Graphene/zinc oxide nanocomposite : a versatile platform for electrochemical-based genosensor

In this work, a versatile electrochemical biosensing platform was developed based on graphene/zinc oxide (G/ZnO) nanocomposite. For the synthesis of G/ZnO nanocomposite, two facile and green approaches were employed to eradicate the issues associated with conventional methods, which use harsh chemicals and high temperature. The G/ZnO nanocomposite synthesised via low temperature hydrothermal growth method exhibited approximate 58 times improvement in terms of sensitivity as compared to the G/ZnO nanocomposite synthesised via the mechanical stirring method. Therefore, the G/ZnO nanocomposite with higher sensitivity was employed for the following work. Results from cyclic voltammetry and amperometry showed that the G/ZnO-modified electrode displayed a wide linear range up to 15 mM for the detection of H2O2 and sensitivity improvements of 200% in comparison to the control sample. Subsequently, an electrochemical genosensor based on G/ZnO nanocomposite was fabricated for the detection of Avian Influenza H5N1 virus. The G/ZnO-based genosensor displayed its potential in replacing the conventional detection method, with result showing higher sensitivity and efficiency. The G/ZnO-based genosensor was further applied for the detection of Coconut Cadang-Cadang Viroid disease (CCCVd) single stranded RNA (ssRNA). Promising results were obtained with high specificity in discriminating the target from mismatched ssRNA sequences

Editing the genome of chicken primordial germ cells to introduce alleles and study gene function

With continuing advances in genome sequencing technology, the chicken genome assembly is now better annotated with improved accuracy to the level of single nucleotide polymorphisms. Additionally, the genomes of other birds such as the duck, turkey and zebra finch have now been sequenced. A great opportunity exists in avian biology to use genome editing technology to introduce small and defined sequence changes to create specific haplotypes in chicken to investigate gene regulatory function, and also perform rapid and seamless transfer of specific alleles between chicken breeds. The methods for performing such precise genome editing are well established for mammalian species but are not readily applicable in birds due to evolutionary differences in reproductive biology. A significant leap forward to address this challenge in avian biology was the development of long-term culture methods for chicken primordial germ cells (PGCs). PGCs present a cell line in which to perform targeted genetic manipulations that will be heritable. Chicken PGCs have been successfully targeted to generate genetically modified chickens. However, genome editing to introduce small and defined sequence changes has not been demonstrated in any avian species. To address this deficit, the application of CRISPR/Cas9 and short oligonucleotide donors in chicken PGCs for performing small and defined sequence changes was investigated in this thesis. Specifically, homology-directed DNA repair (HDR) using oligonucleotide donors along with wild-type CRISPR/Cas9 (SpCas9-WT) or high fidelity CRISPR/Cas9 (SpCas9-HF1) was investigated in cultured chicken PGCs. The results obtained showed that small sequences changes ranging from a single to a few nucleotides could be precisely edited in many loci in chicken PGCs. In comparison to SpCas9-WT, SpCas9-HF1 increased the frequency of biallelic and single allele editing to generate specific homozygous and heterozygous genotypes. This finding demonstrates the utility of high fidelity CRISPR/Cas9 variants for performing sequence editing with high efficiency in PGCs. Since PGCs can be converted into pluripotent stem cells that can potentially differentiate into many cell types from the three germ layers, genome editing of PGCs can, therefore, be used to generate PGC-derived avian cell types with defined genetic alterations to investigate the host-pathogen interactions of infectious avian diseases. To investigate this possibility, the chicken ANP32A gene was investigated as a target for genetic resistance to avian influenza virus in PGC-derived chicken cell lines. Targeted modification of ANP32A was performed to generate clonal lines of genome-edited PGCs. Avian influenza minigenome replication assays were subsequently performed in the ANP32A-mutant PGC-derived cell lines. The results verified that ANP32A function is crucial for the function of both avian virus polymerase and human-adapted virus polymerase in chicken cells. Importantly, an asparagine to isoleucine mutation at position 129 (N129I) in chicken ANP32A failed to support avian influenza polymerase function. This genetic change can be introduced into chickens and validated in virological studies. Importantly, the results of my investigation demonstrate the potential to use genome editing of PGCs as an approach to generate many types of unique cell models for the study of avian biology. Genome editing of PGCs may also be applied to unravel the genes that control the development of the avian germ cell lineage. In the mouse, gene targeting has been extensively applied to generate loss-of-function mouse models to use the reverse genetics approach to identify key genes that regulate the migration of specified PGCs to the genital ridges. Avian PGCs express similar cytokine receptors as their mammalian counterparts. However, the factors guiding the migration of avian PGCs are largely unknown. To address this, CRISPR/Cas9 was used in this thesis to generate clonal lines of chicken PGCs with loss-of-function deletions in the CXCR4 and c-Kit genes which have been implicated in controlling mouse PGC migration. The results showed that CXCR4-deficient PGCs are absent from the gonads whereas c-Kit-deficient PGCs colonise the developing gonads in reduced numbers and are significantly reduced or absent from older stages. This finding shows a conserved role for CXCR4 and c-Kit signalling in chicken PGC development. Importantly, other genes suspected to be involved in controlling the development of avian germ cells can be investigated using this approach to increase our understanding of avian reproductive biology. Finally, the methods developed in this thesis for editing of the chicken genome may be applied in other avian species once culture methods for the PGCs from these species are develope

