from patients with food allergy. 6 Barley Wheat Apple Patient Sensitization No. IgE kU/L 1 1.2 - 6.4 wheat products, nuts, beans 2 9.8 33.1 25.6 egg white 3 15.8 14.3 14.4 wheat products, nuts, beans 4 - - 11.7 apple, pear, carrot, pollen 5 25.6 46.8 19.2 peach, apple, pear, fish, meat 6 9.5 17.8 0.9 nuts, beans 7 5.6 5.7 1.2 grass pollen 8 5.5 5.4 4.7 fish 9 6.4 2.9 - plum, dates 10 - - 11.5 milk products, egg, nuts, beans -: not determined. 7 Figure 2. The reactivity of sera IgE obtained from food allergic patients with proteins in 8 barley extract. (A) SDS-PAGE pattern of molecular mass markers (a), 8 µg of PBS- 9 extracted barley proteins (b), and 2 µg of barley LTP (bLTP) (c). (B) Western blotting 10 pattern of PBS-extracted barley proteins (8 µg) with sera from food allergic patients 11 (No. 1–10). IgE was visualized with alkaline phosphatase-conjugated anti-human IgE. 12 The IgE characteristics of sera from allergic patients are shown in Table 1. 13 Antibodies 2012, 1 263 2.3. Preparation and Characterization of Monoclonal Antibodies to bLTP 1 Thirteen mice were immunized with bLTP, and the spleen cells from mice showed high anti-bLTP 2 titer were fused with myeloma cells. Four positive clones, 5-4, 10-1, 10-4, and 12-3 screened by an 3 indirect ELISA possessed high specificity to bLTP. Using a mouse mAb isotyping kit, it was shown 4 that the mAbs produced by these clones were all IgG1/κ. 5 The specificity of these four anti-bLTP mAbs to LTP from other cereal grains was examined by 6 immunoblotting analysis. After separation by SDS-PAGE, protein bands corresponding to LTP at 7 around 9-kDa were detected in extracts from barley, wheat, adlay, and rye (Figure 3A). Barley LTP 8 was clearly detected by western blotting analysis with all four mAbs (Figure 3B–E). MAbs 5-4, 10-1, 9 and 10-4 showed negative reactivity to the extracts from other sources. While, weak cross-reaction of 10 mAb 12-3 with LTP from rye was observed (Figure 3E). The minor cross-reaction of mAb 12-3 with 11 rye LTP might contribute to homologous amino acid sequences of its epitope in barley and rye. These 12 mAbs exhibited no cross-reactivity against extracts from the major allergenic foods, such as eggs， 13 milk peanuts, shrimp, and crab (data not shown). MAbs reactive with bLTP and their epitopes were 14 previously revealed [29]. However, their cross-reactivity to other LTPs and application for quantitative 15 analysis were unknown. 16 Figure 3. Cross reactivity of anti-bLTP monoclonal antibodies with other cereal grain 17 LTPs. (A) SDS-PAGE pattern of molecular mass markers (a), 2 µg of bLTP (b), and 8 µg 18 of PBS-extracted proteins from barley ①, wheat ②, adlay ③, and rye ④. (B–E) Western 19 blotting pattern of PBS-extracted barley proteins (8 µg) with various established anti-bLTP 20 monoclonal antibodies (mAbs). (B), 5-4; (C), 10-1; (D), 10-4; (E) 12-3. = 21 2.4. Sandwich ELISA for the Determination of bLTP 23 For the quantitative analysis of bLTP with high sensitivity, we developed sandwich ELISA systems 24 with these mAbs. Each established mAb was used as the first antibody and peroxidase-conjugated 25 Antibodies 2012, 1 264 counterparts were used as the second antibody. As shown in Figure 4, six different systems were 1 capable of detecting bLTP. The system that uses mAb 10-4 as the first antibody and peroxidase- 2 conjugated mAb 5-4 as the second antibody was identified as the highest sensitive system for bLTP 3 quantitative analysis (Figure 4C). The practical working range was 0.3–3 ng/mL. The sensitivity of this 4 system to wheat LTP was 1/300,000 times less than that to bLTP (Figure 5). Thus, this sandwich 5 ELISA system is significantly sensitive and specific to bLTP. 6 Figure 4. Sandwich enzyme-linked immunosorbent assays (ELISAs) for the quantitative 7 analysis of bLTP. Sandwich ELISAs were constructed with mAb 5-4 (A), 10-1 (B), 10-4 8 (C), or 12-3 (D) as a solid-phase antibody and peroxidase-conjugated mAb 5-4 (●), 10-1 (○), 9 10-4 (▲), or 12-3 (△) as a second antibody. 10 The 10-4/5-4 system was then applied for the determination of bLTP in several extracts prepared 12 from barley or barley food products (Table 2). Barley is a major food and animal feed source. Two 13 kinds of barley are widely used in food industry, two-row barley (Hordeum distichum) and six-row 14 barley (Hordeum vulgare). The former from which we prepared bLTP is a traditional one for brewing, 15 while the latter is used well as roasted barley grain, a raw material for a popular Asian tea. Comparable 16 bLTP contents were obtained in the materials of both cultivars. However, bLTP could not be 17 determined in leaf by this method probably due to the difference in amino acid sequence between grain 18 and leaf LTP (Figure 1) [2]. Compared to those in raw materials, bLTP contents in processed barley 19 foods were extremely low. In spite of stable structure of LTP suggesting high temperature tolerant 20 characteristic, some denaturation of LTP at 96 ° C was reported by Nierop et al. [23]. In addition, 21 Antibodies 2012, 1 265 extraction efficiency might be lowered by heat-induced aggregation of components. Therefore, the low 1 amount of bLTP detected in such processed barley foods could be contributed to these artificial factors. 2 We have reported allergenicity of heat-denatured ovomucoid, a major egg allergen, which shows rigid 3 conformation due to disulfide bonds and possesses heat stability similar to LTP [30]. Then, allergen 4 had better to be estimated as total amounts irrespective of the degree of heat denaturation [31]. 5 However, the 10-4/5-4 system showed several times lower reactivity to heated bLTP at 100 °C for 6 10 min than native bLTP (data not shown). 7 Figure 5. Reactivity of the 10-4/5-4 sandwich ELISA system to barley and wheat LTPs. 8 Barley (●) or wheat LTP (○) was determined by the sandwich ELISA, constructed with mAb 9 10-4 as a solid-phase antibody and peroxidase-conjugated mAb 5-4 as a second antibody. 10 Table 2. Barley LTP content. 12 Food µg/g (mL) Two-row barley flower 393.7 Two-row barley flower 301.3 Barley leaf - Hattaiko * 0.1 Roasted barley grain ** - Barley miso *** 18.3 Malt vinegar 21.0 beer 2.6 -: not detected; * Roasted and grounded barley flour used for Japanese sweets; ** Raw material for 13 popular Asian tea; *** Soybean paste made with barley malt. 14 ELISA screening is one of the official detection method to identify the allergenic ingredient in food 15 by the Japanese official government [26]. Ovalbumin is selected as the target to detect egg, casein and 16 β-lactoglobulin to detect milk, protein complex containing Ara h2 to detect peanut, and gliadin to 17 detect wheat. In foods such as soybean paste, vinegar, and beer that wheat is used as one of the 18 important ingredients, however, it is a serious problem that gliadin is hydrolyzed by proteolytic 19 Antibodies 2012, 1 266 enzyme during fermenting process [32]. The fact that LTP is highly stable toward proteolysis attracted 1 us an interest in utilization of wheat LTP as a substitute target protein in detection of wheat content in 2 fermented food instead of gliadin [33]. In the same context, it was notable that bLTP was detected in 3 fermented foods such as barley-malt miso, malt vinegar, and beer in Table 2. 