Identification and characterisation of novel shellfish allergens for improved diagnosis

Kamath, Sandip Dayanand (2014) Identification and characterisation of novel shellfish allergens for improved diagnosis. PhD thesis, James Cook University.

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Food allergy is in general defined as a type I, IgE antibody mediated hypersensitivity reaction, which is triggered on exposure to innocuous food sources. Increased consumption of seafood has not only resulted in higher incidences of allergic reactions but also a rise in occupational allergic sensitisation and asthma among workers due to inhalation of air-borne food allergens. The problem of accidental allergen exposure is compounded by the highly cross-reactive nature of major shellfish allergens with dust mite and insect allergens.

A detailed review on the current status of food allergy and diagnostic approaches is provided in Chapter 1. In the case of crustacean and mollusc allergy, allergy testing is mostly performed using whole crude shellfish preparations. The crude protein extracts currently used for such tests are prepared using only a few selected European species which are not commonly consumed in the Asia-Pacific region. Such diagnostic tests may not be accurate enough to detect specific IgE against allergens present in shellfish species specific to Australia. Moreover, since most of the crustaceans are processed by means of heat-treatment, the effects of heat on crustacean allergens and subsequent IgE binding properties are not fully understood.

The work presented in this PhD thesis provides a comprehensive study on the identification and characterisation of major and novel allergens found in shellfish species, specific for the Asia-Pacific region and their relevance among the Australian population. Furthermore, the effects of heat processing on the stability of shellfish allergens and the molecular basis of immunological cross-reactivity were investigated.

In Chapter 2, an extensive panel of raw and heat-treated shellfish allergen extracts were produced which included eleven crustacean and seven mollusc species. Tropomyosin was identified as the major heat-stable allergenic protein in the tested shellfish extracts using specific monoclonal antibodies. Enhanced antibody binding was demonstrated to tropomyosin in all heat-treated crustacean species but only in few selected mollusc species. Based on the antibody binding data and worldwide importance, Black tiger prawn was investigated in further detail for characterisation of the prawn allergen repertoire and the effects of heat-processing on their IgE binding properties.

In Chapter 3, novel mass spectrometric methods were developed and validated for the identification and sequencing of tropomyosin and other shellfish allergens. Subsequently in Chapter 4, IgE binding proteins in the Black tiger prawn raw and heat-treated extracts were detected using sera from a patient cohort. Several IgE binding allergens were identified in addition to tropomyosin such as arginine kinase, myosin light chain, sarcoplasmic calcium binding protein, triose-phosphate isomerase and two putative novel allergens fructose bis-phosphate aldolase and titin. Enhanced IgE binding was observed to prawn allergens in the heat-treated protein extracts. For the first time, IgE binding was demonstrated to a heat-generated tropomyosin fragment which highlighted the stability of the allergenic molecule post-degradation and which may have implications in exposure to heat-processed prawns. Three-dimensional homology modelling of the allergens highlighted the existence of dimeric or oligomeric protein structures; shown to be a strong characteristic of allergenic proteins.

In Chapter 5 and Chapter 6, the immunological cross-reactivity of tropomyosin was analysed in detail between three commonly consumed Australian crustacean species; Black tiger prawn, King prawn and Blue swimmer crab. Differential IgE antibody binding was observed to King prawn tropomyosin as compared to Black tiger prawn tropomyosin thus indicating structural variation of the IgE binding epitopes. Moreover, higher IgE reactivity was observed to the heated prawn or crab extracts in patients sensitised to tropomyosin, thus demonstrating enhanced IgE reactivity to tropomyosin post heat treatment. Basophil activation assays revealed stronger IgE reactivity to raw prawn and crab extracts among patients who lacked IgE binding to tropomyosin, thus indicating reactivity to crustacean allergens other than tropomyosin. To further investigate the differential IgE binding observed for crustacean tropomyosin, a multiple sequence alignment of 60 invertebrate tropomyosins was performed specifically for the previously identified eight IgE binding epitopes. A detailed comparison revealed epitope-specific conservation or variation among the various allergenic tropomyosins. IgE binding epitope regions 2 (amino acid residue 91-101), 4 (187-197) and 5a (251-259) were found to be highly conserved among crustacean and mollusc species and may be responsible for the immunological and clinical cross-reactivity observed.