Detection and thermal stabilization of virus based on surface properties

Viral diseases take the lives of millions of people each year. The most effective methods to prevent viral disease outbreak are viral detection to reduce contact with viral pathogens and vaccines to prevent disease. To reduce the costs of the detection of viruses and improve vaccine formulation, we explored viral surface properties. The properties we have focused on are viral hydrophobicity and surface charge using chemical force microscopy (CFM). CFM is a single-particle technique that measures the adhesion force of a functionalized atomic force microscopy (AFM) probe, and in this study, a virus covalently bound to a surface. The non-enveloped porcine parvovirus (PPV) and enveloped bovine viral diarrhea virus (BVDV) were used to demonstrate the use of CFM for viral particles with different surface properties. The high hydrophobicity of PPV and BVDV by CFM was used for a ligand-free, non-specific virus detection method that relies on the interaction of virus with osmolytes. It was previously found that the osmolyte mannitol can preferentially aggregate viruses while leaving proteins in solution. The virus was incubated with gold nanoparticles (AuNPs), and aggregation of the virus-AuNP complex with mannitol was detected by dynamic light scattering (DLS). The isoelectric point (pI) of PPV and BVDV by CFM was used for a vaccine formulation strategy of virus particle encapsulation by polymers that relies on electrostatic interactions of the virus with polypeptides. The random screen of different ratios of polyelectrolytes to encapsulate viruses could be reduced by knowing the virus pIs. An encapsulated non-enveloped PPV is thermally stabilized, demonstrating that this method is promising for formulating thermostable vaccines. We have developed a unique detection method and can improve vaccine formulation that would reduce the impact of viral diseases worldwide, based on the viral surface properties

Construction and molecular characterisation of an improved chloroplast transformation vector system as a versatile delivery and expression platform for in-vitro propagated Nicotiana benthamiana

The objective of this study is to develop a versatile vector system for the delivery and expression of transgenes in the chloroplast genome of N. benthamiana. The successful advent of such a system would vastly streamline the construction process of chloroplast transformation vectors for the expression of recombinant proteins, such as vaccine candidates, in the chloroplasts of N. benthamiana. Transgenes targeted to the chloroplasts of higher plants are expected to be expressed at considerably higher levels as compared to nuclear expression, resulting in more significant accumulation of recombinant proteins. In this study, a 2-part chloroplast transformation vector system was developed and two new GFP vector prototypes, pEXPR-G and pEXPR-UG were generated for preliminary evaluation of functionality. The aadA and GFP expression cassettes of pEXPR-G and pEXPR-UG were evaluated in E. coli prior to actual delivery into N. benthamiana via particle bombardment. Particle bombardment parameters were optimised with particular emphasis on minimising excessive damage to the target tissue in order to facilitate the recovery of antibiotic resistant shoots and calli following transformation. To further evaluate the versatility of the developed system for the expression of vaccine antigens, recombinant vectors, pEXPR-HA and pEXPR-NA were constructed for the delivery of hemagglutinin (HA) and neuraminidase (NA) genes of avian influenza strain H5N1 into the chloroplast genome of N. benthamiana. Experimental results indicated that pEXPR-G and pEXPR-UG were fundamentally functional in E. coli and both the aadA and GFP expression cassettes were active, allowing the bacteria to withstand 500mg/l spectinomycin and express the transgene of interest at the protein level. Similar results were also observed in transplastomic N. benthamiana transformed with pEXPR-UG and pEXPR-NA. In essence, the developed 2-part chloroplast transformation vector system was found to be highly versatile and could be conveniently applied for the construction of transformation vectors for the delivery and expression of HA and NA in the chloroplast of N. benthamiana

