<p>Version:1.0 StartHTML:0000000178 EndHTML:0000005278 StartFragment:0000002640 EndFragment:0000005242 SourceURL:file://localhost/Volumes/NAMNLOS/Examensarbete%20kortisol.doc</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p>

<p>ABSTRACT</p><p>Allergy to mustard has been reported for many years, in some cases as severe anaphylactic reactions. Recent studies imply that this allergy is increasing. Three major allergens have been isolated and characterised; Sin a 1 and Sin a 2 in yellow mustard (Sinapis alba), and Bra j 1 in oriental mustard (Brassica juncea). Yellow mustard and black mustard (Brassica nigra) are the most common species in Europe, whereas oriental mustard is more frequent outside Europe. Mustard plants belong to the Brassicaceae/Cruciferae family. Mustard is present as an ingredient in different foods, sauces and spices, often in small amounts. According to the European labelling directives, mustard and products thereof must always be declared. To monitor this regulation, methods need to be developed to detect mustard. Polyclonal antibodies, produced in rabbits, against yellow and black mustard were characterised with immunodiffusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and immunoblotting. Rocket-immunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA) were developed for the detection and quantification of mustard protein. With indirect competitive ELISA a concentration of 156ng mustard protein per ml food extract was detected, which is more than enough to cover the lowest reported reactive doses.</p>

ABSTRACT Allergy to mustard has been reported for many years, in some cases as severe anaphylactic reactions. Recent studies imply that this allergy is increasing. Three major allergens have been isolated and characterised; Sin a 1 and Sin a 2 in yellow mustard (Sinapis alba), and Bra j 1 in oriental mustard (Brassica juncea). Yellow mustard and black mustard (Brassica nigra) are the most common species in Europe, whereas oriental mustard is more frequent outside Europe. Mustard plants belong to the Brassicaceae/Cruciferae family. Mustard is present as an ingredient in different foods, sauces and spices, often in small amounts. According to the European labelling directives, mustard and products thereof must always be declared. To monitor this regulation, methods need to be developed to detect mustard. Polyclonal antibodies, produced in rabbits, against yellow and black mustard were characterised with immunodiffusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and immunoblotting. Rocket-immunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA) were developed for the detection and quantification of mustard protein. With indirect competitive ELISA a concentration of 156ng mustard protein per ml food extract was detected, which is more than enough to cover the lowest reported reactive doses.

Taenia solium is a cestode that has a two-hosts life cycle. The adult tapeworm causes an asymptomatic disease known as taeniasis whereas the larval stage causes a disease called cysticercosis. In humans, the most common localization for the larvae is the central nervous system where it produces the neurological disorder neurocysticerco-sis. Previous works by several research groups around the world have shown that T. so-lium is a potentially eradicable parasite. Control programs have included treatment of human and pig populations with antihelmintics in conjunction with health education and are now considering vaccination of naïve piglets. The potential of a live vector vaccine system to deliver Taenia solium Tsol18, a proven protective antigen, to prevent transmission of cysticercosis was investigated. An attenuated strain of Salmonella enterica serovar Typhimurium χ9402 was used to develop an oral delivery system. Tsol18 gene was cloned downstream from the β-lactamase signal sequence in a multicopy asd + plasmid vector pYA3620 to yield plasmid pYA3620/Tsol18 and then transformed into the vaccine strain. The recombinant atte-nuated salmonella vaccine construct was stable for 50 generations and expressed rTsol18. Immunization of mice either with one or two doses of 109 CFU of the recombi-nant vaccine strain carrying plasmid pYA3620/Tsol18 elicited specific antibody response to Salmonella self antigens and to rTsol18. Moreover, oral immunization of piglets with 1012 CFU of the vaccine construction significantly reduced the numbers of viable cysts after challenged. The development of a quantitative assay to detect specific antibodies against Tsol18 is also presented here. The Falcon assay screening test –enzyme linked immu-noabsorbant assay (FAST-ELISA) format was used to develop a quantitative antibody detection assay. We have cloned, expressed and purified rTsol18. With purified porcine IgGs we constructed a standard curve that can be used to quantify the immune re-sponse. Our Fast-ELISA was able to follow the kinetics of the immune response in vac-cinated pigs from an experimental trial. The data we present here provides the basis for a safe, affordable and easy vaccine delivery system that can be used as an adjunct in control programs.

