Staphylococcus aureus is an important human and animal pathogen that causes a wide range of infections. These infections can be very serious and sometimes hard to get rid of, because of the many virulence factors the bacteria produce during infections. This project was a research of the extracellular fibrinogen-binding protein, Efb, which is a 15.9 kDa protein that has been shown to be an important virulence factor during S. aureus infections. The purpose with the project was to find out if the protein has more than one binding site to fibrinogen and if people produce antibodies against Efb. This was performed with methods such as affinity chromatography, ELISA, coagulation test and western blot. It was shown that Efb has two binding sites to fibrinogen. One is placed on the C-terminal part of Efb and the other on the N-terminal. It was also shown that the production of antibodies against Efb rises significantly in people during an ongoing infection.

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Version:1.0 StartHTML:0000000178 EndHTML:0000005278 StartFragment:0000002640 EndFragment:0000005242 SourceURL:file://localhost/Volumes/NAMNLOS/Examensarbete%20kortisol.doc Föreliggande studie syftar till att försöka utläsa skillnader mellan två olika perioder då den personliga stressfaktorn upplevs vara olika intensiv. Undersökningen syftar även till att studera huruvida den mänskliga kortisolutsöndringens diurnala upp - och ned gångar följer en intraindividuell stabilitet av CAR (cortisol awakening responce). Detta skulle innebära ett upprepande mönster av kortisolkoncentrationens magnitud och mätvärde inom varje individ från dag till dag, vid uppvaknandet och 30 minuter efter. Undersökningen har genomförts som en pilotstudie där en försökspersons kortisolkoncentration i saliv har mätts genom enzymkopplad immunabsorberande analys (ELISA). För att jämföra mätserierna inom de olika perioderna med varandra har även en variationsanalys av typen Analysis of variance (ANOVA) utförts med hjälp av programvaran SPSS. Då provernas mätvärde har analyserats och jämförts med varandra har ett resultat kunnat fastställas. Eftersom utsöndringen av den individuella kortisolkoncentrationen lätt påverkas av omgivningsfaktorer användes endast en försöksperson, författaren, vilket underlättade en detaljerad analys där observation av påverkande faktorer lätt kunde tas med i beräkningen för att fastställa ett tillförlitligt resultat. Försökspersonen, kvinna 21 år, utförde 6 provtagningar under två perioder som upplevdes ha olika hög stressfaktor. Perioderna innehöll två arbetsdagar. Parallellt med provtagningen fördes noggranna dagboksanteckningar för att underlätta analyseringsarbetet. Resultatet uppvisar en intraindividuell stabilitet av CAR hos försökspersonen. Studien visar även en skillnad mellan de två perioderna genom en högre procentuell ökning av CAR under den period då stressfaktorn upplevdes som mer intensiv. Den tydliga skillnaden av kortisolkoncentrationens mätvärde mellan de olika dagarna indikerar även att livsstil, fysisk aktivitet och drömmar kan påverka utseendet av kortisolkoncentrationskurvans diurnala upp – och nedgångar.

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

The main objective of this work was to develop an antigen-capture enzyme-linked immunosorbent assay for detection of beta2-toxin in the intestine of neonatal piglets. The format of the assay comprised of capture antibodies (polyclonal), antigen (beta2-toxin), detecting antibody (labeled monoclonal) and a substrate. The ELISA was optimized using recombinant protein. After intestinal content samples were applied, the test protocol needed to be adjusted because of the presence of high background signal in some samples consistent with intestinal proteases. This was overcome by processing the samples at 4oC and using citrate buffer pH 6.1 containing 5% bovine serum albumin. The second objective was to identify cpb2 in Clostridium perfringens type A isolates from selected animal species and to examine genotype-phenotype corelation. The study concluded that consensus cpb2, if present, was almost always expressed. In contrast, only about three-quarters of atypical cpb2, mostly was present in isolates of non-porcine origin, were expressed. / Ontario Ministry of Agriculture, Food and Rural Affairs; The Natural Sciences and Engineering Research Council of Canada

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Elk (Cervus elaphus) were historically present throughout Kentucky, but were extirpated by the mid 19th century. Kentucky Department of Fish and Wildlife Resources initiated elk reintroduction efforts in 1997, resulting in a self-sustaining population. I designed this project to study the effects of a parasitic nematode, meningeal worm (Parelaphostrongylus tenuis), on Kentucky’s elk herd. I examined potential maternal transfer of P. tenuis antibodies to elk calves, and investigated the relationship between elk habitat use and meningeal worm infection. I captured neonatal elk in 2004-06, fitted them with VHF transmitters, and collected blood samples for an enzyme-linked immunosorbent assay (ELISA) to determine P. tenuis infection. I monitored animals to determine habitat use, and attempted to recapture each individual to collect a follow-up blood sample. I found substantial rates of maternal meningeal worm antibody transfer (55%) over the course of the study. Neither sex nor predicted birth weight was associated with increased likelihood of obtaining maternal antibodies. Habitat variables associated with P. tenuis infection included herbaceous, shrub, and bare cover types, herbaceous mean core area, forest edge density, and forest mean core area. Confounding variables complicated habitat data analysis, but high rates of maternal P. tenuis antibody transmission suggested that meningeal worm infection does not threaten the long-term viability of the Kentucky elk herd.

