Characterization of Monoclonal Antibody Specific to Fish Major Allergen Parvalbumin Kamil Gajewski

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2008 Characterization of Monoclonal Antibody Specific to Fish Major Allergen Parvalbumin Kamil Gajewski

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FLORIDA STATE UNIVERSITY

COLLEGE OF HUMAN SCIENCES

CHARACTERIZATION OF MONOCLONAL ANTIBODY SPECIFIC TO

FISH MAJOR ALLERGEN – PARVALBUMIN

KAMIL GAJEWSKI

A Thesis submitted to the Department of Nutrition, Food and Exercise Sciences in partial fulfillment of the requirements for the degree of Master of Science

Degree Awarded: Summer Semester, 2008

The members of the Committee approve the Thesis of Kamil Gajewski defended on June 23, 2008.

______Yun-Hwa Peggy Hsieh Professor Directing Thesis

______Kenneth H. Roux Outside Committee Member

______Cathy W. Levenson Committee Member

Approved:

______Bahram H. Arjmandi, Chair, Department of Nutrition, Food and Exercise Sciences

______Billie J. Collier, Dean, College of Human Sciences

The Office of Graduate Studies has verified and approved the above named committee members.

ACKNOWLEDGEMENTS

I am grateful for all the support I have received during preparation of this thesis. I especially would like to thank my advisor, Dr. Yun-Hwa Peggy Hsieh, for insightful conversations during the development of the ideas in this thesis, for helpful comments on the text, and for keeping me focused in my research. Without her encouragement and contribution this work would not have been completed.

I would also like to thank Dr. Cathy Levenson and Dr. Kenneth Roux for agreeing to be on my thesis committee despite their extremely busy schedule.

I want to thank all of my colleagues in Dr. Hsieh’s laboratory for the support, advice, and friendship. Qinchun Rao, Jack Ofori, Yi-Tien Chen and Marsha Fridie made the laboratory a wonderful place to work.

Special thanks to my mom and uncle who supported my dreams and aspirations. Finally, I also want to thank my wife, Iwona, for her patience and for helping me keep my life in proper perspective and balance. Her faith in me helped me to overcome all difficulties.

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TABLE OF CONTENTS

List of Tables ...... vi List of Figures ...... vii Abstract ...... viii

1. Introduction ...... 1

2. Literature Review...... 4

2.1 Fish Allergy ...... 4 2.2 Major Fish Allergen - Parvalbumin ...... 5 2.3 Other Fish Allergens ...... 6 2.4 Fish Allergen Contamination ...... 7 2.5 The Food Allergen Labeling and Consumer Protection Act ...... 9 2.6 Allergen Detection Methods ...... 10 2.6.1 Allergen protein-based detection methods ...... 10 2.6.1 Allergen DNA-based detection methods ...... 12

3. Hypotheses and Objectives ...... 14

3.1 Hypotheses ...... 14 3.2 Objectives ...... 14

4. Materials and Methods ...... 15

4.1 Materials ...... 15 4.2 Protein extraction from fish and meat samples ...... 16 4.3 Indirect ELISA ...... 16 4.4 Ca2+ dependent binding assay ...... 17 4.4 Epitop comparison ...... 17 4.6 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western blot ...... 18

5. Results and Discussion ...... 19

5.1 Specificity of MAbs 3E1 and PARV-19 ...... 19 5.2 Thermal-stable protein profile in cooked fish extracts ...... 20 5.3 Antigenic protein for MAb 3E1 ...... 21 5.4 Ca2+ sensitive epitopes ...... 22 5.5 Epitope comparison ...... 23

6. Conclusions ...... 25

APPENDICES ...... 26

A Protocols and Procedures ...... 26 B Figures and Tables ...... 30

REFERENCES ...... 38

BIOGRAPHICAL SKETCH ...... 45

LIST OF TABLES

Table 1: Immunoreactivity of MAbs 3E1 and PARV-19 against cooked shellfish, meat, poultry and food additives samples determined by indirect ELISA ...... 30

Table 2: Immunoreactivity of MAbs 3E1 and PARV-19 against cooked fish samples determined by indirect ELISA ...... 31

LIST OF FIGURES

Figure 1: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) profiles of 20 cooked fish species ...... 32

Figure 2: Antigenic protein banding patterns in cooked fish extracts by Western blot analysis, using MAbs 3E1PARV-19 by Western blot analysis ...... 33

Figure 3: Comparison of the antigenic protein banding patterns in cooked fish extracts using MAbs 3E1 and PARV-19 by Western blot ...... 34

Figure 4: Effect of presence and absence of calcium on the immunoreactivity of anti-parvalbumin specific MAbs 3E1 with fish and meat extracts determined by indirect ELISA ...... 35

Figure 5: Effect of presence and absence of calcium on the immunoreactivity of anti-parvalbumin specific MAbs PARV-19 with fish and meat extracts determined by indirect ELISA ...... 36

Figure 6: Epitope comparison of MAbs 3E1 and PARV-19 ...... 37

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ABSTRACT

Fish allergy is a worldwide problem, especially in industrialized countries where fish consumption is high. Fish contains a wide variety of proteins but, only few of them are responsible for triggering an allergic reaction. The major fish allergen, parvalbumin, is a low molecular weight (10-13 kD) heat-stable protein. High homology in amino acid sequences and antibody cross-reactivities have been demonstrated for parvalbumin in different fish species. Although several detection methods based on specific antibodies or DNA amplification are currently employed for detection of allergic components in food products, there is limited number of studies reporting methods for the detection of fish allergens. This study aimed to investigate the antigen binding characteristics of a monoclonal antibody (MAb) 3E1 by comparing its immunoreactivity against various fish and other animal species with a commercially available anti-frog parvalbumin monoclonal antibody (PARV-19) to evaluate the usefulness of MAb 3E1 as a anti-fish parvalbumin reagent. MAb 3E1 was previously developed in our laboratory against heat-treated catfish crude sarcoplasmic protein extract. The antigen binding characteristics of this antibody was investigated by comparing its immunoreactivity against soluble proteins extracts from various cooked fish and other animal meats with MAb PARV-19. Non-competitive indirect ELISA was performed to examine the immunoreactivity of both MAb 3E1 and MAb PARV-19 with sample extracts. Western blot was performed to compare the antigenic protein banding patterns in cooked fish extracts using these two MAbs. Results showed that MAb 3E1 cross reacted with majority of tested fish species and recognized a thermal-stable protein with a molecular weight range of parvalbumin in the extracts. Moreover, ELISA and western blot results revealed that both MAbs 3E1 and PARV-19 had almost identical reaction patterns to the fish species tested. The antigenic protein banding pattern in various fish species blotted by MAb 3E1 corresponds to the molecular weights of parvalbumins recognized by PARV-19. Additionally, both antibodies recognized exactly the same antigenic protein, parvalbumin, but their epitopes (binding sites) overlaped to the extend causing inhibitive binding on the protein. However, screening with non-finfish extracts revealed MAb 3E1 to be strictly finfish specific, while PARV-19 cross-reacted with frog, rat and rabbit extracts. The results obtained in this study clearly indicate that MAb 3E1 is specific to fish parvalbumin. It would, therefore, be a useful probe for investigating the major fish allergen in both raw and processed food.

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1. INTRODUCTION

Fish is becoming an increasingly important food source due to its high consumption and its dietary values, namely high quality proteins, beneficial polyunsaturated fatty acids and lipid- soluble vitamins. However, together with wheat, soy, cow’s milk, peanuts, tree nuts, shellfish, and eggs, fish is one of the eight major allergenic foods or food types that cause immunoglobulin E (IgE)-mediated food allergy in humans (Bahna 2004; FDA 2005; Kobayashi, Tanaka et al. 2006; Sicherer and Sampson 2006). Food allergies are becoming an increasing problem in industrialized countries. The prevalence of fish allergy in the United States was determined to be 0.4% of food-hypersensitive patients among the adult population. Approximately 1.1 million Americans suffer from fish allergy, and this number is increasing (Sampson 1999; Sicherer, Munoz-Furlong et al. 2004). Symptoms of fish allergy, which are similar to those of other IgE- mediated food allergies, usually appear immediately after exposure (minutes to an hour). Clinical symptoms in fish-allergic patients might comprise acute urticaria, atopic dermatitis, asthma, gastrointestinal disorders (diarrhea, vomiting) and in some cases even life-threatening anaphylaxis (O'Neil, Helbling et al. 1993; Swoboda, Bugajska-Schretter et al. 2002; Van Do, Hordvik et al. 2005). Major allergens generally are defined as proteins for which 50% or more of the allergenic patients tested have specific IgE (King, Hoffman et al. 1994). Although fish contains a wide variety of proteins, only few of them cause an allergic reaction (Lehrer, Horner et al. 1996). The first purified and characterized fish allergen was Gad c 1, a codfish parvalbumin (Aas and Elsayed 1975; Elsayed and Bennich 1975). Parvalbumins are considered the major allergen in fish because more than 95% of fish-allergic patients have been found to have specific IgE to this protein and many of the IgE-binding eptiopes on this allergen are present in various fish species (de Martino, Novembre et al. 1990; Bugajska-Schretter, Grote et al. 2000). Parvalbumins are a family of calcium-binding proteins that play an important role in muscle relaxation (Heizmann, Berchtold et al. 1982; Muntener, Kaser et al. 1995). They have low molecular weights of approximately 10-13 kDa and acidic pI values, and are water soluble and resistant to heat treatment as well as enzymatic degradation (Elsayed and Aas 1971; Aas and Elsayed 1975). Parvalbumins are present in relatively high quantities in the muscles of lower vertebrates, such as fish, and in lesser amounts in higher vertebrates, including human (Gerday 1982). The

