Background: As a part of a national health control program, Statens VeterinärmedicinskaAnstalt performs diagnostics to screen flocks for certain pathogens causing high mortality,morbidity and/or serious economical losses. There are several viruses in the programincluding IBDV (infectious bursal disease virus), IBV (infectious bronchitis virus) and NDV(Newcastle disease virus). Method: 96 serum samples were collected from different poultryflocks in Sweden and analyzed by ELISA, which are currently used in the health controlprogram as well as by a commercial prototype of a multiplex immunoassay manufactured byLuminex Corp., which is currently under evaluation at the United States Department ofAgriculture USDA. This 4-plex assay detects antibodies for the three above-mentionedviruses as well as antibodies of avian reovirus. In the context of this study the ELISAs run inroutine diagnostics as well as a REO ELISA were used as the standard for comparison.Result: The antibody concentration in serum from vaccinated chickens was high while theantibody concentration level in serum from not vaccinated chickens was low. There was agood correlation between the multiplex assay and ELISA. Conclusion: The results obtainedin this study indicate that the Luminex multiplex assay tested has the potential to be usedroutinely for screening flocks.

Measuring HPV exposure relies on detection of HPV type-specific antibodies, but methods are not standardized. Additionally, there is little information on the best sample type for HPV antibody detection. This study validated pseudovirion neutralization (PVN) assay for HPV antibody detection and compared it to IgG ELISA. Both assays were applied to paired serum and cervical mucus samples. Additionally, PVN assay was utilized to evaluate the feasibility of oral mucosal transudate (OMT) samples to monitor the HPV immune response. Serum was more likely to be positive on PVN assay than on IgG ELISA (p= 0.025). Both assays correlated with HPV-16 DNA status. HPV-18 PVN assay results correlated with HPV-18 DNA status. Few cervical mucus samples had detectable antibodies; no correlation with HPV DNA status was seen. OMT results were unsatisfactory. PVN assay was more sensitive than IgG ELISA; serum was a more reliable indicator of HPV-16/18 antibody status than cervical mucus.

Accurate diagnosis of active tuberculosis is required to improve treatment, reduce transmission of the disease and control the emergence of drug resistance. A rapid and reliable test would make a considerable contribution to the management of the TB epidemic, especially in HIV-burdened and resource-poor countries where access to diagnostic laboratories are limited. Surrogate marker antibody detection to mycobacterial lipid cell wall antigens gave promising results, in particular with cord factor. The specific advantage of using mycolic acids as lipid antigens in comparison to protein antigens is that mycolic acid is a CD1 restricted antigen with the ability to induce proliferation of CD4/CD8 double negative T-cells, which may explain the sustained antibody production in AIDS patients. Traditional end-point assays to detect anti-MA antibodies showed an unacceptable number of false positive and negative test results. Here a much improved biosensor method (the MARTI-assay, i.e. Mycolic acid Antibody Real-Time Inhibition assay) was developed to detect antibodies to mycolic acid in patient sera as surrogate markers of active tuberculosis. The test was assessed on an IAsys optical biosensor and gave an accuracy of 82%. The technology was transferred to an SPR (ESPRIT) biosensor to economise and simplify the assay. Mycolic acid containing liposomes were immobilized on the SPR gold surface pre-coated with octadecanethiol. The following parameters were optimized on the ESPRIT biosensor to enable reliable TB diagnosis: effect of degassed buffer, saponin blocking, first exposure to serum at low concentration and second exposure to antigen inhibited serum at high concentration. The IAsys biosensor system has a weakness in the double channel cuvette system, in which the channels often do not give matching results, while being ten times more expensive than the gold discs provided for the ESPRIT biosensor. The ESPRIT biosensor is provided with an adjustable laser setting to compensate for differences in the channel readings as well as an automated fluidic system that reduces variance from one sample to the next. First indications are that the test can also be used for prognosis of TB during treatment. It is hoped that the ESPRIT biosensor will improve the accuracy of the test to more than 90%. If the MARTI-assay technology could be made amenable for high throughput screening, it may provide the solution to the serodiagnosis of tuberculosis and monitoring of progress during TB treatment both in adult and children, thereby reducing the spread of TB within the communities. / Thesis (PhD)--University of Pretoria, 2009. / Biochemistry / unrestricted