4 2.5. Quantitative Analysis of bLTP in Beer 5 Barley LTP is a major proteinaceous component in beer [15,22–24]. Besides being an allergenic 6 agent, it is also recognized as a foaming agent in brewing industry. Therefore, routine analysis of bLTP 7 in product might be used to assist the quality controlling of beer production. In this study, we 8 introduced the ELISA 10-4/5-4 system for assessing the quality of beer. 9 Three different brewing processes produce beers of the world. Bottom fermentation, generally used 10 in production of beer in Japan, Germany, and the Netherlands, is performed at 4–13 °C for 4–6 weeks. 11 Beer is brewed at 20 ° C for two weeks in top fermentation, a brewing process widely used in United 12 Kingdom and Belgium. Spontaneous fermentation, widely done in Belgium, is carried out with wild 13 type yeast. Figure 6 shows that the amount of bLTP in beer samples was inconstant (1–11 μg/mL), 14 independently of the brewing process and the producing country. Accordingly, variation in LTP 15 contents is due to the difference in raw materials and detail of processing and might determine the 16 character and quality of each beer. 17 Figure 6. Barley LTP levels in beer samples prepared by three different brewing processes. 18 Concentration of bLTP was determined by the 10-4/5-4 sandwich ELISA system. Ⅰ, bottom 19 fermentation; Ⅱ, top fermentation; Ⅲ, spontaneous fermentation. No. 1–7, Japanese products; 20 8, English product; 9, German product; 10, Irish product; 11–17, Belgian products. 21 Four kinds of beer (A to D) manufactured in four different factory (①–④) and different production 23 days were monitored for bLTP level using the ELISA 10-4/5-4 system (Figure 7). All of the brewing 24 processes in each factory had been performed perfectly in every day that the samples were taken. 25 Analysis of the resultant beers indicated that there was no remarkable difference in the bLTP content 26 of final products from different days in the same factory. The difference between bLTP content in beer 27 from different lots of production is capable of being used to indicate the unusual producing condition 28 Antibodies 2012, 1 267 such as quality of raw materials and is a good predictor of the different taste of beer. After the claim of 1 a customer, a canned beer B was reported for induction of urticaria. However, this ELISA system 2 failed to distinguish between bLTP content in beer of the claimed lot and that of other normal lots 3 (Figure 7B, X). In default of any bizarreness in bLTP level, it is confirmed that the claimed lot of beer 4 was produced by a proper brewing process. Thus, determining bLTP level in beer product by this 5 ELISA gives valuable information for assuring the quality of beer to customer with little investment of 6 time and money. 7 Figure 7. Comparison of bLTP content in beer between different lots of production. Four 8 kinds of beers (A–D) produced in four different factories (①–④) and different days were 9 obtained. The amount of bLTP was determined by the 10-4/5-4 sandwich ELISA system. 10 ① 10/1 indicates that it was produced in factory ① on October 1st. (X) indicates the same 11 lot product that was claimed by a customer. 12 Controlling and monitoring bLTP content in beer is important, as bLTP is a factor for foam 14 formation and stability and also as an allergen. This study introduces the sandwich ELISA using mAbs 15 specific to bLTP as a useful tool for quality control and authenticity assessment of beer. Barley LTP 16 determination by this ELISA might also be used to indicate the physical characteristic and taste of beer 17 as well. For future experiment, the correlation of bLTP content in beer product with wort boiling 18 temperature, fermenting method, addition of malt and brewing process will be determined. Moreover, 19 bLTP might be indicated as a target protein in assessing of barley content in order to assist quality 20 control of barley-fermented foods. Further information in application of bLTP in quality control system 21 could provide the valuable information in novel product development in food and beverage industry. 22 Antibodies 2012, 1 268 3. Experimental Section 1 3.1. Reagents and Apparatus 2 The following materials were obtained from the indicated sources: cell fusion and cell culture 3 reagents, fetal bovine serum (FBS), and polyethylene glycol 1500 (Invitrogen, Carlsbad, California, 4 USA), RPMI 1640 medium (Nacalai Tesque Inc., Kyoto, Japan), hypoxanthine thymidine (HT), 5 hypoxanthine aminopterin thymidine (HAT) reagents and B cell growth factor, BriClone (Dainippon 6 Pharmaceutical Co. Ltd., Osaka, Japan), Freund’s complete and incomplete adjuvants (Difco 7 Laboratories, Detroit, MI, USA), Protein G Sepharose and monoclonal antibody isotyping kit (GE 8 Healthcare, Buckinghamshire, England), Maxisorp microplate for ELISA (Nunc, Roskilde, Denmark), 9 TM DC protein assay kit (BIO-RAD laboratory, Bradford, UK), pristine (Sigma Chemical Corp, St. Louis, 10 MO, USA), alkaline phoshatase-conjugated anti-mouse IgG and anti-human IgE (American Qualex, 11 San Clemente, CA, USA). Sera from 10 patients allergic to food were supplied by PlasmaLab (Everett, 12 WA, USA). Unless specified otherwise, all other chemicals used were guaranteed reagent grade. 13 3.2. Isolation of Barley Grain LTP 14 Barley grain flour (80 g) was mixed with 3 fold volumes of water. The mixture was then stood 15 overnight at room temperature. After centrifugation at 10,000 × g, 4 °C for 20 min, the supernatant 16 was collected as barley extract. Barley extract was further subjected to (NH ) SO fractional 17 4 2 4 precipitation by adding (NH ) SO to 30% saturation, and then the pellet was discarded. Subsequently, 18 4 2 4 (NH ) SO was added to the supernatant to 80%. Following precipitation, the pellet was resuspended in 19 4 2 4 1.2 M (NH ) SO . The pH of the solution was adjusted to 7.0 using 20 mM phosphate buffer, 20 4 2 4 beforehand, and this solution was applied to a Toyopearl butyl column (2.5 × 10 cm) (Tosoh, Tokyo, 21 Japan) equilibrated with 1.2 M (NH ) SO . Proteins were eluted from the column by a linear gradient 22 4 2 4 of 1.2 to 0 M (NH ) SO . Relevant fractions were identified by the presence of a band at 9-kDa on 23 4 2 4 sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels under reduced 24 condition. These fractions were pooled and dialyzed overnight against 20 mM phosphate buffer, pH 25 5.0 (PB) using a dialysis tube (MWCO 3500 Da, Spectrum Laboratories, Rancho Dominguez, CA, 26 USA). Then, the sample was applied to a Toyopearl CM column (1.6 × 5 cm) (Tosoh, Tokyo, Japan) 27 equilibrated with PB. Proteins were eluted with a linear gradient of 0 to 0.3 M NaCl in PB. LTP 28 containing fractions were collected. The purity of protein was determined by SDS-PAGE. Purified 29 LTP was dialyzed against water overnight, lyophilized, and stored at −20 °C, until use. Wheat LTP 30 TM was purified as previously described [33]. Protein concentration was determined by DC protein 31 assay using bovine serum albumin as a standard protein. 