Three protein targets are used in malaria rapid diagnostic tests (RDTs). These are Plasmodium falciparum histidine rich protein 2, Plasmodium lactate dehydrogenase and aldolase. A thrust of research in RDTs is to improve on their specificity and sensitivity. In this study the current diagnostic target, P. falciparum lactate dehydrogenase (PƒLDH) was compared to a new target glyceraldehyde-3-phosphate dehydrogenase (PƒGAPDH) that was identified based on transcriptional data. These proteins are conserved amongst all Plasmodium species, with minor amino acid sequence variations which were evaluated as possible species-specific peptide epitopes for PƒLDH: LISDAELEAIFDRC and PƒGAPDH: CADGFLLIGEKKVSVFA; CAEKDPSQIPWGKCQV, where common peptides were identified as pan-malarial epitopes for pLDH: APGKSDKEWNRDDLC and pGAPDH: CKDDTPIYVMGINH. The chosen peptides were located on the surface of their predicted 3D crystal structure models. Antibodies were raised against these peptides in chickens (IgY) and affinity purified. PƒLDH and PƒGAPDH were recombinantly expressed in E. coli BL21(DE3) cells and their coding inserts confirmed by sequencing. The recombinant proteins were detected in Western blots with specific anti-His₆ tag antibodies at approximately 35 kD (PƒLDH ~ 36 kD and PƒGAPDH ~ 39 kD) which compared with their expected values. Both recombinant proteins were found to form tetramers in solution and were used to raise IgY antibodies for comparison of Pheroids™ and Freund’s adjuvants. Pheroids™, like Freund’s appeared to exhibit a depot effect, however Freund’s adjuvant gave higher affinity purified IgY yields. The anti-recombinant and anti-peptide IgY specifically detected their respective recombinant and native antigens and did not cross-react with other human blood proteins. Immunoprecipitation detected higher levels of PƒGAPDH to PƒLDH in P. falciparum culture lysates. A double antibody sandwich ELISA detected 17.3 ng/ml PƒLDH and 138.5 ng/ml PƒGAPDH at 1% parasitemia in in vitro cultures, however this needs to be further evaluated. These findings suggest PƒGAPDH to be at least as good a protein target as PƒLDH for malaria diagnosis and further trials using it as a target in an RDT format should be considered. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Trypanosoma congolense is a major causative agent of the bovine disease trypanosomosis which has a considerable economic impact on sub-Saharan Africa. Current control methods for trypanosomosis are unsatisfactory and vaccine development has been hampered by antigenic variation. An anti-disease vaccine is based on the idea that disease is caused by the pathogenic factors released by the parasite, rather than by the parasite itself. Therefore, if these pathogenic factors could be neutralised by antibodies produced by vaccination, the disease could be circumvented. The method used here for identification of novel pathogenic factors is based on the concept that trypanotolerant cattle are able to mitigate the disease by generating a specific immune response against a few key antigens (pathogenic factors). Two immuno-affinity columns were therefore prepared: one containing IgG from noninfected sera and a second column containing IgG from trypanotolerant N’Dama cattle serially infected with T. congolense. The differential binding of antigens to the two columns allowed identification of antigens specifically recognised by the immune system of a trypanotolerant animal, i.e. potential pathogenic factors. The most promising antigens identified included several variant cathepsin L-like cysteine peptidases (CPs) and the Family M1 Clan MA aminopeptidases (APs). For the CPs, a study of the genetic organisation was conducted in order to further understand the variability present in this gene family. To this end, two different mini-libraries of cathepsin L-like genes were prepared: one in which genes as different as possible from congopain (the major CP of T. congolense) were selected, and a second which contained all possible genes present in the congopain array. Analysis of the sequences obtained in these two mini-libraries showed that there was significant variability of the genes within the congopain array. Two variants of CPs, chosen for differences in their catalytic triads, were cloned for expression. The recombinantly expressed CP variants differed in substrate preferences from one another and from C2 (the recombinant truncated form of congopain), and surprisingly, all enzymes were active at physiological pH. The two APs were cloned and expressed as insoluble inclusion bodies in an E. coli system, and subsequently refolded. The refolded APs showed a substrate preference for H-Ala-AMC, an optimum pH of 8.0, localisation to the cytoplasm and inhibition by puromycin. The two APs were not developmentally regulated and present in procyclic, metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro. Immunisation of BALB/c mice with the APs did not provide protection when challenged with T. congolense. For an anti-disease vaccine to be protective, it would possibly have to include all pathogenic factors, including the two APs and at least one CP described in the present study. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Alloresponses to blood group antigens result from antigen mismatch between donor and recipient during blood transfusion or transplantation and between mother and fetus during pregnancy. During pregnancy, antigen mismatch can result in haemolytic disease of the newborn (HDN), a disease characterised by the development of potentially harmful alloantibodies, which cross the placenta and mediate the destruction of fetal erythrocytes. This project investigates examples of clinically important alloresponses to blood group antigens and, more specifically, characterises the ymphocytes that either drive or regulate these responses. The main aims or this project were to first map alioreactive T-helper cell epitopes and secondly to clone using a novel method, IL-10 secreting blood group specific regulatory cells. The work focussed on two major antigens, the kell (K) 1 and Rhesus (Rh) D antigens.