quantity of parvalbumin also varies in different types of fish muscles; white muscle generally contains more parvalbumin than dark muscle, which makes the dark muscle tissue of fish much less allergenic than the white muscle tissue (Rehbein and Kundiger 1984; Kobayashi, Tanaka et al. 2006). Based on a comparison of their amino acid sequences, parvalbumins are subdivided into phylogenetic lineages α and β. The α-parvalbumins have isoelectric points at or above pI 5.0, while the β-parvalbumins, containing more acidic amino acids, have a pI value of 4.5 or lower (Pechere, Capony et al. 1973; Goodman, Pechere et al. 1979). Members of both lineages have been identified in a number of fish species, including thornback ray, carp, mackerel, cod, salmon and Alaska pollack (Thatcher and Pechere 1977; Lindstrom, van Do et al. 1996; Swoboda, Bugajska-Schretter et al. 2002; Hamada, Tanaka et al. 2003; Van Do, Elsayed et al. 2005). The Food and Drug Administration (FDA) requires manufacturers to declare on the label of packaged foods the use of any allergenic food or any ingredients derived from these allergenic foods in compliance with the Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA) (FDA, 2005). This new regulation has created the need for a rapid test capable of detecting various fish species as a potential source of allergens in food products, which is the only way to ensure the safety of food products for fish allergic consumers (Vierk, Falci et al. 2002). A number of protein-based immunochemical detection methods have been developed for the detection of these allergenic food or food ingredients. These immunoassays all employ specific polyclonal or monoclonal antibodies against the target allergens (Poms, Klein et al. 2004). Polyclonal antibodies used in immunochemical techniques are either obtained from the sera of food allergic patients [immunoglobulin E (IgE)] or from the sera of animals previously immunized with food derived allergenic protein [immunoglobulin G (IgG)]. Monoclonal antibodies (MAbs) are a homogeneous population of antibodies produced by hybridoma technology that have defined biological activity, consistent specificity, and their production capacity is unlimited. Currently there is no antibody available for the detection of fish allergen, although a commercial anti-frog parvalbumin MAb (PARV-19) originally raised against frog parvalbumins has also been reported to bind fish parvalbumin (Hilger, Thill et al. 2004; Chen, Hefle et al.

2006). However, its specificity against parvalbumin from a wide range of fish species has not yet been reported. A monoclonal antibody (MAb 3E1) initially developed by our group against heat-treated catfish crude sarcoplasmic protein extract was also found to be cross reactive with other fish species. It recognizes a thermal-stable protein with a molecular weight of 10-13 kDa in fish extracts, which corresponds to the molecular weights of the major fish allergen, parvalbumin. We therefore speculated that MAb 3E1 could be specific to fish parvalbumin, which is a heat stable and Ca2+ binding sarcoplasmic protein. In order to verify whether MAb 3E1 does in fact bind to fish parvalbumin, the present study was designed to investigate the antigen binding characteristics of MAb 3E1 by comparing its immunoreactivity against a wide range of fish and non-fish animal species with that of the anti-frog parvalbumin antibody, MAb PARV-19.

2. LITERATURE REVIEW

2.1 Fish allergy

Fish plays an important role in the human diet, but is also one of the most frequent causes of immunoglobulin E (IgE)-mediated food allergy, especially in countries with high fish consumption (Swoboda, Bugajska-Schretter et al. 2002; Bahna 2004; Kobayashi, Tanaka et al. 2006). Due to its high nutritive value and health benefits (high quality proteins, polyunsaturated fatty acids and lipid-soluble vitamins), global fish consumption has increased by 35% between 1973 and 1997 (Delgado, Wada et al. 2003). The recently increased levels of production and consumption of seafood has led to more frequent reporting of allergic reactions in occupational and domestic settings (O'Neil, Helbling et al. 1993; Sicherer, Munoz-Furlong et al. 2004). Numerous researchers around the world have attempted to determinate the prevalence of fish allergy. In Italy, 0.4% to 0.5% of children in the general population were diagnosed to be allergic to codfish (de Martino, Peruzzi et al. 1993). Another research group, also from Italy, showed that among 54 episodes of food-induced anaphylaxis in children, 30% were caused by fish (Novembre, Cianferoni et al. 1998). In a Spanish study evaluating food allergy in children, fish was the third common food allergen after eggs and cow’s milk (Boyano, Martin Esteban et al. 1987). Later, Crespo, Pascual et al. (1995) reported that in a group of 355 children with food allergy in Spain, 30% had fish and 6.8% shellfish allergy (Crespo, Pascual et al. 1995). The frequency of fish allergic individuals in a Norwegian population was estimated to be approximately 0.1% (Aas 1987). Recently, Sicherer, Munoz-Furlong et al. (2004), based on the data obtained from a random telephone survey, estimated that in the United States 0.4% of the general population suffer from fish allergy (Sicherer, Munoz-Furlong et al. 2004). Bjornsson, Janson et al. (1996) reported that 0.3% of an adult population in Sweden was fish-allergic (Bjornsson, Janson et al. 1996). Studies from Finland have estimated that 3% of 3-years old Finnish children were fish-allergic (Saarinen and Kajosaari 1980). Emmett, Angus et al. (1999), based on the data obtain from survey conducted in the UK, reported that the prevalence of fish allergy was about 0.5% of general population in the UK (Emmett, Angus et al. 1999). According to the investigation conducted in 1997 by the Japanese Ministry of Health and Welfare approximately 20% of food allergic adults in Japan are sensitive to fish (Hamada, Nagashima et al. 2001). 4

The prevalence of allergy to particular foods varies geographically and is related with regional dietary practices and the extent of exposure (Hourihane 1998). Fish hypersensitivity is frequently encountered in costal countries, where considerable numbers of population work in fish industry, and fish consumption is high (Van Do, Elsayed et al. 2005; Kobayashi, Tanaka et al. 2006). Fish allergy often appears at early age, and tends to be persistent, in contrast to some other food allergies with similar low ages of onset that are typically resolved at school age because of tolerance development (Eigenmann, Sicherer et al. 1998; Peng, Shyur et al. 2001). In fish allergic patients, ingestion of fish, inhalation of vapors generated during cooking, and skin contact can cause a variety of IgE-mediated symptoms. Symptoms of fish allergy usually appear immediately (minutes to an hour) and can range from urticaria and dermatitis to angiedema, diarrhoea, asthma and, at worst in some cases even fatal anaphylaxis (O'Neil, Helbling et al. 1993; Sampson 1999). Fish sensitive patients are often allergic to multiple fish species and are therefore advised to avoid consuming fish in general (Hansen, Bindslev-Jensen et al. 1997).

2.2 Major fish allergen - parvalbumin

The first purified and characterized fish allergen was Gad c 1, a codfish parvalbumin (Aas and Elsayed 1975; Elsayed and Bennich 1975). Parvalbumins are considered as the main allergen in fish. More than 95% of fish-allergic patients had specific IgE reacted to this protein with multiple IgE-binding eptiopes on this allergen being present in various fish species. Parvalbumin is a small (10-13 kDa) calcium-binding muscle protein that is heat-resistant and highly conserved across fish species and amphibians (Aas and Elsayed 1975; Bugajska-Schretter, Grote et al. 2000; Van Do, Hordvik et al. 2003; Van Do, Elsayed et al. 2005; Van Do, Hordvik et al. 2005; Kobayashi, Tanaka et al. 2006). Parvalbumins are found in all vertebrates, including human, but are present at much higher quantities in the muscles of lower vertebrates, such as fish (Gerday 1982). The quantity of parvalbumin varies in different types of fish muscles. White muscle generally contains more parvalbumin than dark muscle, what makes the dark muscles of fish much less allergenic than the white muscle. In their study Kobayashi, Tanaka et al. 2006 showed that horse mackerel white muscle contains 5-6 times more parvalbumin than dark muscle. In the same study, authors report that parvalbumin content markedly varies among the fish species and

that yellowtail (Seriola quinqueradiata) contains very low amounts of parvalbumin (Kobayashi, Tanaka et al. 2006). Parvalbumin the major fish and frog allergen belongs to a family of calcium-binding proteins (Bugajska-Schretter, Elfman et al. 1998; Hilger, Thill et al. 2004). Although, the physiological functions of parvalbumin are not completely understood it is suspected that this protein play an important role in muscle relaxation (Heizmann, Berchtold et al. 1982; Muntener, Kaser et al. 1995). A few studies investigating properties of fish parvalbumin showed that its binding to specific antibodies is calcium dependent. Quantification by gamma counting revealed that after removing calcium from carp parvalbumin the IgE binding to this protein was reduced by 57% (Swoboda, Bugajska-Schretter et al. 2002). The IgE-binding to carp parvalbumin has been reported to be strongly reduced by treatment with chelating reagents leading to Ca2+- depletion (Bugajska-Schretter, Elfman et al. 1998; Bugajska-Schretter, Grote et al. 2000). Based on the comparison of amino acid sequences, parvalbumins are divided into α- and β-phylogenetic lineages. The α-parvalbumins have a pI of 5.0 or higher, while the β- parvalbumins contain more acidic amino acids, resulting in a pI value of 4.5 or lower (Goodman, Pechere et al. 1979).