32 3.3. Electrophoresis and Western Blotting 33 SDS-PAGE was performed by the method of Laemmli [34] by using 10% gel. Sample was dissolved 34 in 20 mM Tris-HCl buffer (pH 6.8) containing 2% (w/v) SDS and 10 mM 2-mercaptoethanol, and 35 heated for 5 min in boiling water. After electrophoresis, the gel was stained with Commassie Brilliant 36 Blue R-250. The protein with molecular mass of 9-kDa was identified as LTP. 37 Antibodies 2012, 1 269 Extracts from barley, wheat, adlay, and rye, with 8 µg protein content, were applied to gel electrolysis. 1 After been performed on SDS-PAGE, the proteins in a gel were transblotted to a polyvinylidene 2 difluoride (PVDF) membrane. The membrane was transferred to a blocking solution (PBS containing 3 5% skim milk) for 60 min, and then incubated with the anti-bLTP mAb at room temperature for 1 h. 4 After washing with 10 mM Tris buffer, pH 7.4 containing 0.15 M NaCl (TBS) several times, the 5 membrane was incubated with alkaline phosphatase-conjugated anti-mouse IgG at room temperature 6 for 1 h. After extensively washing again with TBS, protein band on the membrane was developed with 7 alkaline-phosphatase kit. When human sera were tested, alkaline phosphatase-conjugated anti-human 8 IgE was used. 9 3.4. N-Terminal Amino Acid Sequencing 10 The N-terminal amino acid sequence of purified LTP electrophoretically transferred to a PVDF 11 membrane was analyzed by Edman degradation using Applied Biosystem Procise 491 HT Protein 12 Sequencer (Foster City, CA, USA). The amino acid sequence obtained was compared with that of 13 bLTP in public database. 14 3.5. Production of Monoclonal Antibody 15 Thirteen eight-week old female BALB/c mice (Nippon SLC Co., Shizuoka, Japan) were immunized 16 by intraperitoneal injection of 100 µg of bLTP emulsified in Freund’s complete adjuvant. At two and 17 four weeks after the initial immunization, the mice were boosted by intraperitoneal injection of 50 µg 18 of bLTP emulsified in Freund’s incomplete adjuvant. The final booster was given by intraperitoneal 19 injection of 10 µg LTP in 10 mM phosphate buffer, pH 7.4 containing 0.15 M NaCl (PBS) at the sixth 20 week. On the third day after the final injection, the spleen cells were harvested from the mice and then 21 fused with the mouse myeloma cell (P3U1) [35]. Fused cells were seeded in 96-well culture plates and 22 incubated at 37 °C in HAT medium. After 2 weeks in a CO incubation, the culture supernatant of each 23 hybridoma was examined for reactivity with bLTP by an indirect ELISA. Hybridoma from the well 24 that perform high reactivity with antigen were subcloned twice by limiting dilution on HT medium 25 containing 5% BriClone. Each clone secreting antibody to bLTP was grown in mass culture. Subclass 26 of immunoglobulin in culture supernatant of each clone was identified by a mouse mAb isotyping kit. 27 Cloned cells were injected intraperitoneally into a 10-week old mouse treated with pristine. The 28 monoclonal antibody was purified by using a protein G affinity column from the ascitic fluid. The 29 purified antibody was conjugated with horseradish peroxidase [36]. Animal experiments were carried 30 out under the guideline of Animal Experiment Committee of Kyoto Women’s University following the 31 bulletin (No.71, 2006) of the Ministry of Education, Culture, Sports, Science, and Technology in Japan. 32 3.6. Preparation of Extract for LTP Determination 33 Milled cereal grain powder (1 g) was briefly homogenized with 19 mL PBS. After being shaken 34 overnight, the homogenate was centrifuged at 3,000 × g, 4 °C for 20 min. The supernatant was 35 collected and further filtrated through a filter paper No.2. The clear filtrate was used as cereal grain 36 extract to determine the LTP content in further experiment. There was no special treat for beer sample. 37 Antibodies 2012, 1 270 3.7. Enzyme-Linked Immunosorbent Assay (ELISA) 1 For indirect ELISA, each well of 96-well flat-bottom polystyrene microplate was coated with 50 µL 2 per well of 2 µg/mL bLTP. After overnight incubation at 4 °C, the unbound site was blocked by 3 incubation with 200 µL per well of blocking reagent (10% BSA in PBS) at 37 °C for 1 h. Media fluid 4 of hybridoma culture (50 µL) was added into wells and incubated at 37 °C for 1 h. After washing with 5 TBS containing 0.02% tween 20 (TBS-T), 100 µL of anti-mouse IgG conjugated to alkaline 6 phosphatase diluted in TBS-T containing 0.1% BSA was added and incubated at 37 °C for 1 h. After 7 washing, 100 µL of chromogenic substrate (1 mg/mL ρ-nitrophenylphosphate and 0.5 mM MgCl in 8 10% diethanlamine buffer, pH 9.8) was added and incubated at 37 °C for 1 h. The intensity of the 9 resulting color was detected at 415 nm by a microplate reader. For sandwich ELISA, each well of the 10 plate was coated with 50 µL of 5 µg/mL of anti-bLTP mAb in coating buffer at 37 °C for 1 h. After 11 blocking and three washing, 50 µL of barley extract (0–100 ng/mL) or beer sample was add to each 12 well of the coated plate, and incubated at 37 °C for 1 h. Then, to each washed well 50 µL of 13 peroxidase-conjugated anti-bLTP mAb was added. After incubated at 37 ° C for 1 h, the substrate 14 solution was added, developed, and finally the absorbance at 450 nm was measured as described above. 15 4. Conclusions 16 A sandwich-type of enzyme-linked immunosorbent assay was developed with mouse monoclonal 17 antibodies raised against LTP purified from barley grain flour. LTP is known to be a factor in foam 18 formation and stability in beer, while also being an allergen. This method was then proposed as a 19 predominant system for the quality control of beer. 20 Acknowledgements 21 The authors thank Mayuko Mishima, Chie Iguchi, and Saori Shibata of Department of Food and 22 Nutrition, Kyoto Women’s University for their helpful technical assistance. 23 Conflict of Interest 24 The authors declare no conflict of interest. 25 References and Notes 26 1. Kader, J.C. Lipid-transfer proteins in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996, 47, 27 627–654. 28 2. Douliez, J.P.; Michon, T.; Elmorjani, K.; Marion, D. Structure, biological and technological 29 functions of lipid transfer protein and indolines, the major lipid binding protein from cereal 30 kernels. J. Cereal Sci. 2000, 32, 1–20. 31 3. Garcia-Olmedo, F.; Molina, A.; Segura, A.; Moreno, M. The defensive role of nonspecific 32 lipid-transfer proteins in plants. Trends Microbiol. 1995, 3, 72–74. 33 4. Kader, J.C. Lipid-transfer proteins: A puzzling family of plant proteins. 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The promoter of a Brassica napus lipid transfer protein 10 gene is active in a range of tissues and simulated by light and viral infection in transgenic 11 Arabidopsis. Plant Mol. Biol. 1999, 41, 75–87. 