SIMPLIFIED NUCLEIC ACID ISOLATION AND DETECTION TECHNIQUES FOR POINT-OF-CARE APPLICATIONS

The first step towards the treatment of any disease is diagnosis; however, a major hindrance towards monitoring diseases remains the availability of rapid diagnostic tests to detect DNA and RNA biomarkers.Unfortunately, traditional assays to test for these analytes are limited to centralized laboratories. For example, the gold-standard sample preparation methods to isolate and purify nucleic acid analytes require multiple hands-on steps, numerous chemicals, and specialized equipment. Recent research endeavors have made incremental progress to simplify these procedures by miniaturizing these steps, but still require the numerous chemicals and bulky peripheral instrumentation for operation. Similarly, gold-standard detection methods, namely quantitative PCR (qPCR), rapidly cycle between near-boiling temperatures, thus mandating the use of sophisticated instrumentation with high power demands. These methods for sample preparation and target detection are overall laborious, time-consuming, and require technical expertise thereby imposing large time, and financial costs for diagnoses. To address these challenges, this dissertation aims to shift the paradigm of how we approach sample preparation for nucleic acid isolation, and to design novel assays for rapid, specific, and multiplexed detection in a single reaction. First, a new sample preparation method is presented that simultaneously accomplishes three time-consuming sample preparation steps (cell lysis, DNA capture, and purification) in less than ten minutes. This platform enables DNA isolation from whole blood droplets, with subsequent amplification and detection of both genomic and pathogenic DNA bound to the microparticle surface. Second, we explore optimizations of a popular isothermal DNA amplification technique that previously demonstrated multiplex detection of bacterial genomes. By developing a triplex assay towards the identification of drug resistant bacteria, we address inhibition that is normally observed with this technique, and present strategies towards achieving amplification within 30 minutes. Lastly, we outline design consideration towards the development of a novel amplification scheme specifically for microRNA targets. This system leverages both DNA and RNA polymerases to achieve positive feedback and thus, requires evaluation of enzymes, sequence design, and buffers to inform assay design. Together, this work advances the development of NAATs towards a simplified, specific and multiplexed system

Construction and molecular characterisation of an improved chloroplast transformation vector system as a versatile delivery and expression platform for in-vitro propagated Nicotiana benthamiana

The objective of this study is to develop a versatile vector system for the delivery and expression of transgenes in the chloroplast genome of N. benthamiana. The successful advent of such a system would vastly streamline the construction process of chloroplast transformation vectors for the expression of recombinant proteins, such as vaccine candidates, in the chloroplasts of N. benthamiana. Transgenes targeted to the chloroplasts of higher plants are expected to be expressed at considerably higher levels as compared to nuclear expression, resulting in more significant accumulation of recombinant proteins. In this study, a 2-part chloroplast transformation vector system was developed and two new GFP vector prototypes, pEXPR-G and pEXPR-UG were generated for preliminary evaluation of functionality. The aadA and GFP expression cassettes of pEXPR-G and pEXPR-UG were evaluated in E. coli prior to actual delivery into N. benthamiana via particle bombardment. Particle bombardment parameters were optimised with particular emphasis on minimising excessive damage to the target tissue in order to facilitate the recovery of antibiotic resistant shoots and calli following transformation. To further evaluate the versatility of the developed system for the expression of vaccine antigens, recombinant vectors, pEXPR-HA and pEXPR-NA were constructed for the delivery of hemagglutinin (HA) and neuraminidase (NA) genes of avian influenza strain H5N1 into the chloroplast genome of N. benthamiana. Experimental results indicated that pEXPR-G and pEXPR-UG were fundamentally functional in E. coli and both the aadA and GFP expression cassettes were active, allowing the bacteria to withstand 500mg/l spectinomycin and express the transgene of interest at the protein level. Similar results were also observed in transplastomic N. benthamiana transformed with pEXPR-UG and pEXPR-NA. In essence, the developed 2-part chloroplast transformation vector system was found to be highly versatile and could be conveniently applied for the construction of transformation vectors for the delivery and expression of HA and NA in the chloroplast of N. benthamiana