Parallel detection of antibodies with different specificity has many potential applications in epidemiological research, vaccine development and in the diagnosis of allergies, autoimmune and infectious diseases. Although ELISA-based tests are suitable for this purpose, available assays can be time consuming and require large quantities of both sample and reagents thus limiting their application for mass screening. / Revisión por pares

La tuberculose (TB) est une maladie infectieuse causée par M. tuberculosis. En 2009, la mortalité était élevée avec 1,7 millions de décès enregistrés dans le monde. La co-infection par le VIH, notamment en Afrique, et les tuberculoses à bacilles multi-résistants aux antibiotiques, rendent la maîtrise de la pandémie encore plus complexe. Les tests actuels de diagnostic présentent des lacunes notamment en terme de sensibilité pour la microscopie qui est le test le plus utilisé, ou en terme de praticité, en ce qui concerne la culture qui est le test de référence. Le développement de nouveaux tests pour le diagnostic de la TB active représente un enjeu majeur de nos sociétés modernes. Les tests immunologiques, en particulier les tests sérologiques actuellement disponibles, présentent une alternative aux tests bactériologiques couramment utilisés mais leurs performances restent faibles. Pour cela, il est indispensable de cibler de nouveaux biomarqueurs pour développer un test efficace, sensible, rapide et peu onéreux. C'est dans de cadre que s'inscrit ce travail de thèse.Nous rapportons dans ce mémoire, la recherche de marqueurs innovants dans le cadre de la mise au point d'un test de diagnostic de la tuberculose. Nous avons ciblé plusieurs marqueurs protéiques (OmpATb, LipY, Rv0183, Rv1984c et Rv3452) et un marqueur glycolipide, le tréhalose-6,6'-dimycolate (TDM). Parmi ces candidats, plusieurs présentent de réelles performances sur le plan diagnostique. Parallèlement à cette étude, nous avons mis au point une technique de séparation des différentes formes de TDM. La séparation des différentes formes de TDM devrait permettre de déterminer de façon précise les fonctions de ces composants majeurs de la paroi de M. tuberculosis et d'évaluer leur potentiel en terme de diagnostic.Mots clés : tuberculose, sérodiagnostic, détection antigène, détection anticorps, enzymes lipolytiques, glycolipide. / Tuberculosis (TB) is an infectious disease caused by M. tuberculosis. In 2009, mortality was high with 1,7 million of recorded deaths in the world. The co-infection by the HIV, in particular in Africa, and tuberculosis with multi-resistant bacilli to antibiotics, make the control of pandemia more complex. The current tests of diagnosis present gaps, in particular, in term of sensitivity for microscopy, which is currently the used test, or in term of praticity, for the culture which is considered as the gold standard.The development of new tests for the diagnosis of TB active represents a major challenge of our modern societies. The immunological tests, in particular the serologic tests available, can present an alternative to the currently bacteriological tests. However, their performances are not sufficient. That's why, it is essential to target new biomarkers which permit to establish an effective, sensitive, fast and cheap test. It is in this context that my study takes place.We report in this memory, the research of innovating markers for the development of a diagnostic test of tuberculosis. We targeted several protenic markers (OmpATb, LipY, Rv0183, Rv1984c and for Rv3452) and a glycolipid marker, the trehalose-6,6'-dimycolate (TDM). Among these candidates, several shown good performances for active TB diagnosis. In parallel to this study, a technique of separation of the various forms of TDM was developed. The separation of the various forms of TDM will permit to study their role on the biological activities and evaluate their potential in term of diagnosis.Key words: tuberculosis, serodiagnostic, antigen detection, antibody detection, lipolytic enzymes, glycolipid.