2.3 Other fish allergens

Recently, fish collagen (~100 kD) has been identified as a fish allergen and found to be a highly cross-reactive allergen among various species of fish. Sakaguchi et al. (1999) reported a single case of a child IgE reactive to fish (salmon and cod) gelatin (Sakaguchi, Hori et al. 1999). Later, Sakaguchi, Toda et al. (2000) reported that some fish-sensitive patients showed IgE reactivity to fish gelatin (heat-degraded type I collagen). In this study the patient’s IgE reacted with both 1 and 2 chains of tuna fish collagen type I. Moreover, strong cross-reactivity among fish gelatins derived from cod, tuna, salmon, saurel, and mackerel was observed (Sakaguchi, Toda et al. 2000). Another research group (Hamada, Nagashima et al. 2001) identified a high molecular weight allergen detected in bigeye tuna muscle as collagen based on findings with SDS-PAGE, immunoblotting and amino acid analysis. The authors of this study reported that IgE from fish allergic patient sera reacted with two bigeye tuna proteins of 120 and 240 kDa corresponded to α-chain and β-chain (dimmer of α-chain) of collagen (probably type I collagen), respectively

(Hamada, Nagashima et al. 2001). Based on these aforementioned reports fish collagen is considered as common allergen which is cross-reactive regardless of fish species. In addition to collagen, aldehyde phosphate dehydrogenase (APDH), a ~41 kDa heat- labile protein has also been reported as a fish allergen. Das Dores et al. (2002) presented characterization of a new allergen from codfish. In this study the authors reported that 4 out of 13 patients had specific IgE to ~41 kDa protein extracted from raw codfish. The 41 kDa protein was purified and identified as APDH (Das Dores, Chopin et al. 2002). Later, Rosmilah, Shahnaz et al. (2005) reported the APDH as a fish allergen in two species of snappers, Lutjanus johnii (golden snapper) and Lutjanus argentimaculatus (red snapper). Additionally they also reported 46 kDa and 51 kDa proteins to react with IgE from patients with fish allergy. The researchers identified a 51 kDa protein as a major allergen in raw and cooked extracts from both fish species in addition to the 12 kDa protein corresponding to parvalbumin. The authors suggested that the 51 kDa protein might be a parvalbumin tetramer, previously described by Das Dores et al. (2002). The 46 kDa protein has been also reported as a potential allergen in different studies, but it is still not well characterized (Rosmilah, Shahnaz et al. 2005).

2.4 Fish allergen contamination

The primary therapy for food allergy is to avoid the casual foods. Experts advise that fish- sensitized subjects should avoid consumption of all species of fish until it is definitely demonstrated that they can safely eat other fish species (Helbling, Haydel et al. 1999). Fish- allergic individuals avoiding all products containing fish should also avoid food ingredients derived from fish. For example, processed fish meat (surimi) is used as a basis for a variety of imitation non-fish products such as beef or pork substitutes (Musmand, Helbling et al. 1996). Several widely used food ingredients are derived from fish. Fish gelatin may be used to encapsulate certain vitamins, and isinglass may be used as a fining agent in beer, wines, and champagnes. Isinglass finings, composed mainly of fish collagen, are protein solutions extracted from swim bladders of certain fish species like: sturgeon, hake, and cod. Isinglass serves several functions in the brewing and wine industries to produce clarification, to enhance physical stabilization of cask-conditioned ales, and improve filtration performance. Isinglass residues in filtered beer ranged from 0.02 ppm to 0.16 ppm (Taylor, Kabourek et al. 2004). Ingestion of fish

maws would represent a high exposure to parvalbumin and fish collagen (Regenstein 2004; Taylor, Kabourek et al. 2004). In addition to exposure to fish allergens from food ingredients of fish origin, foods may become unintentionally contaminated with fish allergens at almost any step of manufacturing prior to final packaging or at any step of household food preparation. Hidden allergens in foods represent a major health problem for sensitized persons. A substance is a hidden allergen when it is unrecognized or not declared on the product label. This omission is not always intentional; and there are many ways for allergens to be hidden in food, for example through misleading labels, allergenic foods that can contaminate other safe foods, carelessness, food that is listed by an uncommon term, and ingredient switching, among others (Anibarro, Seoane et al. 2007). There are many ways for allergens to be hidden in foods. Probably the most common cause is unintentional contamination in the manufacture, handling or cooking process, when common equipment or the same cooking oil are used for different foods. Contamination with fish constituents can occur during shipping and storage, processing or from carry-over due to inadequate cleaning of shared processing equipment (Huggett and Hischenhuber 1998; van Hengel 2007). Visual observation is not always a reliable means to determine whether allergen cross-contact is actually occurring. It is likely that there is a significant risk of allergen cross- contact where appropriate preventive measures are absent. Therefore, a sensitive convenient assay which can detect trace amount of fish protein, regardless of its species, would be important to prevent accidental exposure of fish allergen. Allergic reactions caused by fish allergens hidden in other foods are quite frequent in fish allergic patients. Anibarro et al. (2007) reported that 35.5% of allergic reactions in fish sensitive individuals were caused by hidden fish allergens. These reactions were severe and usually took place during meals or stays away from home. For example, a fish allergic patient suffered an anaphylactic reaction after the ingestion of a canapé containing salmon cream, two patients had symptoms caused by inhalation of fish cooking vapors in a restaurant; another two through contact with a napkin and a fork contaminated with fish; another after the ingestion of a pâté with hidden fish; and another two through the consumption of other safe foods which had been fried in cooking oil previously used to fry fish (Anibarro, Seoane et al. 2007).

2.5 The Food Allergen Labeling and Consumer Protection Act (FALCPA) of 2004

Around 1-2% of adults and up to 5-7% of children suffer from some types of food allergy with foods such as fish and shellfish, peanuts, tree nuts, wheat, soy, cow's milk, and egg. These eight foods or food groups are considered as major allergen foods (Big 8) which account for 90% of all food allergies (Burks, Helm et al. 2001). To protect individuals with food allergies, the FALCPA (Public law 108-282) was enacted in August 2004 and became effective on January 1, 2006. According to the new labeling law, all packaged foods that are labeled on or after January 1, 2006, must comply with FALCPA's food allergen labeling requirements. FALCPA requires food manufacturers to label food products that contain an ingredient that is or contains protein from a major food allergen in one of two ways. The first option for food manufacturers is to include the name of the food source in parenthesis following the common or usual name of the major food allergen in the list of ingredients in instances when the name of the food source of the major allergen does not appear elsewhere in the ingredient statement. The second option is to place the word "Contains" followed by the name of the food source from which the major food allergen is derived, immediately after or adjacent to the list of ingredients, in type size that is no smaller than the type size used for the list of ingredients. The FALCPA also requires that the ingredient list be specific about what type of tree nut, fish, or shellfish is in the product: the type of tree nut (e.g., almonds, pecans, walnuts); the type of fish (e.g., bass, flounder, cod); and the type of Crustacean shellfish (e.g., crab, lobster, shrimp). However, the Act does not require FDA to establish a threshold level for any food allergen. Additionally, the labeling requirements only apply to major food allergens that are intentionally introduced into food products, in contrast to traces of such allergens being the result of cross-contact or accidental contamination during the production process (FDA 2005; Taylor and Hefle 2006). This new regulation has created the need for developing of a rapid test able to detect the eight foods or food groups listed in FALCPA as a potential source of allergens in food products. The food industry and governmental food safety control agencies should join effort to develop a rapid and specific detection method for the enforcement of the FALCPA law as well as the quality control of the food products because this is the only way to assure safety of food products for fish allergic consumers (Vierk, Falci et al. 2002).

2.6 Allergen detection methods

In order to enforce the FALCPA, the availability of methods that are designed to identify traces of food allergens on food production equipment and in food products is required (van Hengel 2007). Several approaches have been designed to detect the presence of allergens in food products. The methods employed are either targeting the allergen (protein) itself or a marker that indicates the presence of the offending food (Poms, Klein et al. 2004). To date there is only a limited number of validated methods available for just few allergens (Besler 2001). The methods used for the detection of food allergens can be generally divided into two groups, namely protein- and DNA-based methods (Goodwin 2004; Poms, Klein et al. 2004). Protein based methods usually involve immunochemical protocols such as the radioallergosorbent test (RAST), enzyme allergosorbent test (EAST), rocket immune-electrophoresis (RIE), enzyme-linked immunosorbent assay (ELISA), immunoblotting, dot immunoblotting. Methods operating on the DNA level are based on an amplification of a specific DNA fragment by polymerase chain reaction (PCR).

