Pectobacterium carotovorum ssp. carotovorum (Pcc) is implicated in the destruction of ornamental plants in greenhouse recirculating systems. PCR-based detection and quantification of Pcc requires expensive instrumentation and knowledgeable users. This thesis describes the production of polyclonal antibodies and a single-domain antibody fragment (VHH) against Pcc lipopolysaccharide (LPS), and the development of user- friendly diagnostic assays for detection and quantification of the pathogen. Polyclonal ELISAs against heat-killed (HK) Pcc (limit of detection (LOD) = 81 CFU/ml; limit of quantitation (LOQ) = 216 CFU/ml) and Pcc LPS (LOD = 23 ng/ml; LOQ = 76 ng/ml) were developed. A preliminary user-friendly dipstick assay was also developed (≥ 105 CFU/ml). A phage display library was constructed (6.0 x 105 clones/ml), yielding one unique anti-Pcc LPS VHH. Using the Pcc LPS-specific VHH to produce affordable, user- friendly diagnostic assays is feasible since antibody fragments can be produced on a large scale through expression in Escherichia coli or Piccia pastoris. / Flowers Canada, CANADA-ONTARIO RESEARCH AND DEVELOPMENT (CORD) PROGRAM, Canada Research Chairs (CRC) Program, NSERC/NRC

Despite individual autoimmune diseases being relatively rare, collectively these diseases afflict 8 % of the population according to the American Autoimmune Related Diseases Association. With over 75 % of those affected being women, autoimmune disease has been recognised, by the World Health Organisation and the US National Institutes of Health, as a major global women's health issue. One third of autoimmune sufferers have a rheumatological disorder, which commonly affect the joints, muscle, skin, salivary glands and kidneys. Antibodies against nuclear antigens are a serological hallmark of these diseases. Detection of these antibodies is used in the diagnosis and prognosis of the disease. The sensitivity and specificity of the test, of which the antigen is a key component, is pivotal to correct disease diagnosis and management. The relationship between circulating autoantibodies and the target antigen is complex. Improving the effectiveness of a test to assist in diagnosis and prognosis comes from characterisation and understanding these complex relationships. This thesis compares bovine spliceosomal U1 small nuclear ribonucleoprotein particle (U1 snRNP) complex with its human equivalent, and examines the validity of using this bovine derived autoantigen in the diagnosis of the human autoimmune diseases, systemic lupus erythematosus and mixed connective tissue disease. Differences between bovine and human U1 snRNP composition were characterised using a combination of electrophoretic, immunoassay and mass spectrometry techniques. Although the U1C protein could not be identified in bovine U1 snRNP, all other specificities were present. U1A remained intact, whilst the U1 snRNP specific 68K protein was dephosphorylated and a large C-terminal domain was removed, such that 68K migrated as a 30-36 kDa cluster on SDS-PAGE. Bovine SmD proteins, present in U1 and non-U1 snRNPs, were unaffected, whereas, SmB'/B was truncated to a 12 kDa peptide, which interestingly, was no longer reactive with anti-RNP sera in western blot. The recognition of human SmB'/B protein by anti-RNP sera in western blot was further examined. A technique was developed to immunoaffinity purify tryptic digests of SmB'/B which could then be analysed by mass spectrometry. Interestingly, the human replication element protein (HREP) was tentatively identified, rather than SmB'/B as expected. It may be possible, therefore, that anti-RNP sera may be reacting with a protein other than SmB'/B. To examine the contribution of the individual U1 snRNP proteins to anti-RNP and anti-Sm sera reactivities, a method was developed to dissociate bovine U1 snRNP and to purify the individual component antigens. It was demonstrated both empirically and through anecdotal feedback from a commercial diagnostic kit producer that patient sera respond better to purified Sm-free 68K than the recombinant 68K antigen. The effect of commercial processing of bovine thymus, the source for U1 snRNP antigen, was determined. In this study, variables that may be controlled during processing, such as temperature, protease activity and pH, were investigated. Hydrolysis of the intact human 68K protein with the necrotic protease, cathepsin L, produced 38 and 25 kDa fragments, whereas exposure to ambient temperature and low pH produced 32 kDa peptide fragments similar to those observed in purified bovine 68K. It was therefore proposed that 68K protein may undergo autocatalytic hydrolysis during necrotic cell death. Thorough characterisation of the bovine spliceosomal U1 snRNP proteins has not only validated their use as diagnostic reagents in autoimmune disease but also provided some insight into the inactivation of U1 snRNP function during early cell death.