Finally, in Chapter 7 a sensitive immunoassay was developed and validated for the quantification of aerosolised crab tropomyosin in crab-processing factories. Using this sensitive assay, tropomyosin was quantified in the personal breathing zones of 80 crab processing workers. High exposure levels to airborne crab allergens were observed among workers involved in crab meat-boiling or heating activities, thus indicating higher generation of bio-aerosols in these areas.

In summary, tropomyosin seems to be a relevant cross-reactive major allergen among the affected Australian population. Heat-processing of shellfish has an enhancing effect on the IgE antibody binding and reactivity of tropomyosin and other heat-stable allergens. Ex vivo IgE reactivity was demonstrated to the identified prawn allergens, highlighting the importance of characterising the whole allergen repertoire in other shellfish species. The work presented in this thesis provides an important contribution towards the development of improved and sensitive allergy diagnostic platforms. Future work involving IgE epitope mapping of the identified shellfish allergens is central to the development of component resolved diagnostics and immunotherapeutic strategies for shellfish allergy.

Item ID: 43779
Item Type: Thesis (PhD)
Keywords: allergens; allergic reactions; antibodies; Asia-Pacific; Australia; crustaceans; food allergies; food sensitivities; hypersensitivity; immune response; molluscs; Oceania; seafood; shellfish; tropomyosin
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Publications arising from this thesis are available from the Related URLs field. The publications are:

Chapter 1: Lopata, Andreas L., and Kamath, Sandip (2012) Shellfish allergy diagnosis: gaps and needs. Current Allergy and Clinical Immunology, 25 (2). pp. 60-66.

Chapter 2: Kamath, Sandip D., Rahman, Anas M. Abdel, Komoda, Toshikazu, and Lopata, Andreas L. (2013) Impact of heat processing on the detection of the major shellfish allergen tropomyosin in crustaceans and molluscs using specific monoclonal antibodies. Food Chemistry, 141 (4). pp. 4031-4039.

Chapter 3: Rahman, Anas M. Abdel, Kamath, Sandip, Lopata, Andreas L., and Helleur, Robert J. (2010) Analysis of the allergenic proteins in black tiger prawn (Penaeus monodon) and characterization of the major allergen tropomyosin using mass spectrometry. Rapid Communications in Mass Spectrometry, 24 (16). pp. 2462-2470.

Chapter 4: Kamath, Sandip D., Rahman, Anas M. Abdel, Voskamp, Asrid, Komoda, Toshikazu, Rolland, Jennifer M., O'hehir, Robyn E., and Lopata, Andreas L. (2014) Effect of heat processing on antibody reactivity to allergen variants and fragments of black tiger prawn: a comprehensive allergenomic approach. Molecular Nutrition & Food Research, 58 (5). pp. 1144-1155.

Chapter 5: Abramovitch, Jodie B., Kamath, Sandip, Varese, Nirupama, Zubrinich, Celia, Lopata, Andreas L., O'Hehir, Robyn E., and Rolland, Jennifer M. (2013) IgE reactivity of blue swimmer crab (Portunus pelagicus) tropomyosin, Por p 1, and other allergens; cross-reactivity with black tiger prawn and effects of heating. PLoS ONE, 8 (6). pp. 1-13.

Chapter 6: Kamath, Sandip D., Thomassen, Marte R., Saptarshi, Shruti R., Nguyen, Hong M.X., Aasmoe, Lisbeth, Bang, Berit E., and Lopata, Andreas L. (2014) Molecular and immunological approaches in quantifying the air-borne food allergen tropomyosin in crab processing facilities. International Journal of Hygiene and Environmental Health, 217 (7). pp. 740-750.

Date Deposited: 17 May 2016 23:49
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110106 Medical Biochemistry: Proteins and Peptides (incl Medical Proteomics) @ 50%
11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110107 Metabolic Medicine @ 50%
SEO Codes: 92 HEALTH > 9202 Health and Support Services > 920203 Diagnostic Methods @ 50%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920108 Immune System and Allergy @ 50%
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