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Analysis of biological samples plays a crucial role in disease diagnostics and monitoring, as well as in research. For the analysis, detection and quantification is of the essence, and can be performed with immunoassays. These immunoassays exploit antibodies, and their characteristic specificity, to target analytes. Furthermore, immunoassays can be either singleplex or multiplex, meaning they assess one single, or multiple analytes in parallel. As of today, singleplex methods such as ELISA dominate the market, favoured for their precision and reliability. However, multiplexing exhibit reductions in time, costs, and materials. Nevertheless, one common challenge is the detection of small amounts of analyte, possibly discovering and monitoring earlier stages of disease. For this purpose, Cavidi AB has developed a signal amplification technology, namely the binding oligo ladder detection (BOLD). Currently, this technique is applicable to the singleplex market, while the implementation on the multiplexing remains. Here,  we present a literature review of a selection of multiplexing techniques, and a thorough investigation of their possible compatibility with Cavidi AB’s signal enhancement technology BOLD. After undergoing a comprehensive analysis to evaluate various multiplexing technologies and theircompatibility with BOLD, a spectrum of compatibility levels were revealed. Some of the multiplexing technologies, exemplified by Luminex’s xMAP technology, demonstrated inherent compatibility. Contrarily, other platforms, including Olink’s PEA technology, exhibited incompatibility. Moreover, certain technologies, such as Standard Biotools’ CyTOF technology, were found to possess potential compatibility if modification to either BOLD or to the respective multiplexing technology were to be implemented. Furthermore, the need for signal amplification was identified to vary amongst the versatile technologies and the different analytes. This stems from the fact that the lowest detectable concentration level fluctuates between different analytes and technologies. Therefore, Cavidi AB should target companies with a generally high limit of detection (LOD). In addition, because of the fluctuations in LOD between analytes, specifying to which analytes an integration of BOLD would be beneficial, is recommended.

Tripeptidyl peptidse II (TPPII) is an enzyme forming a complex of 4,5 MDa. It is located in the cytosol and participates in protein turnover where it degrades oligopeptides to smaller peptides and tripeptides. Moreover, some of its products might take part in antigen presentation through MHC-class 1. High expression of the enzyme is believed to correlate with tumor progression. A decreased concentration seems to contribute to increased susceptibility to infection. The aim of this project was to develop an enzyme linked immusorbent assay (ELISA) specific for TPPII and compare it to two commercial kits. TPPII was detected through western blot and activity assay to ensure which samples were containing the enzyme. Samples with purified enzyme from different species, erythrocyte lysate and plasma were used to develop an indirect ELISA as well as compare all the assays and evaluate which one performed the best. The commercial kits were based on sandwich and competitive techniques. It was observed that the developed ELISA was able to detect the purified enzyme of different concentrations whereas the commercial kits could not. On the other hand, the commercial kits were able to generate their own standard curve which leads to suspicion that these might be non-specific to TPPII. Though, all assays did detect the enzyme in erythrocyte lysate. This study presents that it is possible to develop an ELISA specific for TPPII using an indirect technique which also performed better than commercial kits.