Development and evaluation of QCM sensors for the detection of influenza virus from clinical samples

The Quartz crystal microbalance (QCM) is a very sensitive mass-detecting device which is based on changes in to the vibrational frequency of quartz crystals after adsorption of substances to a modified crystal surface. In this study a QCM-based biosensor was developed for the rapid diagnosis of influenza viruses and its suitability and limitations were compared with currently available diagnostic methods on 67 clinical samples (nasal washes) received during the 2005 Australian winter. The type-specific and conserved viral M1 proteins of both A/PR/8/34 and B/Lee/40 viruses were used to prepare polyclonal antisera for the development of an ELISA. The limits of detection of ELISAs for the detection of purified A/PR/8/34 and B/Lee/40 ƒnviruses were 20ƒÝg/mL ƒnand 14 ƒÝg/mL using polyclonal antibodies, and 30 ƒÝg/mL and 20 ƒÝg/mL for monoclonal antibodies, respectively. The limit for detection of each virus was 104 pfu/mL, irrespective of whether antisera or monoclonal antibodies were used for capture. Non-purified cell culture-grown preparations of either virus could be detected at 103 pfu/mL The QCM utilised the same reagents used in ELISAs. However, a number of parameters were then further optimised to improve the sensitivity of the tests. These included blocking of non-specific binding, examination of the effects of flow-cell compression, the role of pH, flow rate, antibody concentration and the addition of protein A to the crystal surfaces of the biosensor. The lowest virus concentration that could be detected with the QCM was 104 pfu/mL for egg-grown preparations of both A/PR/8/34 and B/Lee/40, which could be detected within 30 min. However, conjugation of 13 nm gold nanoparticles to a second detector antibody resulted in a 10-fold increase in sensitivity and a detection limit of 103 pfu/mL that could be determined within 1 h. The direct detection of the influenza viruses in nasal samples was not possible by QCM because of the significant frequency fluctuation that was probably caused by the viscosity of the samples. Therefore, an additional culture step of 12 h was required, which increased the processing time to 2 days. The QCM/nanoparticle method was shown to be as sensitive as the standard cell culture method, and the QCM method as sensitive as the shell vial method. The QCM and QCM/nanoparticle methods were shown to be 81 and 87% as sensitive and both were 100% as specific as the real-time polymerase chain reaction. However, for use in rapid diagnosis, improvements are required to remove frequency fluctuation resulting from the direct use of nasal samples

Nano-probes for point of care diagnostics

The COVID-19 pandemic has exposed deep health inequalities between more economically developed and less economically developed countries: both in terms of diagnostics and vaccinations. Robust and low-cost point of care devices are needed to ease these diagnostic inequalities. Current point of care lateral flow immunoassays, utilise proteins, such as antibodies, to sense for analytes. This is epitomised by the malaria rapid diagnostic test and archetypal home pregnancy test. Glycans are emerging as alternative detection units due to their fundamental role in biological signalling and recognition events. Furthermore, the increased robustness, low-cost and synthetic possibilities offered by glycan-based systems, especially glycosylated polymers, make them a promising alternative to antibody-based biosensing and diagnostic systems. Chapter 1 discusses the current use of protein-based lateral flow and flow-through devices; their advantages and disadvantages versus non-point of care techniques, and the potential of glycan-based lateral flow devices. The concepts introduced in Chapter 1 are then applied in Chapters 2 through 5. Chapter 2 demonstrates the use of glycosylated polymer-coated nanoparticles, produced by controlled radical polymerisation techniques for the sensitive, label-free detection of lectins in lateral flow and flow-through. The systems produced use only glycans, not antibodies, to provide recognition – a “lateral flow glyco-assay.” The lessons learned in Chapter 2 are applied in Chapter 3 to probe the glycan-binding of the SARS-COV-2 spike protein in a “flow-through glyco-assay” and target a pseudovirus mimic of the target coronavirus in a lateral flow glyco-assay. Chapter 4 builds on Chapters 2 and 3, applying the fledgling glyco-assay technology to the “real-world” by sensing for the SARS-COV-2 virus in patient samples, alongside exploring the robustness of the devices themselves. Having explored the concept of glycosylated polymer-coated nanoparticles in lateral flow and flow-through setups; Chapter 5 changes focus and explores the use of polymeric anchors for the design of all-polymer (“vegan”) lateral flow and flow-through devices. This work completely removes proteins as either detecting units or anchors from lateral flow for the first time. Chapters 6 and 7 explore more fundamental Chemistry than the previous chapters. Chapter 6 considers the use of the Mannich reaction to produce monosaccharides with amine functionality at C2, ideal for polymer conjugation, while maintaining hydroxyl functionality at C2. Although unsuccessful with the reagents used, the chapter highlights a potential avenue of future chemical exploration in novel glycan synthesis. Chapter 7 pulls together the x-ray photoelectron analysis data and spectra collected across a range of studies, including data collected in previous chapters, and considers if x-ray photoelectron spectroscopy can be used to determine relative grafting density in glycosylated polymer-coated nanoparticle systems. In summary, the key components of the emerging technology of lateral flow glycoassays are introduced, interrogated and investigated. The prototype devices tested against model proteins, viral proteins and patient samples, are found to show specificities and sensitivities that rival lateral flow immunoassay systems. The understanding developed in this thesis could pave the way to the first generation of lateral flow glyco-assays that are low-cost, stable in a wide range of conditions, and able to target a wide range of analytes and diseases