2.6.1 Allergen protein-based detection methods Protein-based methods usually involve immunological (antibody-based) techniques employing immunoglobulin G (IgG) antibodies that are raised in animals against purified allergens or crude protein extracts from the allergenic food product. Alternatively, immunoglobulin E (IgE) obtained from food allergic individuals can be also utilized (Goodwin 2004; Poms, Klein et al. 2004). Two serological tests, RAST and EAST, employing specific human serum IgE are wildly used in clinical diagnosis of food allergy. For allergy diagnosis purposes the binding of specific IgE antibodies to food allergens bound to solid phase is measured. In order to identify allergens in food products the RAST/EAST has been modified by preincubation of the human serum with protein extracts from the respective foods. Subsequently preadsorbed IgE antibodies are washed out, resulting in a decreased amount of specific IgE bound to the solid phase (Besler 2001). RAST/EAST inhibition has been applied for allergen detection in a wide range of food products (Herian, Taylor et al. 1993; Mata, Favier et al. 1994; Fremont, Kanny et al. 1996; Koppelman, Knulst et al. 1999).

Although specific IgE is required for allergen characterization it is not suitable for reliable allergen determination in food products, since the specificity of IgE from sensitized individuals differs considerably and the amount of serum is usually limited. Additionally, multiple sensitivities and/or cross-reactivities to more than one allergenic food may be present in human serum IgE. All those properties of sera obtained from individual patients prevent standardization and commercialization of methods based on human IgE (Besler 2001). In order to overcome the disadvantages associated with the use of human serum IgE, immunoassays relying on IgG antibodies raised in animals such as rabbits, mice, goats, sheep or chickens have been developed. The defense mechanism of an animal initiate production of the specific antibodies after immunization of the animal with purified protein or with crude protein extract obtained from allergenic food. Such antibodies may be either monoclonal (reacting to a single antigenic epitope or binding site; usually obtained from cell culture) or polyclonal (reacting to multiple epitopes; obtained from individual, immunized animals and therefore more variable) in nature. A favored immunogen against which the antibodies are developed is a known allergenic protein of high purity with relatively high resistant to food processing condition such as heat, pressure, acidity, and salinity. Antibodies raised using more defined allergens, rather than those produced by using crude protein extracts, will generally give analytical data that are easier to interpret (Goodwin 2004). One-dimensional sodium dodecyl sulphate polyacryl electrophoresis (SDS-PAGE) followed by immunoblotting is the standard protein separation procedure for protein/allergen detection and identification, providing information on the molecular weight of proteins that are bound by the antibodies employed. Major disadvantages of immunoblotting are laborious and time-consuming procedures that are rather inconvenient for routine analysis (Poms, Klein et al. 2004). Detection of food allergens using specific antibodies has also been reported for two additional methods: rocket immune-electrophoresis (RIE) (Holzhauser, Dehne et al. 1998) and dot immunoblotting (Blais and Phillippe 2000). However, due to laborious procedures in the case of RIE or inability to obtain quantitative data using dot immunoblotting, both methods are not widely used for allergen detection (Besler 2001). Enzyme-linked immunosorbent assay (ELISA), employing IgG antibodies, is currently the most commonly used method for determination and quantification of food allergens (Poms, Klein et al. 2004). The ELISA tests allow detecting allergens or specific marker proteins. The

11 detection is based on colorimetric reaction following binding with a specific enzyme-labeled antibody. The allergens or proteins of potentially allergenic food can be detected using different ELISA variants such as indirect, competitive and sandwich ELISA. In contrast to indirect and competitive ELISA, which involve only one antibody reacting with antigen, sandwich ELISA requires two antibodies (capture antibody and detection) antibody that will recognize two different binding sites of the same antigen molecule. A number of ELISA methods have been developed to test many different food products for traces of allergic food ingredients such as peanuts (Yeung and Collins 1996; Holzhauser and Vieths 1999), soybean (Tsuji, Okada et al. 1995; Bando, Tsuji et al. 1998), almonds (Hlywka, Hefle et al. 2000; Scheibe, Weiss et al. 2001), casein components of cow milk (Plebani, Restani et al. 1997), wheat (Skerritt and Hill 1991), crustaceans (Fuller, Goodwin et al. 2006; Werner, Faeste et al. 2007), eggs (Yeung, Newsome et al. 2000; Williams, Westphal et al. 2004). An alternative to ELISA format could be dipstick assays, which are inexpensive, rapid, portable, easy to perform and do not require instrumentation. However, currently this type of assay is not suited for a quantitative detection (van Hengel 2007). Although several protocols are available to diagnose fish allergy in fish allergic individuals, to date only one recent study describe the determination of fish parvalbumin in food products. In their study Faeste and Plassen (2008) reported development of quantitative sandwich ELISA for the determination of fish in foods. Specific antibody used in this assay was produced through immunization of a rabbit with purified cod parvalbumin. Obtained polyclonal rabbit anti-cod parvalbumin antibody was used as a capture and detection (after previous conjugation with biotin) antibody to construct sandwich ELISA. The assay was used for the quantification of 32 fish species in different food matrixes with a limit of detection of 5 mg fish/kg food. The assay showed high specificity to fish with no cross-reactivity with meat, shellfish or food additives. However, from all tested fish species twelve obtained recovery rates lower than 1% in the assay which may be unsuited to trace these kinds of fish (Faeste and Plassen 2008).

2.6.2 Allergen DNA-based detection methods The DNA-based methods relay on the amplification of specific DNA fragments by employing the polymerase chain reaction (PCR). The specificity of PCR method is achieved by

12 the use of short stretches of DNA called primers that facilitate amplification of DNA originating from the offending food. The amplified product is visualized by staining with a fluorescent dye or by southern blotting following electrophoresis in an agarose gel. Additionally, gel free product detection by means of real time PCR where both amplification and detection are performed simultaneously can be performed (Goodwin 2004). PCR methodology is commonly used for detection of food microbial pathogens or genetically modified crops. This technique is now being employed as a sensitive tool for the detection of specific allergenic components in food. PCR technique was applied to detect variety of common allergenic foods in different food matrices, such as peanuts (Hird, Lloyd et al. 2003; Watanabe, Akiyama et al. 2006), hazelnuts (Piknova, Pangallo et al. 2008), soybean (Meyer, Chardonnens et al. 1996; Yamakawa, Akiyama et al. 2007; Gryson, Messens et al. 2008), crustaceans (Brzezinski 2005) and wheat (Allmann, Candrian et al. 1993), and can be suitable for use with raw or processed foods. PCR-based testing, although highly specific and direct (if required, PCR products can be sequenced for absolute confirmation), is only an indicator of the presence of a particular food, rather than the compounds responsible for triggering the allergic (Goodwin 2004; Poms, Klein et al. 2004).

3. HYPOTHESES AND OBJECTIVES

3.1 Hypotheses

1) If MAb 3E1 is specific to fish parvalbumin, the positive or negative reaction pattern against fish species should be comparable with the anti-parvalbumin antibody MAb PARV-19. 2) If MAb 3E1 is specific to fish parvalbumin, the antibody should bind to low molecular weight antigenic proteins (in the range of 10-13 kDa) in fish extracts corresponding to the parvalbumins which recognized by the anti-parvalbumin antibody MAb PARV-19. 3) If MAb 3E1 is specific to fish parvalbumin, the immunoreactivity of this antibody against fish extracts may be Ca2+ dependent. 4) If MAb 3E1 is specific to fish parvalbumin, the MAb 3E1 and MAb PARV-19 will bind to the same protein, parvalbumin, however, on the same or different epitope(s).

3.2 Objectives

The overall goal of this study was to investigate the antigen binding characteristics of MAb 3E1 against various fish and other animal meats to evaluate the usefulness of this antibody as a anti-fish parvalbumin reagent. The specific objectives were a) to examine species specificity of MAb 3E1 and compare with a commercially available anti-frog parvalbumin monoclonal antibody (PARV-19) using indirect ELISA; b) to identify the antigenic proteins for MAb 3E1 in cooked fish extract; c) to study the effects of presence and absence of calcium on the immunoreactivity of MAb 3E1; d) to compare the epitope(s) of MAbs 3E1 with the epitope(s) of MAb PARV-19.