Weaning is defined as the physical separation of the cow-calf pair and the end of milk feeding. Natural weaning occurs between 7 and 14 months and is a gradual process. However, domestic weaning occurs between 6 and 8 months and occurs rapidly. Calves that are abruptly separated from their dam respond with increased vocalization and walking, and decreased eating and resting. The psychological stress the calf undergoes during weaning causes elevated glucocorticoid and catecholamine hormone concentrations that may predispose to increased morbidity and/or mortality from infectious diseases such as Bovine Respiratory Disease Complex. As an attempt to counter these changes, alternative weaning methods have been implemented and normally occur in two stages. Two-stage weaning begins with the cessation of milk feeding for approximately one week with the calf maintaining some contact with their dam and then permanent separation occurs. One of these methods uses a single fence to separate the cow-calf pair; this process allows the calf to see, hear and smell their dam, but does not allow the calf to suckle from its dam. Increases in cortisol, a glucocorticoid, have been linked to immunological alterations. Most notably, elevated cortisol concentrations decrease neutrophil function by down regulating the gene expression of CD62L and Fas. Cortisol also alters lymphocyte phenotype by decreasing ?δ T cells and increasing°? T cells in the circulation. Lastly, increases in cortisol can modify T cell cytokine production. The cytokines IL-12 and IFN? are secreted from T helper 1 cells while T helper 2 cells secrete IL-4 and IL-10; these T cells subsets also inhibit one another. During higher cortisol concentrations, these T cells are biased toward T helper 2 cytokine production. All these changes in immune function can lead to increased susceptibility to disease around the time of weaning. Therefore, two trials were conducted to test the hypotheses that abrupt weaning results in elevated concentrations of cortisol and subsequently alters immunological functions, and that fenceline weaning alleviates the increase in cortisol and alterations to immune function associated with weaning. In the fall of 2008, 12 Angus and Angus-X heifers (186°21 kgs; 174°16 days of age) were blocked by age and weight and randomly allotted into two groups, fenceline and abrupt. Blood samples were taken on day -7, 0, 7, 14, 21, and 42; fecal samples were taken on day -7, 0, and 3. All calves were weighed on day -7, 0, 7, 14, and 42. On day -1 all calves were separated from their dam and transported for 2 hours to another facility. On day 0 all calves were vaccinated with Brucella abortus (strain RB51). Serum was analyzed for IFN? and IL-4 as well as IgG1 and IgG2 specific antibodies to RB51. Fecal samples were analyzed for cortisol metabolites. Both IgG1 and IgG2 antibodies to RB51 increased from day 0 to day 14 (P<0.05), however no differences were detected between treatment groups. Fecal cortisol metabolites were higher on day 0 in abruptly weaned calves (P< 0.001) but did not differ between groups on day -7 or day 3. Fenceline calves had higher concentrations of IFN? in the serum on day -7 and day 0 as compared to the abruptly weaned calves (P<0.04). In the fall of 2009, forty-four Angus and Angus-X calves (19 heifers and 25 steers; 181°27 kgs; 148°17 days old) were blocked by age and gender and randomly allotted within block into two treatment groups, fenceline (FL) and abrupt (AB). Approximately half the fenceline calves were separated from their dams by a single fence at day -7 and the rest of the fenceline group at day -6; all calves were removed from their dam at day 0. Calves were vaccinated with Histophilus somni on day 1. Blood samples were taken at day -6, 1, 3, 8, 15, and 22. Fecal samples were taken on day -7, -6, 1 and 3. All calves were weighed on day -7, 0, 8, and 22. Serum samples were analyzed for IgG1 and IgG2 specific-H. somni antibodies, white blood cells were analyzed for lymphocyte phenotypes, and gene expression using 18S as the housekeeper gene. Fecal samples were analyzed for cortisol metabolites. Abruptly weaned calves had higher concentrations of cortisol metabolites in the feces than fenceline calves at day 1 (P<0.0001). No difference in average daily gain or H. somni specific antibodies between treatment groups was detected. There was a treatment*date interaction in lymphocyte and neutrophil populations (P<0.05); neutrophils from fenceline calves dropped from day -6 to day 1, but increased from day 1 to day 3, while abrupt calves decreased from day -6 to day 3. Lymphocytes from fenceline calves increased from day -6 to day 1, but decreased from day 1 to day 3, while lymphocytes from abrupt calves increased from day -6 to day 3. No difference in treatment groups was detected for lymphocyte phenotypes or gene expression; however, a date effect was detected. The CD4 and CD8 cell populations increased over time (P<0.0001) and WC1 and TcR1 decreased over time (P=0.0243 and P=0.0027 respectively) for both treatment groups. A decrease was detected over time for expression of GAPDH and CD62L (P<0.0001). The gene expression for the cytokines IFN?, IL-4 and IL-10 had no change over time. Results from the two studies suggest that fenceline weaning decreases the cortisol response associated with cow-calf separation, but does not have a significant effect on immunological parameters measured in this study. / Master of Science