Pseudomonas Aeruginosa is a common gram-negative bacterium present in the environment. It causes severe infections in immunosuppressed patients. Cystic fibrosis patients are especially at risk of being infected with Pseudomonas Aeruginosa. Ongoing studies are preformed to find alternative therapies to antibiotics, due to increased resistance. One new treatment is intake of specific IgY antibodies against Pseudomonas Aeruginosa as an oral therapy. The problem today is that IgY solutions must be kept frozen until consumed.  In this study we examined the possibility to freeze-dry specific IgY antibodies without losing any activity or specificity of the antibodies. This would be more convenient of patients, as well as it makes transportation and storage easier.  The methods used were ELISA for control of activity, western blot analysis and SDS-PAGE gel for control of specificity. Three different batches of the IgY anti-Pseudomonas Aeruginosa solution were tested. The results showed that no loss in activity occurred that would affect clinical outcome or change of specificity in the antibodies after freeze-drying appears. This indicates that it is possible to replace the liquid antibody to a freeze-dried powder.

Proteins secreted or shed by tumors can be found in serum. Detecting these proteins by mass spectrometry (MS) is difficult, due to the wide dynamic range of protein concentrations in serum. To circumvent this issue, we mined the conditioned media of epithelial ovarian cancer (EOC) cell lines which is a less complex fluid to work with. We hypothesize that some of the proteins shed or secreted by EOC cell lines are similar to those secreted or shed by EOC tumors and that some of these proteins can be used as biomarkers. We mined the conditioned medium of four ovarian cancer cell lines (HTB75, TOV-112D, TOV-21G and RMUG-S) by two-dimensional liquid chromatography-mass spectrometry. Our study identified 1208, 1252, 885, and 463 proteins from the HTB-75, TOV-112D, TOV-21G, and RMUG-S cell lines respectively. In all, we identified 2039 proteins from which we focused on 420 extracellular and plasma membrane proteins. High abundance proteins such as albumin and immunoglobulins, which are problematic for serum proteomics, did not interfere with our study. Several known markers of EOC including CA-125, HE4, Mesothelin, and KLK6, were identified in this study. The list of 420 extracellular and membrane proteins was cross-referenced with the proteome of ascites fluid to generate a final list of 51 potential candidates. According to Ingenuity Pathway Analysis, two of the top 10 diseases associated with our list of 51 proteins were cancer and reproductive diseases. Of the 51 candidates, 10 proteins were selected for verification in sera from ovarian cancer patients and healthy individuals. Clusterin showed a significant difference between cancer patients and normal, with sera from cancer patients showing higher levels. Another protein, NPC2, did not show a difference in sera between cancer and normals. Protein expression studies using immunohistochemistry showed that NPC2 is highly expressed in ovarian cancer tissue and absent in normal ovarian surface epithelium. In summary, clusterin and NPC2 appear to play a role in ovarian cancer pathobiology and their role in EOC need to be studied further.

Verschiedene ELISA zur Erkennung von Stx1 und Stx2 in Rinderkotproben wurden auf ihrer Anwendbarkeit hin verglichen. Der ELISA nach RICHTER et al. (1997) unter Anwendung von Hydatidenflüssigkeiten von Echinococcus granulosus als Beschichtungssubstanz und monoklonalen Antikörper als Bindungsmolekül stellte sich als die praktikabelste Lösung im Vergleich mit dem ELISA mit monoklonalen Antikörpern (RANDALL et al., 1997) und einem ELISA mit kommerziellem Gb3 heraus. Der ELISA nach RICHTER et al. (1997) wurde gegen den klassischen Vero-Zell- Neutralisationstest validiert. Zu diesem Zweck wurden 100 E. coli-Feldisolate mit beiden Tests ausgewertet. Die zur Quantifizierung von Ergebnissen angewendete Coulter-Methode (Ermittlung der Überlebensraten der Vero-Zellen mit Hilfe des Z2-Partikelzählers) erwies sich als geeignet, während zwei Färbungsmethoden (KV- und MTT-Färbungsmethode) aufgrund von Einstellungsproblemen bei der spektralfotometrischen Messungen nicht zu auswertbaren Ergebnissen geführt hatten. Die Ergebnisse des ELISA und des Vero-Zell-Neutralisationstest wurden in einer „Two- Graphic Receiver-Operating Characteristic“ (TG-ROC)-Analyse verglichen. Die Indexwerte 0,020 und 0,040 wurden als „cut-off“ für den Stx1- bzw. Stx2-ELISA anhand dieser Analyse 79 festgelegt. Die geschätze Sensitivität und Spezifität für den Stx1-ELISA betrugen 100% (pu = 100%; po = 100%), für den Stx2-ELISA betrug die Sensitivität 100% (pu = 100%; po = 100%) und die Spezifität 94% (pu = 88,4%; po = 99,7%). Nach diesen Ergebnissen ist der ELISA ein geeigneter Test und kann den Vero-Zell-Neutralisationstest ersetzen. Der validierte ELISA wurde in einem Feldversuch parallel mit der MK1/MK2-PCR (KARCH und MEYER, 1989) eingesetzt. In einem Zeitraum vom 15.01.97 bis 31.12.99 wurden 1030 Rinderkotproben mit beiden Tests auf das Vorkommen von STEC untersucht. Die PCR wies stx-Gene bei 500 (49%) der Proben nach. Der ELISA wies nur 307 (30%) Stx-positive nach. Bei 264 positiven und 487 negativen Ergebnissen stimmten die Resultate zu 751 Proben (73%) überein. Der berechnete Kappa-Wert war 0,452. Der Kappa-Wert spricht für eine mäßige Übereinstimmung beider Tests. Diesen Ergebnissen nach sollte die PCR als „pre-screening“-Test favorisiert werden, obwohl eine Kombination beider Tests zum Stx-Nachweis am besten geeignet wäre.