Approaches to DIVA vaccination for fish using infectious salmon anaemia and koi herpesvirus disease as models

The expanding aquaculture industry continues to encounter major challenges in the form of highly contagious aquatic viruses. Control and eradication measures targeting the most lethal and economically damaging virus-induced diseases, some of which are notifiable, currently involve ‘stamping out’ policies and surveillance strategies. These approaches to disease control are performed through mass-culling followed by restriction in the movement of fish and fish products, resulting in considerable impacts on trade. Although effective, these expensive, ethically complex measures threaten the sustainability and reputation of the aquatic food sector, and could possibly be reduced by emulating innovative vaccination strategies that have proved pivotal in maintaining the success of the terrestrial livestock industry. DIVA ‘differentiating infected from vaccinated animal’ strategies provide a basis to vaccinate and contain disease outbreaks without compromising ‘disease-free’ status, as antibodies induced specifically to infection can be distinguished from those induced in vaccinated animals. Various approaches were carried out in this study to assess the feasibility of marker/DIVA vaccination for two of the most important disease threats to the global Atlantic salmon and common carp/koi industries, i.e. infectious salmon anaemia (ISA) and koi herpesvirus disease (KHVD), respectively. Antibody responses of Atlantic salmon (Salmo salar L.), following immunisation with an ISA vaccine, administered with foreign immunogenic marker antigens (tetanus toxoid (TT), fluorescein isothiocyanate (FITC) and keyhole limpet hemocyanin (KLH)) were assessed by antigen-specific enzyme linked immunosorbent assay (ELISA). Although antibodies were induced to some markers, these were unreliable and may have been affected by temperature and smoltification. Detectable antibodies to ISAV antigen were also largely inconsistent despite low serum dilutions of 1/20 being employed for serological analysis. The poor antibody responses of salmon to the inactivated ISA vaccine suggested that DIVA vaccination is not feasible for ISA. A similar approach for KHV, utilising green fluorescent protein (GFP) as the marker, similarly failed to induce sufficiently detectable antibody responses in vaccinated carp (Cyprinus carpio L.). However, as high anti-KHV antibody titres were obtained with an inactivated KHV vaccine (≥1/3200), alternative approaches were carried out to assess the feasibility of DIVA vaccination for carp. Investigations of early KHV pathogenesis in vivo and antigen expression kinetics in vitro (0-10 days post infection (dpi)) provided valuable data for the diagnostics necessary for DIVA surveillance strategies. Following viral infection, molecular methods were shown to be the most effective approach for early detection of KHV infected fish prior to sero-conversion, during which time antibodies are not detectable. An experimental immersion challenge with KHV, however, revealed complications in molecular detection during early infection. The KHV DNA was detected in external biopsies of skin and gills, but also internally in gut and peripheral blood leukocytes ≤ 6 hours post infection (hpi), suggesting rapid virus uptake by the host. The gills and gut appeared to be possible portals of entry, supported by detection of DNA in cells by in situ hybridisation (ISH). However, many false negative results using organ biopsies occurred during the first 4 dpi. The gills were the most reliable lethal biopsy for KHV detection by various polymerase chain reaction (PCR) assays, with a PCR targeting a glycoprotein-gene (ORF56) and a real-time PCR assay being the most sensitive of the 7 methods investigated. Importantly, non-lethal mucus samples reduced the number of false negative results obtained by all KHV PCR assays during the earliest infection stages with large levels of viral DNA being detected in mucus (up to 80,000 KHV DNA genomic equivalents 200 μL-1). KHV DNA was consistently detected in the mucus as a consequence of virus being shed from the skin. Determining the expression kinetics of different viral structural proteins can be useful for DIVA serological tests. Analysis of KHV antigen expression in tissues by immunohistochemistry and indirect fluorescent antibody test was inconclusive, therefore 2 novel semi-quantitative immunofluorescence techniques were developed for determining KHV antigen expression kinetics in susceptible cell lines. During the course of KHV infection in vitro, a greater abundance of capsid antigen was produced in infected cells compared to a glycoprotein antigen (ORF56), as determined by detection with antigen-specific monoclonal antibodies (MAbs). The capsid antigen was characterised as a ~100 kDa protein by SDS-PAGE and identified as a product of KHV ORF84 by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/TOF MS). This antigen was subsequently detected in the serum of >25% of KHV infected/exposed carp (6/17), as well as in carp vaccinated with a live attenuated vaccine (3/4), but not with an inactivated vaccine (0/7), by Western blot making it a potential DIVA target for an inactivated vaccine. Attempts were made to improve the sensitivity of KHV serological testing by taking advantage of recombinant proteins specific for KHV (CyHV-3), rORF62 and rORF68 and eliminating any interference by cross-reacting antibodies to carp pox (CyHV-1). These proteins successfully reacted with anti-KHV antibodies. The feasibility of DIVA strategies for KHVD was determined using these recombinant antigens to coat ELISA plates. Differential antibody responses were detected from carp sera to an internal virus tegument protein (rORF62) and external region of a transmembrane protein (rORF68). Fish vaccinated with an inactivated vaccine produced significantly lower antibody responses to rORF62 than to rORF68, whereas infected, exposed and live attenuated vaccinated fish recognised both proteins allowing differentiation between vaccinated and infected carp. However, the sensitivity of the assay was limited, possibly by high levels of natural antibodies detected at the relatively low serum dilutions (1/200) used. As the capsid antigen (ORF84) and tegument protein (ORF62) are derived from internal KHV structural proteins, they induce non-neutralising antibodies, which may be useful for DIVA strategies. Such antibodies are longer lasting than neutralising antibodies and often comprise the majority of fish anti-viral antibodies. This was noted in a fish surviving experimental challenge, which had an antibody titre of 1/10,000, but neutralising titre of 1/45. Such antigens may therefore hold potential for developing effective serological diagnostic tests for KHV and provide the potential for DIVA strategies against KHVD. Natural antibodies will, however, continue to present a challenge to the development of sensitive and reliable KHV serological tests, and hence the application of DIVA strategies