12 10. Park, C.J.; Shin, R.; Park, J.M.; Lee, G.J.; You, J.S.; Paek, K.H. Induction of pepper cDNA 13 encoding a lipid- transfer protein during the resistance response to tobacco mosaic virus. Plant 14 Mol. Biol. 2002, 48, 243–254. 15 11. Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 2002, 415, 389–395. 16 12. Van Loon, L.; Van Strien, E. The families of pathogenesis-related proteins, their activities, and 17 comparative analysis of PR-1 type proteins. Physiol. Mol. Plant Pathol. 1999, 55, 85–97. 18 13. Sanchez-Monge, R.; Lombardero, M.; Garcia-Selles, F. J.; Barber, D.; Salcedo, G. Lipid-transfer 19 proteins are relevant allergen in fruit allergy. J. Allergy Clin. Immunol. 1999, 103, 514–519. 20 14. Palacin, A.; Cumplido, J.; Figueroa, J.; Ahrazem,O.; sanchez-Monge, R.; Carrillo, T.; Salcedo, G.; 21 Blanco, C. Cabbage lipid transfer protein Bra o 3 is major allergen responsible for cross-reactivity 22 between plant foods and pollens, J. Allergy Clin. Immunol. 2006, 117, 1423–1429. 23 15. Curioni, A.; Santucci, B.; Cristaudo, A.; Canistraci, C.; Pietravalle, M.; Simonato, B.; 24 Giannattasio, M. Urticaria from beer: An immediate hypersensitivity reaction due to a 10 kDa 25 protein derived from barley. Clin. Exp. Allergy 1999, 29, 407–413. 26 16. Pastrorello, E.A.; Farioli, L.; Pravettoni, V.; Robino, A.M.; Scibilia, J.; Fortunato, D.; Conti, A.; 27 Borgonovo, L.; Bengtsson, A.; Ortolani, C. Lipid transfer protein and vicilin are important walnut 28 allergens in patients not allergic to pollen. J. Allergy Clin. Immunol. 2004, 114, 908–914. 29 17. Hiller, K.M.; Lubahan, B.C.; Klapper, D.G. Cloning and expression of ragweed allergen Amb a 6, 30 Scand. J. Immunol. 1998, 48, 26–36. 31 18. Charvolin, D.; Douliez, J.P.; Marion, D.; Cohen-Addad, C.; Pebay-Peyroula, E. The crystal 32 structure of a wheat nonspecific lipid transfer protein (ns-LTP1) complexed with two molecules of 33 phospholipids at 2.1 Å resolution. Eur. J. Biochem. 1999, 264, 562–568. 34 19. Lerche, M.H.; Poulsen, F.M. Solution structure of barley lipid transfer protein complexed with 35 palmitate. Two different binding modes of palmitate in the homologous maize and different 36 nonspecific lipid transfer protein. Protein Sci. 1998, 7, 2490–2498. 37 20. Salcedo, G.; Sanchez-Monge, R.; Diaz-Perales, A.; Garcia-Casado, G.; Barber, D. Plant non-specific 38 lipid transfer proteins as food and pollen allergens. Clin. Exp. Allergy 2004, 34, 1336–1341. 39 21. Salcedo, G.; Diaz-Perales, A.; Sanchez-Monge, R. Fruits allergy: Plant defence protein as noval 40 potential panallergen. Clin. Exp. Allergy 1999, 29, 1158–1160. 41 Antibodies 2012, 1 272 22. Lindroff-Larsen, K.; Winther, J.R. Surprisingly high stability of barley lipid transfer protein, LTP1, 1 towards denaturant, heat and proteases. FEBS Lett. 2001, 488, 145–148. 2 23. Van Nierop, S.N.E.; Evans, D.E.; Axcel, B.C.; Cantrell, I.C.; Rautenbach, M. Impact of different 3 wort boiling temperatures on the beer foam stabilizing properties of lipid transfer protein 1. 4 J. Agric. Food Chem. 2004, 52, 3120–3129. 5 24. Garcia-Casado, G.; Crespo, J.F.; Rodriguez, J.; Salcedo, G. Isolation and charasterization of barley 6 lipid transfer protein and protein Z as beer allergens. J. Allergy Clin. Immunol. 2001, 108, 647–649. 7 25. Quercia O; Zoccatelli, G; Stefanini, GF; Mistrello, G; Amato, S; Bolla, M; Emiliani, F; Asero, R. 8 Allergy to beer in LTP-sensitized patients: Beers are not all the same. Allergy 2012, 67, 1186–1189. 9 26. Akiyama, H.; Imai, T.; Ebisawa, M. Japan food allergen labeling regulation—History and 10 Evaluation. In Advances in Food and Nutrition Research; Taylor, S., Ed.; Academic Press: 11 Burlington, MA, USA, 2011; Volume 62, pp. 139–171. 12 27. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin 13 phenol reagent. J. Biol. Chem. 1951, 193, 265–275. 14 28. Van Paridon, P. A.; Gadella, T.W.J., Jr.; Wirtz, K.W.A. The effect of polyphosphoinositides and 15 phosphatidic acid on the phosphatidylinositol transfer protein from bovine brain: Kinetic study. 16 Biochim. Biophys. Acta 1988, 943, 76–86. 17 29. Lusk, L.T.; Navarro, A.L.; Goldstein, H.; Wagner, R.J.; Ryder, D.S. Monoclonal antibodies for 18 assaying lipid transfer proteins. U.S. Patent 6423546, 23 July 2002. 19 30. Hirose, J.; Kitabatake, N.; Kimura, A.; Narita, H. Recognition of native and/or thermally induced 20 denatured forms of the major food allergen, ovomucoid, by human IgE and mouse monoclonal 21 IgG antibodies. Biosci. Biotechnol. Biochem. 2004, 68, 2490–2497. 22 31. Hirose, J.; Murakami-Yamaguchi, Y.; Ikeda, M.; Kitabatake, N.; Narita, H. Oligoclonal enzyme- 23 linked immunosorbent assay capable of determining the major food allergen, ovomucoid, 24 irrespective of the degree of heat denaturation. Cytotechnology 2005, 47, 145–149. 25 32. Honjoh, T.; Muraoka, S.; Mamekoshi, S.; Sakai, M. Detection methods for allergic substances in 26 foods by enzyme linked immune sorbent assay. Foods Food Ingredients J. Jpn. 2002, 206, 13–22. 27 33. Murakami-Yamaguchi, Y.; Hirose, J.; Honjoh, T.; Narita, H. Evaluation of wheat content with 28 monoclonal antibodies against lipid transfer protein. J. Jpn. Soc. Food Ssi. Technol. 2009, 56, 16–24. 29 34. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage 30 T4. Nature 1970, 227, 680–685. 31 35. Kohler, G.; Milstein, C. Continuous cultures of fused cells secreting antibody of predefined 32 specificity. Nature 1975, 256, 495–497. 33 36. Nakane, P.K.; Kawaoi, A. Perioxidase-labelled antibody. New method of conjugation. 34 J. Histochem. Cytochem. 1974, 22, 1084–1091. 35 © 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article 36 distributed under the terms and conditions of the Creative Commons Attribution license 37 (http://creativecommons.org/licenses/by/3.0/). 38 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antibodies Multidisciplinary Digital Publishing Institute

Quality Control System for Beer Developed with Monoclonal Antibodies Specific to Barley Lipid Transfer Protein