Les glycanes présents à la surface des cellules cancéreuses sont souvent modifiés par rapport à ceux d’une cellule saine. Or ces antigènes glucidiques n’induisent pas de réponse immune efficace. La GalNAc est le premier sucre fixé lors de la O-glycosylation de type mucine et ainsi ce sucre entre dans la composition de nombreux antigènes tumoraux. Le but de notre travail était de préparer des analogues synthétiques de la GalNAc susceptibles d’être incorporés à la surface de cellules cancéreuses et dans les mucines synthétisées par les tumeurs, afin d’augmenter la réponse immune vis-à-vis des glycanes tumoraux. Nous avons synthétisé chimiquement des analogues de la GalNAc afin de les tester in vitro en tant que substrats de la voie de « sauvetage » de la GalNAc chez les mammifères et donc d’enzymes impliquées dans cette voie : une kinase (GK2) et une UDP-pyrophosphorylase (AGX1) humaines. Les meilleurs candidats ont permis la synthèse de différents UDP-sucres et une GalNAc-transférase (ppGalNAc T1) bovine a pu être utilisée in vitro pour transférer certains de ces analogues, à partir de leur forme activée en UDP-sucre, sur des peptides. Nous avons donc pu montrer que certains des analogues synthétisés étaient capables de s’intégrer dans la voie de sauvetage et d’être incorporés dans des peptides. Le pouvoir immunologique des glycoconjugués de type mucine ainsi formés a été étudié chez la souris après couplage de ces glycoprotéines à une protéine immunostimulante (KLH). D’autre part, des cellules de mammifères ont également été cultivées en présence de ces analogues afin de vérifier leur incorporation au niveau des glycoconjugués de la surface des cellules. / Glycans are often present at the cancerous cell surface in a modified form compared to healthy cells. However, these carbohydrate antigens don’t lead to an effective immune response. GalNAc is the first sugar attached to mucin type O-glycans and is thus a component of numerous tumor antigens. The aim of our work was to prepare synthetic GalNAc analogs able to be incorporated at the surface of cancer cell and into mucins synthesized by tumors in order to increase the immune response toward tumor glycans. We chemically synthesized GalNAc analogs to test them in vitro as substrates of enzymes involved in the mammalian GalNAc salvage pathway: a human galactokinase (GK2) and a human UDP-pyrophosphorylase (AGX1). The best candidates allowed the synthesis of the corresponding UDP-sugars further used to test the transfer of those analogs onto peptides using a bovine GalNAc transferase (ppGalNAc T1). We have shown that some synthetic analogs could be integrated in the GalNAc salvage pathway and O-linked to peptides. Immunological properties of the glycoconjugates thus formed were studied in mice after coupling to an immunostimulant protein (KLH). Moreover, mammalian cells were cultivated in the presence of these analogs in order to check their incorporation into glycoconjugates at the cell surface.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma subtype with heterogeneous clinical outcome and a significant number of patients still die of their disease. Characterising lymphoma patients’ autoantibody repertoires represent one approach to improve the understanding of their disease biology. Our hypothesis was that characterisation of antigens eliciting humoural immune responses in lymphoma patients may provide insights into mechanisms of lymphomagenesis and identify novel diagnostic/therapeutic targets. HIP1R was validated as a novel B-NHL autoantigen by immunoblotting with patients’ sera. No response was identified to the related HIP1 protein. Consistent with this finding, more widespread expression of HIP1R, compared to HIP1, was observed in lymphoma cell lines. Expression studies, at both transcript (qRT-PCR and analysis of microarray datasets) and protein levels (blotting and immunolabelling), identified abundant HIP1R in normal B cells and low level expression in a poor prognosis activated B-cell (ABC)-like DLBCL subtype. Upregulation of HIP1R expression was observed in activated non-malignant B cells at both transcript and protein levels, suggesting that downregulation of HIP1R expression in ABC-DLBCL might be a disease-related process. Despite its potential for DLBCL subtyping, HIP1R protein expression was not statistically significantly associated with patients’ survival in a series of 256 DLBCL. Short FOXP1 isoforms were identified as one mechanism repressing HIP1R transcription in an ABC-DLBCL cell line. Phenotypic analysis of HIP1R-depleted B-cell lymphoma cells indicated that HIP1R silencing could increase the surface levels of B-cell receptor (BCR) components such as IgM and CD79b. HIP1R represents a novel lymphoma autoantigen and cell-of-origin marker for distinguishing germinal centre- versus ABC-DLBCL subtypes. As ABC-DLBCL survival is dependent on chronic BCR signalling, future studies will address whether HIP1R silencing plays a fundamental role in disease pathogenesis by promoting BCR signalling.