Rapid methods for the detection of anti-parasitic drugs

The benzimidazoles (BZD), thiabendazole (TBZ), triclabendazole (TCB) and albendazole (ABZ), are veterinary agents employed for the eradication of parasitic infections in ruminant animals. Treatment with these drugs commonly results in trace residues remaining in the food product, thus requiring the development of tests capable of detecting below maximum acceptable residue levels (MRLs) as determined by European legislation. This research describes the development of novel, rapid, antibody-based methods for benzimidazole residue detection below the required MRL. A recombinant antibody fragment (Fab) generated from a hybridoma secreting an anti-TBZ monoclonal antibody was utilised for the development of an ELISA for TBZ with a limit of detection (LOD) of 2.5 ng/mL. Thee sensitivity of this Fab was improved two fold by generation of a modified plasmid vector which converts the Fab from a mono-valent to a bivalent antibody. An avian polyclonal antibody (pAb) was raised against TCB and ABZ. This antibody was incorporated into competitive ELISA and Biacore-based assay with LODs for TCB in spiked pasteurised organic milk samples of 609 and 203 pg/mL, respectively. A short chain fragment (scFv) antibody library of 5.9 x 1011 was also constructed, bio-panned and screened for clones specific for TCB and ABZ. Finally, the anti-TCB IgY was incorporated into a lateral flow immunoassay (LFIA) and a novel TCB-green fluorescent protein (GFP) LFIA assay with a detection threshold of 75-100 ng/ml of TCB in milk, which is at or below the required MR