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Antibodies 2012, 1, 259-272; doi:10.3390/antib1030259 1 OPEN ACCESS antibodies 3 ISSN 2073-4468 4 www.mdpi.com/journal/antibodies 5 Article 6 Quality Control System for Beer Developed with Monoclonal 7 Antibodies Specific to Barley Lipid Transfer Protein 8 1 2 3 4 Yukie Murakami-Yamaguchi , Junko Hirose , Kumiko Kizu , Fumiko Okazaki , 9 5 4, Wataru Fujii and Hiroshi Narita * 10 Kyoto College of Nutritional & Medical, Setogawa-cho 18, Saga Tenryuji, Ukyo-ku, 11 Kyoto 616-8376, Japan; E-Mail: yukie.yamaguchi@taiwa.ac.jp 12 Department of Food and Nutrition, School of Human Cultures, The University of Shiga Prefecture, 13 Hassaka-cho 2500, Hikone, Shiga 522-8533, Japan; E-Mail: jhirose@shc.usp.ac.jp 14 Department of Life and Living, Osaka Seikei College, Aikawa 3-10-62, Higashiyodogawa, 15 Osaka 533-0007, Japan; E-Mail: kizu@osaka-seikei.ac.jp 16 Department of Food and Nutrition, Kyoto Women’s University, Kitahiyoshi-cho 35, Imakumano, 17 Higashiyama-ku, Kyoto 605-8501, Japan; E-Mail: k1134201@kyoto-wu.ac.jp 18 Suntory Business Expert Limited, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, 19 Japan; E-Mail: Wataru_Fujii@suntory.co.jp 20 * Author to whom correspondence should be addressed; E-Mail: narita@ kyoto-wu.ac.jp; 21 Tel./Fax: +81-75-531-7154. 22 Received: 17 September 2012; in revised form: 28 September 2012 / Accepted: 9 October 2012 / 23 Published: 16 October 2012 24 Abstract: Non-specific lipid transfer protein (LTP) in barley grain reacted with the IgE in 26 sera drawn from food allergy patients. A sandwich-type of enzyme-linked immunosorbent 27 assay (ELISA) was developed with mouse monoclonal antibodies raised against LTP 28 purified with barley flour. This ELISA showed a practical working range of 0.3–3 ng/mL 29 and no cross-reactivity with wheat, adlay and rye. Using this ELISA, LTP was determined 30 in several types of barley-foods, including fermented foods such as malt vinegar, barley- 31 malt miso and beer. LTP content in beer of the same kind was approximately constant, 32 even if manufacturing factory and production days were different. Not only as a factor of 33 foam formation and stability but also as an allergen, controlling and monitoring of LTP in 34 beer should be considered. Taken together, our LTP-detecting ELISA can be proposed as 35 an appropriate system for the quality control of beer. 36 37 Antibodies 2012, 1 260 Keywords: lipid transfer protein; beer; ELISA; food allergy; monoclonal antibody 1 1. Introduction 3 Non-specific lipid transfer protein (LTP) is an ubiquitous 9-kDa protein widely present in the plant 4 kingdom. Its lipid binding property primary postulates the role of LTP in the intracellular transport of 5 lipids [1,2]. However, it is now known that LTP is synthesized as a precursor with a typical N-terminal 6 signal peptide and the mature LTP is secreted outside the cell [1,3,4]. Therefore, the former idea about 7 lipid transportation by LTP has been abandoned. While LTP 2 with a size around 7-kDa is also present 8 as a minor component, we will treat LTP 1 as LTP in this study, hereafter [2]. The highest expression of 9 LTP has been found in peripheral cell layers surrounding aerial organs, associated with the cell wall 10 and cuticle of epidermal tissues. Stress factors, such as drought, cold and high salinity have been described 11 to upregulate the expression of LTP in plants [1,5–7]. Many reports have mentioned the physiological 12 role of LTP in the protective mechanism against the attack of bacteria, fungi and viruses [8–11]. 13 Accordingly, LTP has been classified as pathogenesis-related protein 14 (PR-14) [12]. 14 LTP has been confirmed as to its involvement in the human allergic reaction to fruits, vegetables, 15 nuts, and even pollens [13–17]. LTP is composed of eight cysteine residues forming a network of four 16 disulfide bridges. These disulfide bonds provide stability in the structure of LTP to heat treatment and 17 proteolytic digestion, which is the characteristic feature of food allergen. In addition, the lipid binding 18 domain of LTP also plays an important role in its allergenic property [2,18,19]. Furthermore, LTP is 19 known to be a panallergen according to its highly homologous nature in amino acid sequence and 3D 20 structure among various plants [2,20,21]. Since barley LTP (bLTP) is also resistant to heat treatment 21 and proteolytic digestion, bLTP, as well as protein Z, remains in beer and contributes to foam 22 formation and stability of beer [22,23]. It is noteworthy that LTP performs higher allergenicity than 23 protein Z according to the result of prick test [15,24,25]. 24 Globally, food manufacturers have faced the growing of consumers’ concerns regarding the 25 presence of allergens in their products. Japanese Ministry of Health, Labor and Welfare made the first 26 decision in the world for the mandatory labeling of several foods containing allergenic ingredients in 27 2002 from the viewpoint of preventing the occurrence of health hazard [26]. Development of the 28 proper analytical method for the determination of allergens in foods is necessary in order to ensure the 29 quality, safety and fair trade of foods for consumers. Enzyme-linked immunosorbent assay (ELISA) is 30 well known, and is widely utilized as a method for determining the presence of specific proteins in 31 processed foods [26]. In this study, we have developed ELISA with monoclonal antibodies (mAbs) 32 specific to bLTP and proposed its application in quality controlling of beer production. 33 2. Results and Discussion 34 2.1. Barley Grain LTP 35 Barley LTP (bLTP) was purified from commercial barley grain flour by using (NH ) SO 36 4 2 4 precipitation, hydrophobic chromatography and cationic exchange chromatography. Corresponding to 37 Antibodies 2012, 1 261 the position on SDS-PAGE, the homogenous protein with a molecular mass about 9-kDa was obtained. 1 To clearly identify whether this protein is bLTP, the N-terminal amino acid sequence of this protein 2 was analyzed. The result showed the eight amino acid sequence LN(X)GQVDS, that was identical to 3 the first eight amino acid sequence of bLTP reported by Douliez et al. [2]. Thus, the purified 9-kDa 4 protein was bLTP, as confirmed by its apparent molecular mass and a part of amino acid sequence 5 (Figure 1). The concentration of purified bLTP was determined by absorbance assay (280 nm) and 6 TM TM DC protein assay [27] using BSA as a standard protein. According to the result of DC protein 7 assay, concentration of bLTP was three times higher than that determined by measuring absorbance at 8 280 nm (E = 10). The difference in concentration determined by these two methods might be due to 9 1% the lack of tryptophan and small amount of tyrosine (three residues) in the amino acid sequence of 10 bLTP. The purified bLTP showed lipid transfer activity measured according to the Paridon’s 11 method [28], suggesting no changing in bLTP structure by purification process (data not shown). This 12 pure bLTP, obtained as described, was used here for further investigation. 13 Figure 1. Deduced amino acid sequences of barley seed and leaf lipid transfer proteins 14 (LTPs) and wheat seed LTP from their nucleotide sequences (NLT1_HORVU, 15 NLT3_HORVU, and NLTA_WHEAT). The blue highlight indicated the homologous 16 amino acid in the alignment of these amino acid sequences. 17 5 10 15 20 barley （seed） L N C G Q V D S K M K P C L T Y V Q G G barley （leaf） A I S C G Q V S S A L S P C I S Y A R G N wheat （seed） I D C G H V D S L V R P C L S Y V Q G G 25 30 35 40 barley （seed） - P G P S G E C C N G V R D L H N Q A Q barley （leaf） G A K P P V A C C S G V K R L A G A A Q wheat （seed） - P G P S G Q C C D G V K N L H N Q A R 45 50 55 60 barley （seed） S S G D R Q T V C N C L K G I A R G I H barley （leaf） S T A D K Q A A C R C L K S L A T S I K wheat （seed） S Q S D R Q S A C N C L K G I A R G I H 65 70 75 80 barley （seed） N L N L N N A A S I P S K C N V N V P Y barley （leaf） G I N M G K V S G V P G K C G V S V P Y wheat （seed） N L N E D N A R S I P P K C G V N L P Y 85 90 barley （seed） T I S P D I D C S R I Y barley （leaf） P I S M S T D C N K V H wheat （seed） T I S L N I D C S R V 2.2. Allergenicity of bLTP 19 Human sera possessing IgE of barley, wheat, or apple obtained from 10 food allergy patients (Table 1) 20 were tested individually to determine IgE reactivity to bLTP by immunoblotting analysis (Figure 2). 