4. MATERIALS AND METHODS 4.1 Materials

Tris-buffered saline, 0.5 M Tris-HCl buffer (pH 6.8), 1.5 M Tris-HCl (pH 8.8), TEMED (N,N,N,N′-tetra-methyl ethylenediamine), Precision Plus Protein Kaleidoscope Standards, 30% acrylamide/ bis solution, Tris/glycine buffer, 10 × Tris/glycine/SDS buffer, supported nitrocellulose membrane (0.2 µm), and thick blot paper were purchased from Bio-Rad Laboratories Inc., Hercules, CA. Hydrogen peroxide, horseradish peroxidase conjugated goat antimouse IgG (Fc specific), ABTS (2,2′-azino-bis 3-ethylbenzthiazoline- 6-sulfonic acid), and β-mercaptoethanol were purchased from Sigma-Aldrich Co., St. Louis, MO. Brom phenol blue sodium salt was purchased from Allied Chemical Corporation, New York. Sodium chloride

(NaCl), sodium phosphate dibasic anhydrous (Na2HPO4), sodium phosphate monobasic anhydrous (NaH2-PO4), bovine serum albumin (BSA), sodium bicarbonate (NaHCO3), sodium

carbonate (Na2CO3), citric acid monohydrate, sodium dodecyl sulfate, Tween 20 and all other chemicals, reagents, filters (Whatman No. 1 paper), 96 well polystyrene microplate (Costar 9018) were purchased from Fisher Scientific, Fair Lawn, NJ. All solutions were prepared using distilled deionized pure water (DD water) from the NANOpure DIamond ultrapure water system (Barnstead International, Dubuque, IA). All chemicals and reagents were analytical grade. Twenty five (25) authentic fish samples were obtained from the Florida Department of Agriculture and Consumer Services, Tallahassee, FL. An additional 26 different fish samples were obtained from reliable seafood distributors and from local fish markets. Crab, shrimp, scallop, frog legs, fresh beef loin, lamb shoulder, pork loin, frozen dressed rabbit, whole turkey and chicken were purchased from local supermarkets. Horse meat was obtained from a private source. Rat muscles were obtained from the Biological Facilities, Florida State University. Monoclonal mouse anti-frog parvalbumin IgG (PARV-19) was obtained from Sigma-Aldrich. Initial screening of five cross-reactive fish-specific MAbs, previously developed in our laboratory using cooked catfish crude extract as the immunogen, led to selection of MAb 3E1. MAb 3E1 was chosen for further characterization in this study because it is crossreactive with different fish species, and reacts with a group of 10-13 kDa thermal stable proteins, likely parvalbumin, in fish extracts. Supernatant of MAb 3E1 obtained from the propagated cell cultures was used in this study. The isotype of Mab 3E1 was determined with a mouse MAb

isotyping kit (ISO-2 1 kit, Sigma) as IgG1 according to the manufacturer’s protocol (Appendix A.2).

4.2 Protein extraction from fish and meat samples

Approximately ten grams of half thawed frozen fish samples from each species were accurately weighed into beakers. The beakers were covered with aluminum foil, sealed with adhesive tape, and heated in a boiling water bath for 20 min. The cooked samples were then cooled at room temperature and mashed into fine particles using a glass rod, after which a three fold (3:1 mL/g) of saline (0.15 M NaCl) was added to the mashed samples, and the mixture homogenized for 1 min at 11,000 rpm. The homogenized samples were allowed to stand at 4°C for 2 h followed by centrifuging at 5,000×g for 30 minutes at 4°C. Supernatants obtained after centrifugation were then filtered through Whatman No. 1 filter paper and stored at -20°C until use. The non-fish lean muscle samples, including both poultry and other meats, were ground and then prepared in the same manner as the fish samples. Protein concentration of every sample was determined with Bio-Rad Protein Assay according to the manufacturer’s protocol (Appendix A.1).

4.3 Indirect ELISA

Properly diluted sample protein extracts in 0.06 M carbonate buffer (pH 9.6) was coated (2 µg/100 µL per well) onto the wells of a 96-well polystyrene microplate (Costar 9018, Fisher) and incubated at 37°C for 2 h. The plate was then washed three times with PBST [0.05% v/v Tween-20 in 10 mM PBS, pH 7.2] and incubated with 200 µL/well blocking solution (3% NFDM in PBS) at 37°C for 2 h, followed by another washing step. Supernatant of MAbs 3E1 or PARV-19 ascites fluid appropriately diluted in antibody buffer [1% w/v BSA in PBST] was added to each well (100 µL) and incubated at 37°C for 2 h. After washing three times with PBST, 100 µL of the secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG-Fc specific diluted 1:3000 in antibody buffer) was added to each well and the plate incubated at 37°C for 2 h. It was then washed five times before the addition of the substrate solution (22 mg of ABTS and 15 µL of 30% hydrogen peroxide in 100 mL of 0.1 M phosphate- citrate buffer, pH 4.0) for color development at 37°C for 10-35 min. The enzyme reaction was

stopped by adding 0.2 M citric acid solution, and the absorbance measured at 410 nm using a microplate reader (Model MQX200R, BioTek).

4.4 Ca2+dependent binding assay

++ In order to add or remove Ca from the reaction system 10 mM CaCl2 or 10 mM ethylene glycol tetraacetic acid (EGTA), respectively, was added to 0.06 M carbonate buffer (pH 9.6) and the pH of the buffer adjusted back to pH 9.6. Each sample extract was then diluted in the prepared buffers to obtain a concentration of 2 µg/100 µL and coated on the plate to perform the indirect ELISA, as described above.

4.5 Epitope comparison

In order to test if the antibodies used in this study bind to the same epitop the additivity test as described by (Friguet, Djavadi-Ohaniance et al. 1983) was performed with modifications. The microplate wells were coated with 100l of cooked fish extract containing 0.5g of soluble proteins diluted in carbonate buffer (pH 9.6). The plate was incubated for 2 hours at 37ºC followed by washing 3 time with PBS containing 0.05% Tween-20 (PBST). After washing step each well of the microplate was blocked with 200l of nonfat dry milk solution (3% w/v in 10mM PBS) an incubated at 37ºC for 2 hours. During incubation three solutions of primary antibody were prepared: MAb 3E1 diluted 1:3 in antibody buffer [1% w/v BSA in PBST]; MAb PARV-19 diluted 1:7500 in antibody buffer; and both the diluted antibodies together in the ratio 1:1. After two further washings with PBST 100l of each of the primary antibody solution was added into the microplate followed by incubation at 37º C for 2 hours. After washing three times with PBST, 100µl of the secondary antibody (horseradish peroxidase-conjugated goat anti- mouse IgG-Fc specific diluted 1:3000 in antibody buffer) was added to each well and the plate was incubated at 37°C for 2 h. It was then washed five times before the addition of the substrate solution (22 mg of ABTS and 15 µL of 30% hydrogen peroxide in 100 ml of 0.1 M phosphate- citrate buffer, pH 4.0) for color development at room temperature for 10 min. The enzyme reaction was stopped by adding 0.2 M citric acid solution, and the absorbance was read at 410 nm for the MAb 3E1 alone, the MAb PARV-19 alone and the two antibodies together using microplate reader (Model MQX200R, BioTek).

4.6 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot

SDS-PAGE was performed to resolve the soluble proteins in different sample extracts according to the method of (Laemmli 1970) with modifications. Briefly, soluble proteins (3 µg of protein in 10 µL per lane) from the samples were loaded on 5% stacking gel (pH 6.8) and separated on 14% polyacrylamide separating gel (pH 8.8). The gel was subjected to electrophoresis at 200 V for 45 min using a Mini-Protein 3 electrophoresis cell (Bio-Rad, 161- 3301) connected to a power supply (Model 3000, Bio-Rad) (Appendix A.3). Western blot assay was carried out according to the method of (Towbin, Staehelin et al. 1979) with modifications in order to determine the molecular weights of the immunogenic components that reacted with each MAb. After separation of the proteins on the polyacrylamide gel by means of SDS-PAGE, protein bands were transferred electrophoretically (1 h at 100 V) from the gel to nitrocellulose membranes using a MiniTrans- Blot unit (Bio-Rad). Upon completion of the transfer, the membrane was washed with TBST (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5), and then blocked with 1% BSA in TBS for 2 hours. After another washing step, the membrane was incubated with the MAb 3E1 supernatant or PARV-19 ascites fluid diluted 1:1 and 1:5000, respectively, in antibody buffer for 2 hours at room temperature. The excess antibody reagent was removed by washing with TBST, and the membrane was then incubated for 1 hour at room temperature with the secondary antibody (goat anti-mouse IgG- alkaline phosphatase conjugate diluted 1:3000 in antibody buffer). After washing, the membrane was incubated with 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride (BCIP/NBT) in 0.1 M Tris buffer (pH 9.5) for about 3 minutes to develop the color. The color reaction was stopped by washing the membrane with distilled water. The appearance of a dark purplish band indicated an antibody binding site (Appendix A.4). Prestained broad range protein standards (Precision Plus Protein Kaleidoscope Standards, Bio-Rad, 161-0375) were used as molecular weight markers for both the SDS-PAGE and Western blot.

