Characterization of Monoclonal Antibody Specific to Fish Major Allergen â•fi Parvalbumin

54 Pages·2015·1.75 MB·English

by  Kamil Gajewski

Florida State University Libraries
Electronic Theses, Treatises and Dissertations The Graduate School
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

By

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.

ii
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.
iii
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
iv
APPENDICES .................................................................................................... 26

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

REFERENCES ................................................................................................... 38

BIOGRAPHICAL SKETCH .............................................................................. 45
v
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
vi
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

vii
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.
viii
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
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