21 The sera No. 5 and 10 showed no reactivity to bLTP in the extract. On the other hand, the high 22 Antibodies 2012, 1 262 reactivity to bLTP in the extract was detected in sera No. 1–4 and 6–9. Despite other protein bands at the 1 molecular mass of 14, 28 and 60-kDa also were present in the extract and showed similar band intensity 2 as that of bLTP on protein staining; a high intensity of the IgE-binding band was only observed at the 3 position of bLTP. This result indicates the importance of bLTP as the major allergen in barley food 4 products. Thus, preventing bLTP contamination in food processing system should be seriously concerned. 5 Table 1. Characteristics of sera from patients with food allergy. 6 Barley Wheat Apple Patient Sensitization No. IgE kU/L 1 1.2 - 6.4 wheat products, nuts, beans 2 9.8 33.1 25.6 egg white 3 15.8 14.3 14.4 wheat products, nuts, beans 4 - - 11.7 apple, pear, carrot, pollen 5 25.6 46.8 19.2 peach, apple, pear, fish, meat 6 9.5 17.8 0.9 nuts, beans 7 5.6 5.7 1.2 grass pollen 8 5.5 5.4 4.7 fish 9 6.4 2.9 - plum, dates 10 - - 11.5 milk products, egg, nuts, beans -: not determined. 7 Figure 2. The reactivity of sera IgE obtained from food allergic patients with proteins in 8 barley extract. (A) SDS-PAGE pattern of molecular mass markers (a), 8 µg of PBS- 9 extracted barley proteins (b), and 2 µg of barley LTP (bLTP) (c). (B) Western blotting 10 pattern of PBS-extracted barley proteins (8 µg) with sera from food allergic patients 11 (No. 1–10). IgE was visualized with alkaline phosphatase-conjugated anti-human IgE. 12 The IgE characteristics of sera from allergic patients are shown in Table 1. 13 Antibodies 2012, 1 263 2.3. Preparation and Characterization of Monoclonal Antibodies to bLTP 1 Thirteen mice were immunized with bLTP, and the spleen cells from mice showed high anti-bLTP 2 titer were fused with myeloma cells. Four positive clones, 5-4, 10-1, 10-4, and 12-3 screened by an 3 indirect ELISA possessed high specificity to bLTP. Using a mouse mAb isotyping kit, it was shown 4 that the mAbs produced by these clones were all IgG1/κ. 5 The specificity of these four anti-bLTP mAbs to LTP from other cereal grains was examined by 6 immunoblotting analysis. After separation by SDS-PAGE, protein bands corresponding to LTP at 7 around 9-kDa were detected in extracts from barley, wheat, adlay, and rye (Figure 3A). Barley LTP 8 was clearly detected by western blotting analysis with all four mAbs (Figure 3B–E). MAbs 5-4, 10-1, 9 and 10-4 showed negative reactivity to the extracts from other sources. While, weak cross-reaction of 10 mAb 12-3 with LTP from rye was observed (Figure 3E). The minor cross-reaction of mAb 12-3 with 11 rye LTP might contribute to homologous amino acid sequences of its epitope in barley and rye. These 12 mAbs exhibited no cross-reactivity against extracts from the major allergenic foods, such as eggs， 13 milk peanuts, shrimp, and crab (data not shown). MAbs reactive with bLTP and their epitopes were 14 previously revealed [29]. However, their cross-reactivity to other LTPs and application for quantitative 15 analysis were unknown. 16 Figure 3. Cross reactivity of anti-bLTP monoclonal antibodies with other cereal grain 17 LTPs. (A) SDS-PAGE pattern of molecular mass markers (a), 2 µg of bLTP (b), and 8 µg 18 of PBS-extracted proteins from barley ①, wheat ②, adlay ③, and rye ④. (B–E) Western 19 blotting pattern of PBS-extracted barley proteins (8 µg) with various established anti-bLTP 20 monoclonal antibodies (mAbs). (B), 5-4; (C), 10-1; (D), 10-4; (E) 12-3. = 21 2.4. Sandwich ELISA for the Determination of bLTP 23 For the quantitative analysis of bLTP with high sensitivity, we developed sandwich ELISA systems 24 with these mAbs. Each established mAb was used as the first antibody and peroxidase-conjugated 25 Antibodies 2012, 1 264 counterparts were used as the second antibody. As shown in Figure 4, six different systems were 1 capable of detecting bLTP. The system that uses mAb 10-4 as the first antibody and peroxidase- 2 conjugated mAb 5-4 as the second antibody was identified as the highest sensitive system for bLTP 3 quantitative analysis (Figure 4C). The practical working range was 0.3–3 ng/mL. The sensitivity of this 4 system to wheat LTP was 1/300,000 times less than that to bLTP (Figure 5). Thus, this sandwich 5 ELISA system is significantly sensitive and specific to bLTP. 6 Figure 4. Sandwich enzyme-linked immunosorbent assays (ELISAs) for the quantitative 7 analysis of bLTP. Sandwich ELISAs were constructed with mAb 5-4 (A), 10-1 (B), 10-4 8 (C), or 12-3 (D) as a solid-phase antibody and peroxidase-conjugated mAb 5-4 (●), 10-1 (○), 9 10-4 (▲), or 12-3 (△) as a second antibody. 10 The 10-4/5-4 system was then applied for the determination of bLTP in several extracts prepared 12 from barley or barley food products (Table 2). Barley is a major food and animal feed source. Two 13 kinds of barley are widely used in food industry, two-row barley (Hordeum distichum) and six-row 14 barley (Hordeum vulgare). The former from which we prepared bLTP is a traditional one for brewing, 15 while the latter is used well as roasted barley grain, a raw material for a popular Asian tea. Comparable 16 bLTP contents were obtained in the materials of both cultivars. However, bLTP could not be 17 determined in leaf by this method probably due to the difference in amino acid sequence between grain 18 and leaf LTP (Figure 1) [2]. Compared to those in raw materials, bLTP contents in processed barley 19 foods were extremely low. In spite of stable structure of LTP suggesting high temperature tolerant 20 characteristic, some denaturation of LTP at 96 ° C was reported by Nierop et al. [23]. In addition, 21 Antibodies 2012, 1 265 extraction efficiency might be lowered by heat-induced aggregation of components. Therefore, the low 1 amount of bLTP detected in such processed barley foods could be contributed to these artificial factors. 2 We have reported allergenicity of heat-denatured ovomucoid, a major egg allergen, which shows rigid 3 conformation due to disulfide bonds and possesses heat stability similar to LTP [30]. Then, allergen 4 had better to be estimated as total amounts irrespective of the degree of heat denaturation [31]. 5 However, the 10-4/5-4 system showed several times lower reactivity to heated bLTP at 100 °C for 6 10 min than native bLTP (data not shown). 7 Figure 5. Reactivity of the 10-4/5-4 sandwich ELISA system to barley and wheat LTPs. 8 Barley (●) or wheat LTP (○) was determined by the sandwich ELISA, constructed with mAb 9 10-4 as a solid-phase antibody and peroxidase-conjugated mAb 5-4 as a second antibody. 