5. RESULTS AND DISCUSSION

5.1 Species specificity of MAbs 3E1 and PARV-19

In order to study the species specificity of MAbs 3E1 and compare it with PARV-19, indirect ELISA was performed using these two antibodies with fish and meat cooked extracts. The immunoreactivity of MAbs 3E1 and PARV-19 against cooked protein extracts from 51 different fish species, along with 16 non-finfish and animal samples including shellfish (shrimp, crab, scallop), poultry (chicken breast, chicken thigh, turkey breast, turkey thigh, pork, beef, lamb, rabbit, horse, deer, elk, rat thigh, frog legs), and 5 food protein additives (gelatin, egg albumin, soy concentrate, non fat dry milk, bovine serum albumin), was examined. The results are summarized in Table 1 and 2. The ELISA results showed that MAb 3E1 had an identical reaction pattern to that of the anti-frog parvalbumin MAb, PARV-19, when reacted with all the fish species tested. Both MAbs reacted strongly with the majority of the fish species tested, with the exceptions being swordfish, yellowfin tuna, pollock, cod, idaho rainbow trout, wild salmon, whiting, and haddock. This indicates that the antigen recognized by these two antibodies is likely to be the same. Although there was no difference between the reaction patterns of MAbs 3E1 and PARV- 19 for the fish species, screening with non-fish extracts revealed that MAb 3E1 reacted only with finfish, while PARV-19 also reacted with frog, rat and rabbit extracts. As noted earlier, the MAb anti-frog parvalbumin (PARV-19) obtained from Sigma-Aldrich Corp. was previously reported to bind and -frog parvalbumins (Hilger, Thill et al. 2004; Chen, Hefle et al. 2006). According to the product datasheet, this antibody was produced using purified frog muscle parvalbumin as the immunogen, so the reaction with frog parvalbumin was expected. PARV-19 has also been reported to cross-react with parvalbumin in different fish, including Pacific and horse mackerel, red sea bream, sardine, carp, catfish, cod, and tilapia (Chen, Hefle et al. 2006; Kobayashi, Tanaka et al. 2006). Although the structure of parvalbumins is well conserved throughout the animal kingdom, the homology decreases with decreasing zoological relationship. Comparison of amino acid sequences showed 60-70% amino acid identity between frog and fish parvalbumin (Hilger, Thill et al. 2004). MAb 3E1 was developed using fish thermal stable protein; during the

19 hybridoma screening stage only finfish-specific clones were selected, therefore this MAb is finfish specific. The indirect ELISA experiment revealed that some fish species do not react with either MAb 3E1 or PARV-19. Eight out of the fifty one fish species tested showed little or no reaction for both antibodies. Recently Chen and others (2006) reported that PARV-19 did not react with extracts from yellowfin tuna and our result for yellowfin tuna confirms this observation. Moreover, screening with a large number of fish species showed that in addition to yellowfin tuna several other fish species also failed to react with PARV-19, as described above. The lack of a reaction with those fish species could indicate that either skeletal muscles from those fish species do not contain parvalbumin or the amount of parvalbumin is too small to be detected. It is also possible that minor differences in the structure of parvalbumin among various fish species can affect the binding characteristics for the anti-parvalbumin antibody (Chen, Hefle et al. 2006). However, an analysis of the variations in the concentration of parvalbumin among different fish species, as well as an investigation of parvalbumin’s structure, is beyond the scope of the present study.

5.2 Thermal-stable protein profile in cooked fish extracts

SDS-PAGE was performed to obtain the thermal-stable protein banding pattern of cooked fish extracts. The representative SDS-PAGE protein profiles of twenty fish species are shown in Figure 1. Two major groups of proteins (appearing as 36 kDa and 10-13 kDa bands on the gel) remained soluble after heat-treatment of the fish samples. The 36 kDa protein presented in all the extracts of cooked fish species tested, while the 10-13 kDa band appeared in the majority of the fish species tested but was missing in three species assayed on the gel, namely pollock, yellowfin tuna and farm-raised salmon, indicating the lack of a detectable quantity of the 10-13 kDa protein in these three fish species. This result agrees with that of the indirect ELISA, which found a negative reaction with anti-parvalbumin antibodies in these species. This provides further evidence that the 10-13 kDa proteins are parvalbumins. Either one or two distinct bands of this group of 10-13 kDa proteins appeared on the gel for most of the species was observed. The presence of more than one band in some fish species suggests the existence of different isoforms of this protein. Van Do and others (2005) reported that more than one parvalbumin isotype are generally present in the muscle tissue of fish. Some fish species have 20

been shown to display two to five isotypes of parvalbumin (Van Do, Hordvik et al. 2005). The differential expression of this protein is most probably related to the physiological requirements of the developmental stages of the growing fish (Huriaux, Vandewalle et al. 2002). Considering the quantity present in fish muscle, the low molecular weight, the thermal stability, the existence of different isoforms, and the immunoreactivity with the antiparvalbumin antibody of the 10-13 kDa protein, it is reasonable to assume that this group of proteins are fish parvalbumins. The 36 kDa protein that appeared in all the fish species tested corresponds to the molecular weight of fish tropomyosin. Because tropomyosin has also been reported as a thermal- stable protein (Naqpal, Rajappa et al. 1989), it is reasonable to speculate that this 36 kDa protein could be tropomyosin. Tropomyosin, one of the striated muscle regulatory proteins, forms a family of highly conserved actin-binding proteins (Crick 1953). Fish tropomyosin has been reported in several studies, where the existence of multiple isoforms of this protein was also observed (Heeley and Hong 1994; Heeley, Bieger et al. 1995). Recently Huang and Ochiai (2005) reported isolation of tropomyosin from the fast skeletal muscle of six different fish species in their study on thermal stability of this protein (Huang and Ochiai 2005). Tropomyosin is a major allergen in many shellfish, especially crustacean and mollusks, but to date there have been no reports that fish tropomyosin could cause allergic reactions (Daul, Morgan et al. 1993; Leung, Chen et al. 1998). Because MAb 3E1 does not recognize this 36 kDa protein, the further characterization of this protein is not relevant to this study.

5.3 Antigenic protein for MAb 3E1

Following the SDS-PAGE analysis, protein bands were further characterized by Western blot using MAb 3E1 in order to reveal the specific antigenic protein reacting with the antibody. In this experiment MAb 3E1 was tested against cooked extracts of twenty selected fish species, (Figure 2). All protein bands in the molecular weight range of 10-13 kDa were recognized by MAb 3E1. However, no bands in the range of 10-13 kDa appeared in pollock, yellowfin tuna and farm raised salmon. The immunoreactivity of MAb 3E1 against these 20 fish species determined by both indirect ELISA and Western blot agreed closely with each other. These results provide further evidence that MAb 3E1 binds fish parvalbumin. To confirm the above observation that the antigen recognized by MAb 3E1 is fish parvalbumin, a side-by-side comparison of the antigenic proteins profile recognized by MAb

3E1 and MAb PARV-19 in nine randomly selected cooked fish extracts was also made (Figure 3). Both MAbs tested in this experiment had almost identical reaction patterns with all the fish samples tested. The molecular weight of parvalbumin recognized by PARV-19 matched the size of the 10-13 kDa protein bound by MAb 3E1. The results from the Western blot, together with the results from SDS-PAGE and indirect ELISA, demonstrate clearly that the protein recognized by MAb 3E1 is indeed fish parvalbumin.

5.4 Ca2+ sensitive epitopes

The anti-parvalbumin MAb PARV-19 has been reported to bind parvalbumin in a calcium dependent manner (Hilger and others 2004). To investigate whether the binding of the antigen with MAb 3E1 is also calcium dependent, indirect ELISA was performed with both MAbs against cooked fish and meat extracts which had been diluted in coating containing buffer (the control), or coating buffer containing either the calcium chelating reagent EGTA (to remove 2+ 2+ Ca ) or CaCl2 (to add Ca ). The indirect ELISA results for cooked extracts from rat, frog and twenty-four representative fish species in the presence and absence of calcium using MAbs 3E1 and PARV-19 are shown in Figure 4 and 5, respectively. Comparing the readings for the control samples, there were no observable changes in the

immunoreactivity of both antibodies against all the samples tested with added CaCl2. However, an increase was observed in the immunoreactivity of all the samples reacted with MAb 3E1 after removing calcium by adding EGTA. Similar results, but with slightly enhanced reaction signals, were observed in samples reacted with the MAb PARV-19. Three fish species (cod, whiting and haddock), none of which reacted with PARV-19 in the control samples, showed a dramatic increase in absorbance under calcium depleted conditions; removal of calcium from these three fish extracts, however, had no effect on their immunoreactivity when the samples were reacted with MAb 3E1. In general, MAb 3E1 appeared to be less calcium sensitive than PARV-19. Parvalbumins belong to a family of calcium binding proteins and are characterized by the presence of three typical helix-loop-helix Ca2+ binding domains, termed EF hands (Berchtold 1989; Ikura 1996). Seiberler and others (1994) reported that the IgE recognition of EF-hand pollen allergens can be modulated by the presence or absence of protein-bound calcium (Seiberler, Scheiner et al. 1994). A number of studies have been conducted to investigate the effect of calcium on the immunoreactivity of parvalbumins. The IgE binding to carp parvalbumin