IgM monoclonal gammopathy is a common age-related demyelinating sensory and motor polyneuropathy. It has been shown to be associated with antibodies against myelin-associated glycoproteins (MAG/SGPG). The HNK-1 carbohydrate epitope is a terminal 3-sulfo-glucuronyl residue attached to lactosamine structures and it is shared both in MAG and SGPG (SO4-3-GlcA(β1-3)Gal-(β1-4)GlcNAc(β1-3)Gal-(β1-4)Glcβ(1-1′)Cer). It is mostly expressed in the nervous system and plays an important role in preferential motor reinnervation. Nevertheless, the HNK-1 epitope is difficult to be isolated and synthesized and diagnostic assays used in the clinics are not always reproducible and reliable. Therefore in our study, our goal is to identify a simple synthetic diagnostic tool (peptide or monosaccharide), mimetic of the HNK-1 epitope, able to recognize antibodies in neurogammopathies sera by Surface Plasmon Resonance to be used in earlier stage patients and possibly to monitor disease activity. For this reason, we firstly tried to synthesize this trisaccharide and then we achieved the synthesis of its terminal monosaccharides with different function groups (octyl glucopyranoside, octyl glucuronic acid, octyl 3-O-sulfo-glucuronic acid and 8-amino octyl 3-O-sulfo-glucuronic acid). Then 10 linear and cyclic peptides conformationally and/or structurally mimicking HNK-1 were also synthesized (LSETTI, LSETTl, cyclo(-TTILSE-), cyclo(-TTlLSE-), cyclo(-TKTlLSE-), cyclo(-TETKlLSE-), TYTKlLSE, TY(SO3)TKlLSE, cyclo(-TYTKlLSE-) and cyclo(-TY(SO3)TKlLSE-)). The SPR kinetic binding affinities of all these sugar and peptide mimics were studied with commercial anti HNK-1 antibody using Biacore. Moreover, mimics with highest binding affinities were chosen for antigen-antibody interaction study in IgM gammopathy patients' serum.

La gammopathie monoclonale IgM est une polyneuropathie démyélinisante sensorielle et motrice. Il a été montré qu'elle est associée à des anticorps contre des glycoprotéines associées à la myéline (MAG/SGPG). L'épitope HNK-1 est un résidu 3-sulfo-glucuronyle lié à des structures lactosamine et il est présent aussi bien dans MAG que dans SGPG (SO4-3-GlcA(β1-3)Gal-(β1-4)GlcNAc(β1-3)Gal-(β1-4)Glcβ(1-1′)Cer). Il est exprimé principalement dans le système nerveux et joue un rôle important dans la réinnervation motrice préférentielle. Toutefois, l'épitope HNK-1 est difficile à isoler et à synthétiser et les essais diagnostiques cliniques ne sont pas toujours reproductibles et fiables.Le but de notre étude est d'identifier un outil synthétique simple de diagnostic (peptide ou monosaccharide), mimétique de l'épitope HNK-1, capable de reconnaître les anticorps dans les sera des neurogammapathies par Surface Plasmon Resonance (SPR) afin qu'il soit utilisé chez des patients à l'état précoce et qu'il puisse éventuellement permettre le suivi de l'évolution de la maladie. Pour cela, nous avons essayé de synthétiser ce trisaccharide, puis nous avons réalisé la synthèse de ses monosaccharides terminaux avec différents groupements fonctionnels (glucopyranoside d'octyle, acide 1-O-octylglucuronique, acide 1-O-octyl-3-O-sulfoglucuronique et acide 8-aminooctyl-3-O-sulfo-glucuronique).Puis 10 peptides linéaires et cycliques mimant conformationellement et/ou structuralement le HNK-1 ont également été synthétisés (LSETTI, LSETTl, cyclo(-TTILSE-), cyclo(-TTlLSE-), cyclo(-TKTlLSE-), cyclo(-TETKlLSE-), TYTKlLSE, TY(SO3)TKlLSE, cyclo(-TYTKlLSE-) et cyclo(-TY(SO3)TKlLSE-)). Les cinétiques d'affinité de ces mimes sucres et peptides ont été étudiées avec un anticorps anti HNK-1 commercial en utilisant le Biacore. De plus, les mimes avec les plus fortes affinités ont été choisis pour des études d'interaction antigène-anticorps dans des sera de patients atteints de gammapathie IgM. / IgM monoclonal gammopathy is a common age-related demyelinating sensory and motor polyneuropathy. It has been shown to be associated with antibodies against myelin-associated glycoproteins (MAG/SGPG). The HNK-1 carbohydrate epitope is a terminal 3-sulfo-glucuronyl residue attached to lactosamine structures and it is shared both in MAG and SGPG (SO4-3-GlcA(β1-3)Gal-(β1-4)GlcNAc(β1-3)Gal-(β1-4)Glcβ(1-1′)Cer). It is mostly expressed in the nervous system and plays an important role in preferential motor reinnervation. Nevertheless, the HNK-1 epitope is difficult to be isolated and synthesized and diagnostic assays used in the clinics are not always reproducible and reliable. Therefore in our study, our goal is to identify a simple synthetic diagnostic tool (peptide or monosaccharide), mimetic of the HNK-1 epitope, able to recognize antibodies in neurogammopathies sera by Surface Plasmon Resonance to be used in earlier stage patients and possibly to monitor disease activity. For this reason, we firstly tried to synthesize this trisaccharide and then we achieved the synthesis of its terminal monosaccharides with different function groups (octyl glucopyranoside, octyl glucuronic acid, octyl 3-O-sulfo-glucuronic acid and 8-amino octyl 3-O-sulfo-glucuronic acid). Then 10 linear and cyclic peptides conformationally and/or structurally mimicking HNK-1 were also synthesized (LSETTI, LSETTl, cyclo(-TTILSE-), cyclo(-TTlLSE-), cyclo(-TKTlLSE-), cyclo(-TETKlLSE-), TYTKlLSE, TY(SO3)TKlLSE, cyclo(-TYTKlLSE-) and cyclo(-TY(SO3)TKlLSE-)). The SPR kinetic binding affinities of all these sugar and peptide mimics were studied with commercial anti HNK-1 antibody using Biacore. Moreover, mimics with highest binding affinities were chosen for antigen-antibody interaction study in IgM gammopathy patients' serum.