Through chemical analysis and ion exchange chromatography of watersoluble antigens, this investigation supports the view that the majority of differences between the biotypes are quantitative. It was also found that strains demonstrate distinct, qualitative differences when compared to the attenuated strain 19 by immunodiffusion and thin-layer polyacrylamide gel, isoelectric focusing. These differences include the presence of antigens on virulent strains that are absent on strain 19. In addition, one antigen absent on strain 19, was found common to each virulent biotype. Finally, the results from immunodiffusion experiments, employing adsorbed and non-adsorbed immune globulins, indicate that at least some water-soluble antigens are exposed on the cell surface and that their distribution among the biotypes varies.

8 ABSTRACT Author: Bc. Lucie Moravcová Supervisor: MUDr. Vít Řeháček Charles University, Faculty of Pharmacy in Hradec Králové Title of master's thesis: Kidney transplantation: donor-recipient pairing in University Hospital Hradec Králové Background: The aim of this study was to determine HLA, blood group, age and sex match in donor-recipient pairing in kidney transplantation. HLA alleles of deceased donors were typed in Transfusion Department of the University Hospital Hradec Králové. Methods: Donor's HLA-A*, HLA-B* and HLA-DRB1* alleles were typed by PCR - SSP method. Complex data evaluating and processing was then performed. Results: 97 deceased donors were tested between 2013 and 2018. A total of 98 kidneys received from them were subsequently transplanted to 98 recipients in University Hospital Hradec Králové. 60,2 % of the donors were men, 63,3 % of the recipients were men. Most of the donors, as well as the recipients, were 51-70 years old (50,0 % and 59,2 %, respectively). The most common diagnoses in the group of deceased donors were associated with brain damage (66,3 %), the most common cause of renal failure in the group of recipients was chronic inflammatory kidney disease (41,8 %). All 98 transplantations (100,0 %) were AB0 compatible. 74 transplantations (75,5 %) were RhD compatible. 5...

Evidências epidemiológica e imunológica sugerem que o desenvolvimento da imunidade natural contra doença meningocócica pode está associado com a reação cruzada de antígenos em comuns com Neisseria meningitidis e outras bactérias comensais, como Neisseria lactamica. O Objetivo deste trabalho foi de investigar a imunogenicidade de antígenos de vesículas de membrana externa (OMV) de N. lactamica, com ou sem a presença de Bordetella pertussis (BP), utilizada como adjuvante. Grupos de camundongos neonatos da linhagem BALB/c foram imunizados com antígenos de N. lactamica. Os resultados de nossos estudos mostraram o predomínio de altos títulos de anticorpos dos isótipos IgG e IgM com alta e intermediária avidez, depois das imunizações pela via (i.n) com N. lactamica. A análise do soro por immunoblot mostrou proteínas com reatividade cruzada entre as espécies do gênero Neisseria e os anticorpos monoclonais utilizados neste trabalho. Estes resultados sugerem que antígenos de N. lactamica e N. meningititdis em comum, possam ser importantes na imunidade natural contra doença meningocócica, e no desenvolvimento de vacina. / Immunological and epidemiological evidences suggest that the development of natural immunity to meningogoccal disease may be associated with crossreactive antigens together with Neisseria meningitidis and other commensal bacteria, like Neisseria lactamica. The present study aimed to investigate the immunogenicity of antigens of outer membrane vesicles (OMV) of N. lactamica with or without the presence of Bordetella pertussis (BP) used as an adjuvant. Groups of neonate BALB/c mice were immunized intranasally antigens of N. lactamica. The results of our studies showed the predominance of high titers of antibodies of IgG and IgM isotipes with high and intermediate avidity after intranasal immunization with N. lactamica. Immunoblot analysis of serum showed cross-reactivity proteins between the species of the gender Neisseria and the monoclonal antibodies used in this study. These results suggest that antigens of N. lactamica and N. meningitidis in common may be important in natural immunity against meningogoccal disease and in the development of vaccine.