was greatly reduced after the removal of calcium in a Western blot experiment (Bugajska- Schretter, Grote et al. 2000; Swoboda, Bugajska-Schretter et al. 2002), while a significantly reduced or even totally abolished IgE binding was observed with frog muscle extract in the presence or absence of calcium during IgE immunoblotting. MAb PARV-19 was also reported to bind parvalbumin in a calcium dependent manner. The recognition of frog parvalbumin by mouse monoclonal anti-frog parvalbumin antibody PARV-19 has been reported to decrease for samples treated with EGTA (Hilger, Thill et al. 2004). Our ELISA result showed an increase in the immunoreactivity of MAb PARV-19 with fish extracts after the removal of calcium, which disagrees with previously reported studies investigating the Ca-dependence of parvalbumin with IgE. Several factors should be considered when comparing these seemingly contradictory results, however. First of all, the antibody binding characteristics are determined by the location and shape (linear or conformational) of the epitope, and the presence or absence of Ca2+ may or may not affect the epitope binding of the antigen. Secondly, the effect of calcium depletion has predominately been studied for IgE antibodies. The allergen binding sites recognized by the IgE antibodies naturally found in fish- sensitive patients’ serum may not be the same as the IgG antibodies which are produced by immunizing animals with an antigen. The form of antigen preparation (native or heat-treated), as well as the methods of clone selection in the case of MAb development, also greatly affects the binding characteristics of the antibodies. Moreover, all previous studies employed Western blot analysis to study the Ca2+ dependency of the antibody, while in the current study we have tested the effect of calcium using ELISA. Swoboda and others (2002) suggested that removal of free Ca2+ -ions can cause changes in the conformation of IgE-epitopes and/or unfolding of the antigenic protein, both of which would result in a reduction of IgE-binding in the sera of fish allergic patients (Swoboda, Bugajska-Schretter et al. 2002). The same conformational changes may also take place after removal of calcium in our experiment, but in contrast to the previously reported results for IgE-epitopes, removal of calcium may expose more hidden IgG-epitopes and lead to an increase in the epitopes of MAbs 3E1 and PARV-19 for fish parvalbumin.

5.5 Epitope comparison

If MAbs 3E1 and PARV-19 both bind to parvalbumin, then binding sites may or may not be overlapping. The epitope comparison was therefore performed to determine whether the

MAbs 3E1 and PARV-19 bind to the same epitope on the antigenic protein-parvalbumin. The additivity index (A.I.) was calculated to be 36.27%, based on the equation by (Friguet, Djavadi- Ohaniance et al. 1983), shown below.

Where A1, A2 and A1+2 are the absorbance readings reached, in the additivity test, with the first antibody alone, the second antibody alone, and the two antibodies together. The absorbance readings for MAb 3E1 alone (A1), the MAb PARV-19 alone (A2) and the two antibodies together

(A1+2) are shown in Figure 6.

If the antibodies bind to the same epitope of the common antigenic protein, A1+2 should

be equal to the mean value of A1 and A2 and A.I. will be equal to zero. If, on the contrary the two antibodies bind to different non-overlapping epitopes on the antigenic protein, A1+2 should be the sum of A1 and A2 and A.I. will be equal to 100%. Generally, it is considered that the antibodies share the same binding side or their binding sides overlap to some degree if A.I. is below 50%. For the tested antibodies, it appeared that the binding of the first MAb inhibited the binding of the second MAb. The A.I. value obtained in this experiment was below 50% thus, we can conclude that both antibodies recognized exactly the same protein but their epitopes (binding sites) overlap to the extend causing inhibitive binding on the parvalbumin. Moreover, knowing that the antigenic protein for MAb PARV-19 is parvalbumin the result of epitope comparison further supports the hypothesis that MAb 3E1 is also parvalbumin specific.

6. CONCLUSIONS

MAb 3E1 recognizes a group of thermal stable proteins with a molecular weight between 10 and 13 kDa in fish samples, with each antigenic protein corresponding to the molecular weight of each parvalbumin recognized by the anti-frog parvalbumin MAb PARV-19. MAb 3E1 and MAb PARV-19 have almost identical immunoreaction patterns to all the fish species tested using both indirect ELISA and Western blot. The antibody binding of the antigen was Ca- dependent for both MAbs, and the ELISA response was enhanced with the removal of calcium in the reaction system. Screening with non-finfish extracts revealed that MAb 3E1 only reacts with fish proteins and is strictly finfish specific, while MAb PARV-19 cross-reacts with frog, rat and rabbit. Based on the thermal stability, molecular weight of the antigen, Ca-dependent binding characteristics, epitope comparison, and immunoreactivity with various fish species, it is clear that MAb 3E1 is fish parvalbumin specific. This antibody has the potential to be a useful tool in the detection and investigation of the major fish allergen, parvalbumin.

APPENDIX A. PROTOCOLS AND PROCEDURES

Protocol 1. Determination of protein concentration with Bio-Rad Protein Assay

1. The dye reagent was prepared by dilution one part of Day Reagent Concentrate with four parts of DDI water. The diluted dye was then filtered through a Whatman No.1 filter to remove particles. 2. Four dilutions of the protein standard, Bovine Serum Albumin (BSA), which is representative to the protein solution tested, were calculated to obtain linear range of 0.05 mg/ml to approximately 0.5 mg/ml. The original concentration of the protein standard was determined by the spectrophotometer (SmartSpec 3000, Bio-Rad).

3. Four different amounts of BSA were diluted up to 200 ul in 0.01 M phosphate buffer saline (PBS) based on calculations from step 2. PBS without any addition of BSA was used as blank.

4. The cooked samples to be tested were diluted in PBS about 1-5 times to the concentration range described in step 2.

5. 25 l of the blank, the diluted samples and the diluted protein standards was added into centrifuge tubes containing 500 l of diluted dye reagent. All the tubes were mixed before adding into the wells of microplate.

6. 200 l/well of the standards and the samples prepared in step 5 was added in duplicate into a microplate and incubated at room temperature for 5-60 min.

7. The absorbance was read at 595 nm with the micorplate reader (Model MQX200R, BioTek).

8. The concentration of protein standards was plotted vs. the absorbance reading and the protein concentration of tested samples was determined from the standard curve.

Protocol 2. Determination of the isotype of Mab 3E1 with a mouse MAb isotyping kit (ISO- 2 1 kit, Sigma)

The isotype of mouse MAb 3E1 which was in the form of culture supernatant was determined with a mouse MAb isotyping kit (ISO-2 1 kit, Sigma) using “Capture ELISA” procedure described in the manufacture manual.

1. The isotype specific antibodies were diluted 1:1000 in PBS 2. 0.1 ml of each of the diluted antibodies was added into 2 wells of a microplate. 3. The plate was incubated for 1 hour at 37 ºC. 4. After removing the coating solution, the plate was washed 3 times with washing buffer (PBS containing 0.05% Tween-20). 5. 0.1 ml of the undiluted culture supernatant was added into each of the wells. 6. The plate was incubated at room temperature for 1 hour. 7. The plate was washed 3 times with washing buffer. 8. Peroxidase labeled Goat Anti-Mouse IgG (Fab Specific) antibody was diluted 1:600 in washing buffer 9. 0.1 ml of the diluted enzyme conjugated antibody was added into each well. 10. The plate was incubated at room temperature for 30 minutes. 11. The substrate was prepared as follows: a. 5-Aminosalicylic acid was dissolved at 1 mg/ml in substrate buffer (0.02 M sodium phosphate, pH 6.8) b. 1% hydrogen peroxide in water solution was added into the 5-Aminosalicylic acid prepared at the ratio of 0.1 ml per 10 ml. 12. At the end of the 30 minutes incubation, the plate was washed 5 times with washing buffer. 13. 0.1 ml of freshly prepared substrate solution was added into each well. 14. The plate was incubated at room temperature for 20-30 minutes. Brown color developed in a well indicates a positive result. 15. The reaction was stopped by adding 50 l of 3M NaOH to each well. 16. The plate could be inspected visually or by a microplate reader at 550 nm.

Protocol 3. Procedures for SDS-PAGE

1. The glass sandwich was assembled according to the guide of the manufacture; 2. The glass sandwich was filled with 14% separation gel and 5% stacking gel; 3. Sample buffer was prepared by mixing 0.5M Tris-HCl (pH 6.8), 2% (wt/vol) SDS, 25% (vol/vol) glycerol, 0.01% (wt/vol) bromophenol blue, and 5% (vol/vol) -mercaptoethanol which was added prior to use; 4. The protein extracts were diluted 1:2 with sample buffer; 5. 3 g of protein extract was loaded to each well of the gel; 6. The gel was run at constant voltage setting of 200 V for approximately 45 min at room temperature; 7. The gel was removed for electrophoretic transfer.

Protocol 4. Procedures for Western blot

1. The gel was rinsed with DI water; 2. The gel was equilibrated for 5-10 minutes in transfer buffer (10X Tris/Glycine Buffer, Bio-Rad) before blotting to remove electrophoretic salts and detergents; 3. The nitrocellulose membrane was wetted by slow sliding it at 45º into the transfer buffer for 15-30 minutes; 4. The filter paper and fiber pads were soaked in the transfer buffer avoiding entrapment of bubbles; 5. The gel holder cassette was assembled to complete the blotting sandwich; 6. The electrophoretic transfer was performed at 100V/350mA for 1 hour; 7. After the transfer, the membrane was washed with TBS buffer (Bio-Rad), and the membrane was blocked with the blocking buffer (1% (w/v) BSA in TBS) for 2 hours at room temperature; 8. The membrane was washed twice with washing buffer (0.05% (v/v) Tween-20 in TBS); 9. The membrane was incubated with MAb 3E1 or PARV-19 diluted 1:1 and 1:5000 respectively, in antibody buffer (1% (w/v) BSA in washing buffer) for 2 hours at room temperature; 10. The membrane was washed 4 times with washing buffer;

11. The membrane was incubated with second antibody – Goat Anti-Mouse IgG (H+L)-AP Conjugate (Bio-Rad), diluted 1:3000 in antibody buffer, for 1 hour at room temperature; 12. The membrane was washed 4 times with washing buffer and 3 times with TBS prior color development; 13. The membrane was stained by soaking it in 15 ml of color development buffer with addition of 150 l of AP reagent A and 150 l reagent B (Bio-Rad) at room temperature; 14. After approximately 3 minutes the staining was stopped by washing the membrane with DI water for about 10 min to remove residual color development solution; 15. The membrane was dried on a filter paper.