Kidney transplantation is the treatment of choice for patients with end stage renal failure and is associated with prolonged survival of patients and better quality of life than long-term dialysis. Simultaneously, however, transplantation carries the risk of immunological complications leading to graft rejection. A serious problem in patients after organ transplantation is the development of humoral rejection, which is most often associated with the presence of antibodies specific to HLA antigens, particularly against mismatched HLA antigens of the organ donor. In certain cases antibodies may be specific to antigens expressed on endothelial cells, not on lymphocytes, like MICA, MICB, ICAM, and up till now unidentified tissue-specific antigens. Humoral rejection has significantly worse prognosis for the transplanted kidney than cellular rejection, and therefore its timely diagnosis is of great importance for the subsequent choice of appropriate therapy. The diagnosis of humoral rejection is based on the simultaneous detection of C4d deposits in the peritubular capillaries of the transplanted kidney and the finding of antibodies specific to the mismatched antigens of the donor (donor specific antibodies, DSA). The aim of our retrospective study was to contribute to improvement of the diagnosis of acute and...

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IntroduktionDe humana blodgrupperna består av A, B, AB samt O. Antigen kan detekteras av alloantikroppar, att en reaktion sinsemellan kan ske är ett krav för att en molekyl ska kunna kallas antigen. Antigen och antikroppar är vitala för analys inom transfusionsmedicin och möjliggör transfusioner av blod samtidigt som man förhindrar allvarliga transfusionsreaktioner. För att effektivisera manuella serologiska analyser används saltlösning med låg jonstyrka. Syftet med arbetet var att verifiera lösningen MLB 2 (Bio-Rad Medical Diagnostics GmbH, Dreieich, Tyskland). Denna lösning ska förstärka reaktioner i antikroppsidentifiering, blodgruppskontroll med antikropps-screening samt förenlighetsprövning, men även för fenotypning av erytrocyter gällande antigenen s, Fya, Fyb, Kpa, Kpb samt Lua. MetodTotalt fenotypades 61 prover varav 31 var positiva och 30 var negativa för sökt fenotyp. Av de 31 positiva var 17 fenotyper av känt heterozygot anlag. Vid varje analystillfälle utfördes kontroller som bestod av heterozygot positiva samt negativa celler från Örebropanelen för vardera fenotyp. För antikroppsidentifiering ämnade två antikroppar, anti-E samt anti-Lea, att analyseras från två prover där de tidigare identifierats. Beträffande blodgruppskontroll med antikroppsscreening samt förenlighetsprövning analyserades tre prover från patienter som varit i behov av transfusioner och som tidigare påvisat antikroppar. Resultat och slutsatsSamtliga provers resultat överensstämde fullständigt vid fenotypning. För antikroppsidentifiering bekräftas tidigare påvisad anti-E för ett av proven, men för det andra provet erhölls inte resultat fullständigt överensstämmande med patientens anti- Lea. För blodgruppskontroller med antikropps-screen samt förenlighetsprövning överensstämde samtliga resultat. Effekten av förstärkningslösningen MLB 2 bedöms som god. Tydligt blir dock att gelteknik, som idag används som golden standard, är överlägsen de äldre rörteknikerna. / BackgroundHuman blood groups are defined as A, B, AB and O. Antigens are detectable by alloantibodies and a reaction between them is necessary in order to denominate as such. Antigens and antibodies respectively are essential within immunohematology to enable transfusion therapy while evading severe transfusion reactions. To improve the efficiency of manual serological test, low ionic strength saline is used. The purpose of this study was to verify MLB 2 (Bio-Rad Medical Diagnostics GmbH, Dreieich, Germany) for use as low ionic saline solution (LISS) in indirect agglutination technique (IAT) tube-tests for antibody identification, blood group verification with antibody-screen and compatibility test, also for phenotyping antigens s, Fya, Fyb, Kpa, Kpb and Lua. MethodsA total of 61 samples, of which 31 were previously known positive and 31 negative were tested. Of the 31 positive, 17 were heterozygous. At every assay, positive heterozygous and negative cells for each of the phenotype was applied as control, from the Örebro panel. For antibody identification, two previously positive samples were used. For blood group verification with antibody-screen and compatibility test, three samples with previous need for transfusion therapy and positive antibody screen were used. Results and conclusionAll samples phenotyped present fully consistent results compared to the previous ones. For antibody identification the results for one of the samples are completely confirmed. The second sample showed inconsistencies to the previous result. For blood group verification with antibody-screen and compatibility test all samples had equivalent results. The effect from MLB 2 in IAT-LISS tube tests are adequate. A distinct observation can be made; gel column technology is superior to the tube tests.