Thesis (MTech (Chemistry))--Cape Peninsula University of Technology, 2015. / Immunosensors are analytical devices comprising antibody (Ab) molecules intimately integrated with electronic physicochemical transducers. Abs are responsible for specific recognition of an analyte so called antigen (Ag) while transducers are responsible for the conversion of chemical changes brought about by Ab-Ag interactions into measurable and processable signal. Amongst the many analytical tools, immunosensors have shown outstanding performance in applications in fields such as clinical diagnostics, agricultural purposes and environmental monitoring. They have come in place of the many conventional analytical methods which showed a number of disadvantages; high cost and longer time of operation, and requirement of highly knowledgeable personnel. On the other hand, immunosensors have shown potential to overcome these constraints. Their advantages include possibilities of portability, miniaturization, and simplified procedures. Of the possible fields of immunosensor applications, this study focussed on the environmental aspect. The safety of the environment is good for the well-being even though there are still some environmental threats that exist. Polychlorinated biphenyls (PCBs) have reportedly been found to be some of the potential substances to pose such threats due to their toxic and persistent behaviour. In this study, we have developed an electrochemical immunosensor as an analytical tool for the analysis and monitoring of PCBs. The development was based on the use of silver nanoparticles-doped polyaniline (PANI/Ag NPs) for modification of an electrode as a process for fabrication of the transducer. The PANI/Ag NPs composite was deposited on the glassy carbon (GC) and platinum (Pt) electrodes by oxidative electropolymerization of aniline in the presence of Ag NPs in 1 M HCl using cyclic voltammetry (CV) by ramping the potential from -0.1 to 1.4 V at 50 mV/s. The composite was then characterized and evaluated as a potential material for electrochemical transduction. Evaluation was on electroactivity, which is the main property of interest for materials used in the fabrication of electrochemical devices. The PANI composites were characterized using spectroscopic (FTIR), microscopic (TEM) and electrochemical CV techniques. Results confirmed the formation of PANI in its emeraldine form and the presence of Ag NPs. Characteristic functional groups and peaks of PANI were observed in FTIR and CV respectively. TEM micrograms showed one dimensional nanofibric tubes and crystalline-like structure of the composite. The incorporation of Ag NPs was indicated by the transition from the amorphous (PANI) to crystalline (PANI/Ag NPs) structure accompanied by increase in size as well as smoothness of the tubes. EDS-TEM counts increase of the chlorine (Cl) peaks is due to the closeness of these peaks to those of Ag, thus confirming incorporation of Ag NPs.

Tsang Kam Sze, Kent. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 155-174). / Abstract --- p.i / Acknowledgements --- p.v / List of Abbreviations --- p.viii / Table of Contents --- p.x / List of Figures --- p.xvi / List of Tables --- p.ixx / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Idiotype Network --- p.2 / Chapter 1.2. --- Anti-idiotype Classification --- p.8 / Chapter 1.3. --- Blood Transfusion Effect --- p.11 / Chapter 1.4. --- Transfusion Protocol --- p.12 / Chapter 1.5. --- Mechanism of Beneficial Transfusion Effect --- p.15 / Chapter 1.5.1. --- Donor Selection --- p.15 / Chapter 1.5.2. --- Clonal Deletion --- p.16 / Chapter 1.5.3. --- Suppressor Cells Induction --- p.18 / Chapter 1.5.4. --- Prostaglandins Mediation --- p.19 / Chapter 1.5.5. --- Mixed Chimerism Motivation --- p.20 / Chapter 1.5.6. --- Fc-receptor Blocking Antibodies Stimulation --- p.22 / Chapter 1.5.7. --- Anti-idiotypic Antibodies Instigation --- p.23 / Chapter 1.6. --- Study Aims --- p.25 / Chapter 1.7. --- Technical Strategy --- p.26 / Chapter Chapter 2. --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.31 / Chapter 2.1.1. --- Patient Population --- p.31 / Chapter 2.1.2. --- Normal Control Group --- p.31 / Chapter 2.1.3. --- Serum Samples --- p.32 / Chapter 2.1.4. --- Additional Specimens --- p.32 / Chapter 2.1.5. --- Chemicals --- p.32 / Chapter 2.1.6. --- Antisera --- p.34 / Chapter 2.1.7. --- Buffers --- p.35 / Chapter 2.1.8. --- Consumables --- p.38 / Chapter 2.1.9. --- Apparatus and Equipment --- p.39 / Chapter 2.2. --- Methods --- p.40 / Chapter 2.2.1. --- Purification of Human Polyclonal Anti-HLA Antisera --- p.40 / Chapter 2.2.1.1. --- Affinity Chromatography --- p.41 / Chapter 2.2.1.2. --- Dialysis --- p.41 / Chapter 2.2.1.3. --- Concentration --- p.42 / Chapter 2.2.1.4. --- Quantitation --- p.42 / Chapter 2.2.2. --- Generation of F(ab')2 fragments from the Purified Human Anti-HLA Antibodies --- p.42 / Chapter 2.2.2.1. --- Buffer Exchange --- p.43 / Chapter 2.2.2.2. --- Pepsin Digestion --- p.43 / Chapter 2.2.2.3. --- Purification of (ab')2、 --- p.43 / Chapter 2.2.3. --- Enzyme-Linked Immunosorbent Assay for anti-Idiotypes against anti-HLA antibodies --- p.44 / Chapter 2.2.3.1. --- Optimization --- p.44 / Chapter 2.2.3.2. --- Quality Control --- p.45 / Chapter 2.2.3.2.1. --- F(ab')2 Specificity --- p.45 / Chapter 2.2.3.2.2. --- Fc Contamination --- p.46 / Chapter 2.2.3.2.3. --- Precision Test --- p.47 / Chapter 2.2.4. --- Anti-Casein Interference --- p.47 / Chapter 2.2.5. --- Test Protocol --- p.48 / Chapter 2.3. --- Statistical Analysis --- p.48 / Chapter Chapter 3. --- Purification of Anti-HLA IgG and F(ab')2 --- p.50 / Chapter 3.1. --- Immunoglobulin Concentration --- p.51 / Chapter 3.2. --- F(ab')2 Specificity --- p.51 / Chapter 3.3. --- Fc-fragments Contamination --- p.53 / Chapter 3.4. --- Discussion --- p.56 / Chapter Chapter 4. --- ELISA Optimization --- p.57 / Chapter 4.1. --- Coating F(ab')2 Quantitation --- p.58 / Chapter 4.2. --- Blocking and Diluting Agent Concentration --- p.61 / Chapter 4.3. --- Serum Analyte Dilution --- p.61 / Chapter 4.4. --- Conjugated Detector Antibody Titration --- p.64 / Chapter 4.5. --- Discussion --- p.66 / Chapter Chapter 5. --- Quality Control --- p.70 / Chapter 5.1. --- Avoidance of Prozone Phenomenon --- p.71 / Chapter 5.2. --- Inter-assay and Intra-assay Precision --- p.71 / Chapter 5.3. --- Discussion --- p.74 / Chapter Chapter 6. --- Adjustment of Anti-casein Interference --- p.77 / Chapter 6.1. --- Casein Allergy --- p.78 / Chapter 6.2. --- Prevalence of Anti-casein --- p.80 / Chapter 6.3. --- Discussion --- p.81 / Chapter Chapter 7. --- Prevalence of Anti-idiotypic Antibodies --- p.86 / Chapter 7.1. --- Formation Kinetics --- p.87 / Chapter 7.2. --- Occurrence in Transplant Patients --- p.87 / Chapter 7.3. --- Transfusion Effect --- p.101 / Chapter 7.3.1. --- Comparison between Transfused Transplant Patients and Normal Controls --- p.103 / Chapter 7.3.2. --- Comparison between Transfused Transplant Patients and Non-transfused Transplant Patients --- p.116 / Chapter 7.3.3. --- Association with Graft Survival --- p.117 / Chapter 7.4. --- Discussion --- p.128 / Chapter Chapter 8. --- Correlation of Transfusion with the Outcome of Transplant --- p.137 / Chapter 8.1. --- Rejection Episode --- p.138 / Chapter 8.2. --- Graft Survival --- p.139 / Chapter 8.3. --- Discussion --- p.142 / Chapter Chapter 9. --- General Conclusions --- p.149 / References --- p.153