APPENDIX B. TABLES AND FIGURES

Table 1. Immunoreactivity of MAbs 3E1 and PARV-19 against cooked shellfish, meat, poultry and food additives samples determined by indirect ELISA. Absorbance readings at 410 nm: <0.15 — “-“; 0.15-0.199 — “+/-“; 0.2-0.499 — “+“; 0.5-0.999 — “++“; >1 — “+++“; Not tested — “?“.

Abbreviation Market name 3E1 PARV-19

Crab CRAB - - Sco SCALLOP - - Shp SHRIMP - - Cb CHICKEN BREAST - - Ct CHICKEN THIGH - - Tb TURKEY BREAST - - Tt TURKEY THIGH - - B BEEF - - D DEER - - E ELK - - Frog FROG LEGS - + H HORSE - - L LAMB - - PO PORK - - R RABBIT - +/- Rat RAT THIGH - +++ BSA BOVINE SERUM - ? Egg EGG ALBUMIN - ? NFDM NON FAT DRY MILK - ?

Soy SOY CONCENTRATE - ?

Table 2. Immunoreactivity of MAbs 3E1 and PARV-19 against cooked fish samples determined by indirect ELISA. Absorbance readings at 410 nm: <0.15 — “-“; 0.15-0.199 — “+/-“; 0.2-0.499 — “+“; 0.5-0.999 — “++“; >1 — “+++“.

Abbreviation Market name 3E1 PARV-19 Abbreviation Market name 3E1 PARV-19 AJ AMBERJACK ++ + P POLLOCK - - BA BASA ++ ++ POM POMPANO ++ + BC BLUE CATFISH +++ +++ RG RED GROUPER +++ +++ BGC BLACK GROUPER (CARBO) +++ +++ RMG REDMOUTH GROUPER ++ +++ BSB BLACK SEA BASS ++ +++ RS RED SNAPPER ++ +++ CG CAMOUFLAGE GROUPER ++ ++ RT IDAHO RAINBOW TROUT - - CHC CHANNEL CATFISH +++ +++ S SCAMP ++ +++ CO COBIA ++ ++ SB STRIPED BASS +++ +++ COD COD - - SF SOUTHERN FLOUNDER ++ ++ CRS CARIBBEAN RED SNAPPER ++ +++ SG SQUARETAIL GROUPER ++ +++ CS CUBERA SNAPPER ++ +++ SH SHEEPHEAD ++ +++ CT CORAL TROUT ++ ++ SUF SUNFISH +++ + DM DOLPHIN ++ + SV VERMILION SNAPPER ++ +++ DTG DUSKY TAIL GROUPER ++ ++ SWF SWORDFISH - - FS FARM SALMON - - T TRA +++ +++ GG GAG GROUPER +++ +++ TC TROUT COD ++ +++ GS GRAY SNAPPER +++ +++ TH TOMATO HIND + ++ HC HYBRID CATFISH +++ +++ TIL TILAPIA +++ +++ HD HADDOCK - - W WAHOO + +/- HS HOG SNAPPER + + WH WHITING - - LS LANE SNAPPER ++ +++ WLG WAVY LINED GROUPER ++ ++ M MULLET +++ +++ WS WILD SALMON - - MS MANGROVE SNAPPER +++ +++ YEG YELLOW EDGE GROUPER +++ +++ OP OCEAN PERCH +++ +++ YS YELLOWTAIL SNAPPER +++ +++ OR ORANGE ROUGHY ++ +++ YT YELLOWFIN TUNA - - ORANGE SPOTTED OSG +++ +++ GROUPER

Figure 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles of 20 cooked fish species. There are two groups of major protein bands (36 kDa and 10-13 kDa) shown on the gel. The 36 kDa protein appears in all extracts of cooked fish species tested, while the 10-13 kDa band (parvalbumin) is missing in three species: Pollock, yellowfin tuna and farm-raised salmon. B — Basa; SF — Southern Flounder; SH — Sheephead; CRB — Caribbean Red Snapper; LS — Lane Snapper, BSB — Black Sea Bass; GS — Gray Snapper; SB — Striped Bass; OR — Orange Roughy; RS — Red Snapper; P — Pollock; HC — Hybrid Catfish; ChC — Channel Catfish; BC — Blue Catfish; YT — Yellowfin Tuna; BG — Black Grouper; C — Cobia; FS — Farm Salmon; GG — Gag Grouper; RG — Red Grouper; — very low amount or no parvalbumin.

Figure 2. Antigenic protein banding patterns in cooked fish extracts by Western blot analysis, using MAbs 3E1. This MAb reacts with the 10-13 kDa protein in all fish species except these three species: Pollock, yellowfin tuna and farm-raised salmon. B — Basa; SF — Southern Flounder; SH — Sheephead; CRB — Caribbean Red Snapper; LS — Lane Snapper, BSB — Black Sea Bass; GS — Gray Snapper; SB — Striped Bass; OR — Orange Roughy; RS — Red Snapper; P — Pollock; HC — Hybrid Catfish; ChC — Channel Catfish; BC — Blue Catfish; YT — Yellowfin Tuna; BG — Black Grouper; C — Cobia; FS — Farm Salmon; GG — Gag Grouper; RG — Red Grouper; — very low amount or no parvalbumin.

Figure 3. Comparison of the antigenic protein banding patterns in cooked fish extracts using MAbs 3E1 and PARV-19 by Western blot analysis. YT — Yellowtail Snapper; B — Basa; FS — Farm Salmon; YT — Yellowfin Tuna; ChC — Channel Catfish; P — Pollock; RS — Red Snapper; SB — Striped Bass; SH — Sheephead; — very low amount or no parvalbumin.

Figure 4. Effect of presence and absence of calcium on the immunoreactivity of anti-parvalbumin specific MAbs 3E1 with fish and meat extracts determined by indirect ELISA. Absorbance readings at 410 nm: S — Scamp; SUF — Sunfish; M — Mullet; SB — Striped Bass; CF — Catfish; POM — Pompano; RG — Red Grouper; CO —Cobia; SH — Sheephead; TIL — Tilapia; RS — Red Snapper; BA — Basa; T — Tra; AJ — Amberjack; W — Wahoo; SWF — Swordfish; AH — Alaskan Halibut; YT — Yellowfin Tuna; COD — Codfish; WH — Whiting; HD — Haddock; SWF — Swordfish; P — Pollock; FS — Farm Salmon; Rat — Rat thigh; Frog — Frog leg.

Figure 5. Effect of presence and absence of calcium on the immunoreactivity of anti-parvalbumin specific PARV-19 with fish and meat extracts determined by indirect ELISA. Absorbance readings at 410 nm: S — Scamp; SUF — Sunfish; M — Mullet; SB — Striped Bass; CF — Catfish; POM — Pompano; RG — Red Grouper; CO —Cobia; SH — Sheephead; TIL — Tilapia; RS — Red Snapper; BA — Basa; T — Tra; AJ — Amberjack; W — Wahoo; SWF — Swordfish; AH — Alaskan Halibut; YT — Yellowfin Tuna; COD — Codfish; WH — Whiting; HD — Haddock; SWF — Swordfish; P — Pollock; FS — Farm Salmon; Rat — Rat thigh; Frog — Frog leg.

Figure 6. Epitope comparison of MAbs 3E1 and PARV-19. Absorbance readings at 410 nm of immunoreactivity of cooked tra extract containing 0.5l of soluble proteins, against MAb PARV-19, MAb 3E1 and the two antibodies together (3E1+PARV-19) in the ratio 1:1; BLK – blank.

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BIBLIOGRAPHIC SKETCH

I was born in Gdansk, Poland on October 3, 1980. I attended a high school with gastronomic profile where my interest in food related disciplines had its beginning. I went to University of Warmia and Mazury in Olsztyn, Poland in 2001, and received a master’s degree in food technology in 2006. In 2005, during my master’s program, I was invited by Dr. Yun-Hwa Peggy Hsieh to come to Tallahassee, Florida for summer internship in her laboratory. After graduation I decided to continue my education in the United States. I was accepted to the Food Science master’s program, in the Department of Nutrition, Food and Exercise Sciences, at Florida State University, in the fall of 2006. I joined Dr. Hsieh’s lab and focused on food safety research using immunochemical and biochemical techniques.