10 Table 2. Barley LTP content. 12 Food µg/g (mL) Two-row barley flower 393.7 Two-row barley flower 301.3 Barley leaf - Hattaiko * 0.1 Roasted barley grain ** - Barley miso *** 18.3 Malt vinegar 21.0 beer 2.6 -: not detected; * Roasted and grounded barley flour used for Japanese sweets; ** Raw material for 13 popular Asian tea; *** Soybean paste made with barley malt. 14 ELISA screening is one of the official detection method to identify the allergenic ingredient in food 15 by the Japanese official government [26]. Ovalbumin is selected as the target to detect egg, casein and 16 β-lactoglobulin to detect milk, protein complex containing Ara h2 to detect peanut, and gliadin to 17 detect wheat. In foods such as soybean paste, vinegar, and beer that wheat is used as one of the 18 important ingredients, however, it is a serious problem that gliadin is hydrolyzed by proteolytic 19 Antibodies 2012, 1 266 enzyme during fermenting process [32]. The fact that LTP is highly stable toward proteolysis attracted 1 us an interest in utilization of wheat LTP as a substitute target protein in detection of wheat content in 2 fermented food instead of gliadin [33]. In the same context, it was notable that bLTP was detected in 3 fermented foods such as barley-malt miso, malt vinegar, and beer in Table 2. 4 2.5. Quantitative Analysis of bLTP in Beer 5 Barley LTP is a major proteinaceous component in beer [15,22–24]. Besides being an allergenic 6 agent, it is also recognized as a foaming agent in brewing industry. Therefore, routine analysis of bLTP 7 in product might be used to assist the quality controlling of beer production. In this study, we 8 introduced the ELISA 10-4/5-4 system for assessing the quality of beer. 9 Three different brewing processes produce beers of the world. Bottom fermentation, generally used 10 in production of beer in Japan, Germany, and the Netherlands, is performed at 4–13 °C for 4–6 weeks. 11 Beer is brewed at 20 ° C for two weeks in top fermentation, a brewing process widely used in United 12 Kingdom and Belgium. Spontaneous fermentation, widely done in Belgium, is carried out with wild 13 type yeast. Figure 6 shows that the amount of bLTP in beer samples was inconstant (1–11 μg/mL), 14 independently of the brewing process and the producing country. Accordingly, variation in LTP 15 contents is due to the difference in raw materials and detail of processing and might determine the 16 character and quality of each beer. 17 Figure 6. Barley LTP levels in beer samples prepared by three different brewing processes. 18 Concentration of bLTP was determined by the 10-4/5-4 sandwich ELISA system. Ⅰ, bottom 19 fermentation; Ⅱ, top fermentation; Ⅲ, spontaneous fermentation. No. 1–7, Japanese products; 20 8, English product; 9, German product; 10, Irish product; 11–17, Belgian products. 21 Four kinds of beer (A to D) manufactured in four different factory (①–④) and different production 23 days were monitored for bLTP level using the ELISA 10-4/5-4 system (Figure 7). All of the brewing 24 processes in each factory had been performed perfectly in every day that the samples were taken. 25 Analysis of the resultant beers indicated that there was no remarkable difference in the bLTP content 26 of final products from different days in the same factory. The difference between bLTP content in beer 27 from different lots of production is capable of being used to indicate the unusual producing condition 28 Antibodies 2012, 1 267 such as quality of raw materials and is a good predictor of the different taste of beer. After the claim of 1 a customer, a canned beer B was reported for induction of urticaria. However, this ELISA system 2 failed to distinguish between bLTP content in beer of the claimed lot and that of other normal lots 3 (Figure 7B, X). In default of any bizarreness in bLTP level, it is confirmed that the claimed lot of beer 4 was produced by a proper brewing process. Thus, determining bLTP level in beer product by this 5 ELISA gives valuable information for assuring the quality of beer to customer with little investment of 6 time and money. 7 Figure 7. Comparison of bLTP content in beer between different lots of production. Four 8 kinds of beers (A–D) produced in four different factories (①–④) and different days were 9 obtained. The amount of bLTP was determined by the 10-4/5-4 sandwich ELISA system. 10 ① 10/1 indicates that it was produced in factory ① on October 1st. (X) indicates the same 11 lot product that was claimed by a customer. 12 Controlling and monitoring bLTP content in beer is important, as bLTP is a factor for foam 14 formation and stability and also as an allergen. This study introduces the sandwich ELISA using mAbs 15 specific to bLTP as a useful tool for quality control and authenticity assessment of beer. Barley LTP 16 determination by this ELISA might also be used to indicate the physical characteristic and taste of beer 17 as well. For future experiment, the correlation of bLTP content in beer product with wort boiling 18 temperature, fermenting method, addition of malt and brewing process will be determined. Moreover, 19 bLTP might be indicated as a target protein in assessing of barley content in order to assist quality 20 control of barley-fermented foods. Further information in application of bLTP in quality control system 21 could provide the valuable information in novel product development in food and beverage industry. 22 Antibodies 2012, 1 268 3. Experimental Section 1 3.1. Reagents and Apparatus 2 The following materials were obtained from the indicated sources: cell fusion and cell culture 3 reagents, fetal bovine serum (FBS), and polyethylene glycol 1500 (Invitrogen, Carlsbad, California, 4 USA), RPMI 1640 medium (Nacalai Tesque Inc., Kyoto, Japan), hypoxanthine thymidine (HT), 5 hypoxanthine aminopterin thymidine (HAT) reagents and B cell growth factor, BriClone (Dainippon 6 Pharmaceutical Co. Ltd., Osaka, Japan), Freund’s complete and incomplete adjuvants (Difco 7 Laboratories, Detroit, MI, USA), Protein G Sepharose and monoclonal antibody isotyping kit (GE 8 Healthcare, Buckinghamshire, England), Maxisorp microplate for ELISA (Nunc, Roskilde, Denmark), 9 TM DC protein assay kit (BIO-RAD laboratory, Bradford, UK), pristine (Sigma Chemical Corp, St. Louis, 10 MO, USA), alkaline phoshatase-conjugated anti-mouse IgG and anti-human IgE (American Qualex, 11 San Clemente, CA, USA). Sera from 10 patients allergic to food were supplied by PlasmaLab (Everett, 12 WA, USA). Unless specified otherwise, all other chemicals used were guaranteed reagent grade. 