H. somni is capable of forming a prominent biofilm, and luxS is known to play an important role in biofilm formation through quorum sensing, but has also been postulated to function in gene regulation. In order to further study the function of H. somni LuxS, mutants 2336::TnluxS and 2336::TnuspE were identified from a bank of mutants generated with EZ-Tn5 <KAN-2>Tnp transposome (EpiCentre). The 2336::TnluxS and 2336::TnuspE mutants were highly attenuated in mice, but only 2336::TnuspE was deficient in biofilm formation. However, the electrophoretic profiles of the LOS and serum sensitivity of both mutants were substantially altered compared to the parent strain, but exopolysaccharide production during biofilm formation also only decreased in 2336::TnuspE. The altered phenotypes were partially restored in complemented recombinant clones obtained using shuttle vector pHS649S. To clarify whether luxS regulates the expression of various virulence genes, mRNA from both the parent strain and 2336::TnluxS was sequenced. It was determined that the transcription level of 53 genes in 2336::TnluxS and 42 genes in 2336::TnuspE in planktonic form were changed. In biofilm, 320 genes in 2336::TnluxS and 230 genes in 2336::TnuspE were differentially regulated compared to biofilm formed by strain 2336. The immunogloblin binding protein A (IbpA) of H. somni is known to be cytotoxic to phagocytic cells. In this study, we found that strains with a mutation in ibpA were less capable of early replication in monocytes. The IbpA protein concentrated from the culture supernatant of strain 2336 facilitated the intracellular survival of strain 129Pt, which lacks IbpA. However, the ability of several ibpA mutants to resist intracellular killing was not significantly impaired by 48 h post-infection. Two transposon mutants 2336::TnluxS and 2336::TnuspE replicated in monocytes in a similar manner as the ibpA mutants. Confocal microscopy revealed that the intracellular-replicable strains (2336, 64Vc, 2336::TnluxS, 2336::TnuspE and the ibpA mutants) prevented the acidification of the bacterial-containing phagosome and the expression of lysosome marker LAMP-2, which may facilitate survival of H. somni in monocytes. An enzyme-linked immunosorbent assay was developed to detect bovine antibodies to the H. somni exopolysaccharide that is formed during biofilm formation. When an index value of 0.268 was used the sensitivity of the assay for experimentally- and naturally-infected calves was 90.5% at 3 weeks post-infection, and the specificity of the assay for healthy calves was 92.5%. The EPS ELISA may aid in identifying calves with diseases due to H. somni. / Ph. D.

No / Yellow leaf syndrome (YLS) of sugarcane is associated with sugarcane yellow leaf virus (SCYLV), a member of the family Luteoviridae. A fragment of the coat protein and readthrough domain of SCYLV was expressed in a bacterial expression system. The resulting protein was purified and used to immunize mice for monoclonal antibody (MAb) production. Two MAbs, 3A2E3 and 2F7H5, were selected following the screening of hybridoma cells using both plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) and tissue blot immunoassay (TBIA). These MAbs can be incorporated into the TBIA assay currently used for the routine detection of SCYLV but could not be used in triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The two antibodies selected have slightly different specificities. Antibody 3A2E3 gave equivalent results to a polyclonal antiserum (raised to purified virus) in comparative testing using TBIA. The MAbs produced should provide a widely available, uniform reagent for SCYLV diagnosis with the potential to help manage YLS.

Amyloidosis is a disease caused by misfolding of proteins that accumulate in different organs in the body. One of the most common forms of amyloidosis is Transthyretin amyloidosis (ATTR-amyloidosis). Diagnostic of amyloidosis is done by Congo red staining and immunohistochemistry where high-affinity monoclonal antibodies are preferred. For treatment to be effective it is necessary to obtain the diagnosis at an early stage. The aim of this study was to produce monoclonal antibodies against transthyretin for diagnostic purposes of ATTR-amyloidosis. To produce monoclonal antibodies, the spleen from a mouse immunized with transthyretin was collected. Antibody producing cells from the spleen were fused with myeloma cells deficient of their own antibody production. After 2 weeks culture in HAT selection media cells were screened for antibody production with ELISA and immune-histochemistry. Recombinant transtyretin (TTR) was produced and used as antigen in the ELISA. All 65 hybridomas tested were negative on ELISA. Three out of 25 hybridomas tested in immunohistochemistry showed visible staining against muscle cells in tissue from patients with TTR amyloid deposits. No antibody could be produced that detected ATTR-amyloidosis deposits.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第25140号 / 理博第5047号 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 哲, 教授 中務 真人, 准教授 城野 哲平 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

