A New Immunodiagnostic Test for the Detection of Southern Hemisphere Fish Residues in Processed Foods Ji Liang A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Chemical Engineering Faculty of Engineering The University of New South Wales Sydney Australia August 2018 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Liang First name: Ji Other name/s: Abbreviation for degree as given in the University calendar: PhD School: School of Chemical Engineering Faculty: Faculty of Engineering Title: A New Immunodiagnostic Test for the Detection of Southern Hemisphere Fish Residues in Processed Foods Abstract 350 words maximum: (PLEASE TYPE) Fish products are regulated for mandatory allergen labelling in many countries including Australia. However, the commercially available fish ELISA kits have been developed using parvalbumin (PAV) or fish protein extracts from northern hemisphere fish species. This study, therefore, aims to develop an immunodiagnostic test for the detection and quantification of (commercially important) southern hemisphere fish residues in processed foods, by developing antibodies with broad species specificity for southern hemisphere fish species. In order to quantitatively estimate and rank the cross-reactivity between fish species, the cross-reactivity indices were developed using two model antibodies specific to fish parvalbumin (anti-cod PoAb and anti-carp MoAb). Compared with the crude extracts, improved correlation between the PAV content and the immunoreactivity of heated PAV was observed for both antibodies (i.e., R2=0.82 for the anti-cod PoAb and R2=0.55 for the anti-carp MoAb). There was a strong phylogenetic relationship, showing strong immunoreactivity amongst fish species from the order of Perciformes; and weak or no immunoreactivity amongst species from Salmoniformes, Ophiidiformes, Scombriformes, Scorpaeniformes, and Tetraodontiformes. Polyclonal antibodies were developed using a heat-treated immunogen prepared from thirteen selected fish species. Two sensitive sandwich ELISAs, differing in their capture-detection antibody combinations, were developed. The (HM)S627#4 – (HM)RB#2 assay yielded an average detection limit of 0.08 μg/L; and the (HM)RB#2 – (HM)S627#4 assay yielded an average detection limit of 0.59 μg/L. The newly developed assays showed significantly improved detection of fish species from Salmoniformes, Ophidiiformes, and Scopaeniformes, which previously showed weak or no immunoreactivity. The assay buffer was optimised with addition of various additives (e.g., glycerol, glycine and NaCl) to increase the protein recovery from < 10% to 85 – 122%. The spike and recovery study of SM PAV in four blank food matrices achieved good protein recovery of 87 – 117%, with satisfactory assay precision (<18%). A product survey of Thai-manufactured foods showed positive results in 50% of the products. Amongst the negative products were the fish sauce and the yellow curry paste, which did not show quantifiable fish residues, possibly due to the extensive protein hydrolysis. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). ………………………............. ……….……………………...……. 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I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed....................................... Date........................................ COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed Date ........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed Date ........................... Abstract Fish products are regulated for mandatory allergen labelling in many countries including Australia. However, the four commercially available fish ELISA kits have been developed using parvalbumin (PAV) or fish protein extracts from northern hemisphere fish species. This study, therefore, aims to develop an immunodiagnostic test for the detection of (commercially important) southern hemisphere fish residues in processed foods, by developing antibodies with broad species specificity for southern hemisphere fish species. In order to quantitatively estimate and rank the cross-reactivity between fish species, the cross-reactivity indices were developed using two model antibodies specific to fish parvalbumin (anti-cod PoAb and anti-carp MoAb). Sixteen of the thirty-seven species (43%) showed a positive correlation (R2=0.74) between the PAV content in crude fish extracts and the immunoreactivity of PAV with the anti-cod PoAb; while no correlation between the same was established with the anti-carp MoAb. The thermal treatment of the crude extracts, further improved the correlation between the PAV content and the immunoreactivity of heated PAV for both antibodies (i.e., R2=0.82 for the anti-cod PoAb and R2=0.55 for the anti-carp MoAb). There was a strong phylogenetic relationship, showing strong immunoreactivity amongst fish species from the order of Perciformes; and weak or no immunoreactivity amongst species from Salmoniformes, Ophiidiformes, Scombriformes, Scorpaeniformes, and Tetraodontiformes. The polyclonal antibodies were developed using a heat-treated immunogen prepared from thirteen selected fish species. Two sensitive sandwich ELISAs, differing in their capture-detection antibody combinations, were developed. The (HM)S627#4 – (HM)RB#2 assay yielded an average detection limit (LLOD, determined as C15 (a i concentration of fish protein produce 15% of colour relative to the maximum colour development) of 1.7 ± 0.9 μg/L; and the (HM)RB#2 – (HM)S627#4 assay yielded an average LLOD of 1.6 ± 0.4 μg/L. The detection limit calculated as the mean measure value of 10 blank sample replicates plus 3 times of the standard deviation (SD) of the mean value (LLOD’) for the (HM)S627#4 – (HM)RB#2 assay was 0.08 μg/L, and the LLOD’ for the (HM)RB#2 – (HM)S627#4 assay was 0.59 μg/L. The (HM)S627#4 – (HM)RB#2 assay showed a wider species specificity, ranging from 0% for swordfish to 686% for eastern school whiting. While the (HM)RB#2 – (HM)S627#4 assay exhibited a narrower species specificity ranging from 0.0% for swordfish to 122.2% for Spanish mackerel. The newly developed assays showed significantly improved detection of fish species from Salmoniformes, Ophidiiformes, and Scopaeniformes, which previously showed weak or no immunoreactivity. The assay buffer was optimised with addition of various additives (e.g., glycerol, glycine and NaCl) to increase the protein recovery from less than 10% to 84.8 – 122.1%. Food matrices (i.e., rice cake and corn flour) with high carbohydrate contents had detectable immunological responses with the (HM)RB#2 – (HM)S627#4 assay, due to the