13 3.2. Isolation of Barley Grain LTP 14 Barley grain flour (80 g) was mixed with 3 fold volumes of water. The mixture was then stood 15 overnight at room temperature. After centrifugation at 10,000 × g, 4 °C for 20 min, the supernatant 16 was collected as barley extract. Barley extract was further subjected to (NH ) SO fractional 17 4 2 4 precipitation by adding (NH ) SO to 30% saturation, and then the pellet was discarded. Subsequently, 18 4 2 4 (NH ) SO was added to the supernatant to 80%. Following precipitation, the pellet was resuspended in 19 4 2 4 1.2 M (NH ) SO . The pH of the solution was adjusted to 7.0 using 20 mM phosphate buffer, 20 4 2 4 beforehand, and this solution was applied to a Toyopearl butyl column (2.5 × 10 cm) (Tosoh, Tokyo, 21 Japan) equilibrated with 1.2 M (NH ) SO . Proteins were eluted from the column by a linear gradient 22 4 2 4 of 1.2 to 0 M (NH ) SO . Relevant fractions were identified by the presence of a band at 9-kDa on 23 4 2 4 sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels under reduced 24 condition. These fractions were pooled and dialyzed overnight against 20 mM phosphate buffer, pH 25 5.0 (PB) using a dialysis tube (MWCO 3500 Da, Spectrum Laboratories, Rancho Dominguez, CA, 26 USA). Then, the sample was applied to a Toyopearl CM column (1.6 × 5 cm) (Tosoh, Tokyo, Japan) 27 equilibrated with PB. Proteins were eluted with a linear gradient of 0 to 0.3 M NaCl in PB. LTP 28 containing fractions were collected. The purity of protein was determined by SDS-PAGE. Purified 29 LTP was dialyzed against water overnight, lyophilized, and stored at −20 °C, until use. Wheat LTP 30 TM was purified as previously described [33]. Protein concentration was determined by DC protein 31 assay using bovine serum albumin as a standard protein. 32 3.3. Electrophoresis and Western Blotting 33 SDS-PAGE was performed by the method of Laemmli [34] by using 10% gel. Sample was dissolved 34 in 20 mM Tris-HCl buffer (pH 6.8) containing 2% (w/v) SDS and 10 mM 2-mercaptoethanol, and 35 heated for 5 min in boiling water. After electrophoresis, the gel was stained with Commassie Brilliant 36 Blue R-250. The protein with molecular mass of 9-kDa was identified as LTP. 37 Antibodies 2012, 1 269 Extracts from barley, wheat, adlay, and rye, with 8 µg protein content, were applied to gel electrolysis. 1 After been performed on SDS-PAGE, the proteins in a gel were transblotted to a polyvinylidene 2 difluoride (PVDF) membrane. The membrane was transferred to a blocking solution (PBS containing 3 5% skim milk) for 60 min, and then incubated with the anti-bLTP mAb at room temperature for 1 h. 4 After washing with 10 mM Tris buffer, pH 7.4 containing 0.15 M NaCl (TBS) several times, the 5 membrane was incubated with alkaline phosphatase-conjugated anti-mouse IgG at room temperature 6 for 1 h. After extensively washing again with TBS, protein band on the membrane was developed with 7 alkaline-phosphatase kit. When human sera were tested, alkaline phosphatase-conjugated anti-human 8 IgE was used. 9 3.4. N-Terminal Amino Acid Sequencing 10 The N-terminal amino acid sequence of purified LTP electrophoretically transferred to a PVDF 11 membrane was analyzed by Edman degradation using Applied Biosystem Procise 491 HT Protein 12 Sequencer (Foster City, CA, USA). The amino acid sequence obtained was compared with that of 13 bLTP in public database. 14 3.5. Production of Monoclonal Antibody 15 Thirteen eight-week old female BALB/c mice (Nippon SLC Co., Shizuoka, Japan) were immunized 16 by intraperitoneal injection of 100 µg of bLTP emulsified in Freund’s complete adjuvant. At two and 17 four weeks after the initial immunization, the mice were boosted by intraperitoneal injection of 50 µg 18 of bLTP emulsified in Freund’s incomplete adjuvant. The final booster was given by intraperitoneal 19 injection of 10 µg LTP in 10 mM phosphate buffer, pH 7.4 containing 0.15 M NaCl (PBS) at the sixth 20 week. On the third day after the final injection, the spleen cells were harvested from the mice and then 21 fused with the mouse myeloma cell (P3U1) [35]. Fused cells were seeded in 96-well culture plates and 22 incubated at 37 °C in HAT medium. After 2 weeks in a CO incubation, the culture supernatant of each 23 hybridoma was examined for reactivity with bLTP by an indirect ELISA. Hybridoma from the well 24 that perform high reactivity with antigen were subcloned twice by limiting dilution on HT medium 25 containing 5% BriClone. Each clone secreting antibody to bLTP was grown in mass culture. Subclass 26 of immunoglobulin in culture supernatant of each clone was identified by a mouse mAb isotyping kit. 27 Cloned cells were injected intraperitoneally into a 10-week old mouse treated with pristine. The 28 monoclonal antibody was purified by using a protein G affinity column from the ascitic fluid. The 29 purified antibody was conjugated with horseradish peroxidase [36]. Animal experiments were carried 30 out under the guideline of Animal Experiment Committee of Kyoto Women’s University following the 31 bulletin (No.71, 2006) of the Ministry of Education, Culture, Sports, Science, and Technology in Japan. 32 3.6. Preparation of Extract for LTP Determination 33 Milled cereal grain powder (1 g) was briefly homogenized with 19 mL PBS. After being shaken 34 overnight, the homogenate was centrifuged at 3,000 × g, 4 °C for 20 min. The supernatant was 35 collected and further filtrated through a filter paper No.2. The clear filtrate was used as cereal grain 36 extract to determine the LTP content in further experiment. There was no special treat for beer sample. 37 Antibodies 2012, 1 270 3.7. Enzyme-Linked Immunosorbent Assay (ELISA) 1 For indirect ELISA, each well of 96-well flat-bottom polystyrene microplate was coated with 50 µL 2 per well of 2 µg/mL bLTP. After overnight incubation at 4 °C, the unbound site was blocked by 3 incubation with 200 µL per well of blocking reagent (10% BSA in PBS) at 37 °C for 1 h. Media fluid 4 of hybridoma culture (50 µL) was added into wells and incubated at 37 °C for 1 h. After washing with 5 TBS containing 0.02% tween 20 (TBS-T), 100 µL of anti-mouse IgG conjugated to alkaline 6 phosphatase diluted in TBS-T containing 0.1% BSA was added and incubated at 37 °C for 1 h. After 7 washing, 100 µL of chromogenic substrate (1 mg/mL ρ-nitrophenylphosphate and 0.5 mM MgCl in 8 10% diethanlamine buffer, pH 9.8) was added and incubated at 37 °C for 1 h. The intensity of the 9 resulting color was detected at 415 nm by a microplate reader. For sandwich ELISA, each well of the 10 plate was coated with 50 µL of 5 µg/mL of anti-bLTP mAb in coating buffer at 37 °C for 1 h. After 11 blocking and three washing, 50 µL of barley extract (0–100 ng/mL) or beer sample was add to each 12 well of the coated plate, and incubated at 37 °C for 1 h. Then, to each washed well 50 µL of 13 peroxidase-conjugated anti-bLTP mAb was added. After incubated at 37 ° C for 1 h, the substrate 14 solution was added, developed, and finally the absorbance at 450 nm was measured as described above. 15 4. Conclusions 16 A sandwich-type of enzyme-linked immunosorbent assay was developed with mouse monoclonal 17 antibodies raised against LTP purified from barley grain flour. LTP is known to be a factor in foam 18 formation and stability in beer, while also being an allergen. 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