A detecção de antígenos em amostras de líquido cefalorraquiano (LCR) de pacientes com neurocisticercose (NC) foi realizada empregando-se o teste ELISA com soros policlonais de coelhos imunizados com os antígenos total de Taenia solium (T-Tso), líquido vesicular de Taenia crassiceps (LV-Tcra) e peptídeos &#60;30kDa de LV-Tcra. Os soros policlonais foram fracionados para a obtenção da fração IgG - IgG anti-Tso, IgG antiTcra e IgG anti-Tcra&#60;30kDa. Também foi empregado o anticorpo monoclonal específico para o antígeno de excreção e secreção de Taenia crassiceps (ES-Tcra). A seleção dos clones foi realizada por ELISA empregando-se os antígenos T-Tso e LV-Tcra. Foram analisados diferentes grupos de amostras divididos em: grupo de pacientes com NC (grupo NC), incluindo pacientes em diferentes fases evolutivas da doença, grupo de pacientes \"suspeito\" de NC (grupo \"suspeito\" NC), grupo controle (grupo C) e grupo de outros patologias (grupo OP). No teste ELISA empregando-se as frações IgG anti-Tso, IgG anti-Tcra e IgG anti-Tcra&#60;30kDa, a sensibilidade obtida foi de 70%, 82,5% e 95,8% e a especificidade de 82,5%, 98% e 100%, respectivamente nas amostras do grupo NC e grupo C. Nas amostras do grupo diferentes fases evolutivas da doença, não houve diferença significativa de reatividade entre as amostras com as frações empregadas. Para as 21 amostras do grupo NC - fase ativa da doença, com a fração IgG anti-Tcra e o anticorpo monoclonal anti-ES-Tcra, respectivamente, 13 e 16 amostras foram positivas para a pesquisa de antígenos. As amostras de LCR do grupo C não apresentaram reatividade com os anticorpos empregados nos ensaios. Também foram ensaiadas 68 amostras de LCR de pacientes com \"suspeita\" de NC. De acordo com as características citoprotéicas além da reatividade para a pesquisa de anticorpos anti-T solium, as amostras de LCR apresentaram, neste grupo, padrão de reatividade para a pesquisa de antígenos que variou também de acordo com a presença de anticorpos, além das alterações de proteína e/ou células, que as amostras apresentavam ou não. Frações de 14 e 18kDa foram identificadas pelo teste imunoblot somente nas amostras de LCR de pacientes com NC utilizando as frações IgG anti-Tso, IgG anti-Tcra e anticorpo monoclonal anti-ES-Tcra. O anticorpo monoclonal anti-ES-Tcra mostrou-se eficiente para a pesquisa de antígenos no teste de competição por ELISA nas amostras de LCR de pacientes com NC e o antígeno ES-Tcra. / Antigen detection in cerebrospinal fluid (CSF) samples of patients with neurocysticercosis (NC) was performed through ELISA test using rabbits polyclonal sera immune with total antigens of Taenia solium (T-Tso) cysticercus, vesicular liquid of Taenia crassiceps (VL-Tcra) and vesicular liquid peptides (VLP-Tcra&#60;30kDa) cysticercus. The polyclonal sera were separated obtaining IgG-lgG anti-Tso, IgG anti-Tcra and IgG anti-Tcra&#60;30kDa fractions. A specific monoclonal antibody was also applied for reaching the excretion and secretion antigen of the T. crassiceps (ES-Tcra) larvae culture. Clones selection was performed through Elisa test applying the T-Tso and VL-Tcra antigens. When IgG anti-Tso, IgG anti-Tcra and IgG anti-Tcra&#60;30kDa fractions were applied in the ELISA test, the accuracy obtained was 70%, 82,5% and 95,8% and the specificity of 82,5%, 98% and 100%, respectively, in samples of both NC and C groups. In the samples of the group in different stages of the disease there was no significant difference on reactivity between the samples when the fractions were applied. For the 21 samples of the NC group - active stage of the disease with IgG anti-Tcra fraction and the anti-ES-Tcra monoclonal antibody respectively, 13 and 16 samples were positive for antigen analyses. The CSF samples of the C group did not present reactivity with the antibodies applied in the tests. Tests for 68 CSF samples of \"suspected\" NC group, were also conducted. According to the cytoproteic characteristics besides the reactivity for the anti-T. solium antibody study, the CSF samples of this group showed standard reactivity for antigen detection ranging also in accordance with the presence of antibodies and of the protein and/or cell alterations that the samples would or not present. Fractions of 14 and 18kDa were identified by immunoblot test only in the CSF samples of patients with NC using the IgG anti-Tso IgG anti-Tcra fractions and the anti-ES-Tcra monoclonal antibody. The anti-ES-Tcra monoclonal antibody has shown to be efficient for analyzing antigens by a comparing method of the ELISA test in CSF samples of patients with NC and the ES-Tcra antigen.