Alzheimer’s disease is neurodegenerative with amyloid plaque and neurofibrillary tangles as pathological hallmarks. The most abundant component in the amyloid plaque is the amyloid-β (Aβ) peptide, with presence of both isoform Aβ40 and Aβ42. In immunological methods studying the Aβ peptide a specific monoclonal antibody, 6E10, is routinly being used. In this master thesis work unspecific binding of 6E10 antibody to the blood coagulating protein factor X has been investigated. Factor X is a protein in the blood coagulation cascade where it forms protein complex that activates thrombin. Non-hemostatic functions with connections to nerves and Aβ peptide are also known. Studies with Western blot show clear binding of 6E10 to denatured factor X. Interaction studies with ELISA gives uncertain results, where binding is found but no clear binding curve is obtained. Studies with native factor X in real time measurements with SPR gave no binding at all. These results suggest binding to denatured factor X. Immunohistochemistry studies of colocalisation of factor X and Aβ peptide gave clear evidence that factor X and Aβ are found near each other in mouse brain tissue. Factor X is located outside the blood vessels and Aβ is located at the inside.

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.

To induce the production of antibodies, exogenous antigens are taken up and degraded in antigen presenting cells in vivo. Since this process inevitably lead to distort antigen¡¦s structure, it is likely that some arising antibodies following immunization may not react appropriately with native protein. In the present study, comparative studies on the reactivity of cobrotoxin and recombinant cobrotoxin toward anti-cobrotoxin antibodies were carried out. CD spectra and acrylamide quenching of Trp fluorescence showed that global structure of recombinant cobrotoxin was different from that of native toxin. Results of ELISA and dot blotting assay revealed that recombinant cobrotoxin had a superior reactivity toward anti-cobrotoxin antibodies than native toxin did. Reactivity with antibody fractions specifically against N-terminal region or C-terminal region of cobrotoxin also showed the same results. The binding of recombinant cobrotoxin with antibodies was stronger than that of cobrotoxin as revealed by ammonium thiocyanate elution assay. Recombinant protein was susceptible to reduce its antigenicity after tryptic digestion compared to cobrotoxin. Distorting disulfide linkages at C-terminus caused a marked decrease in immunoreactivity of recombinant cobrotoxin, indicating that anti-cobrotoxin antibodies mostly recognized conformation-dependent epitopes. Moreover, cobrotoxin and recombinant cobrotoxin showed a similar immunoreactivity under denaturing condition. Taken together, these results suggest that native conformation with cobrotoxin may unfavorably impede the interaction of some epitope(s) with anti-cobrotoxin antibodies.

Reproduction de : Thèse doctorat : Médecine. Immunologie et biologie parasitaire : Reims : 2005. / Titre provenant de l'écran-titre. Bibliogr. p.121-141.

A novel capture matrix utilizing human serum albumin (HSA) and streptococcal Protein G (PG), which possesses an albumin binding domain (ABD), was used to immobilize antibodies for improved bacterial capture efficiency in immunoassays. Enzyme linked immunosorbent assays (ELISA) were used to characterize and optimize a specific protocol for the HSA-PG capture matrix; which revealed several critical factors that should be considered. The Fc binding domain, on PG, should have high affinity for the species of capture antibody used in the assay. Goat and rabbit species antibodies bound strongly to the Fc binding domain of PG. Displacement of the capture antibody, by the detector antibody should be avoided to reduce background signals. The Fc binding domain on PG should have equivalent or lower affinity for the detector antibody, when compared to the capture antibody. Goat species antibody, used as a detector antibody, did not displace the same-species capture antibody. ELISA analysis showed detection of Escherichia coli O157:H7 cells at 1.0 x 104 CFU/ml using HSA-PG and goat antibody raised against Escherichia coli O157:H7; unlabeled antibody was used for capture while HRP labeled antibody was used for detection. Studies were performed on an automated fiber optic biosensor, RAPTOR, which was used for the rapid detection of pathogens. Biosensor assays showed detection of E. coli O157:H7 at 1.0 x 10³ CFU/ml in PBS and 1.0 x 105 CFU/ml in homogenized ground beef supernatant. Capture efficiency of the HSA-PG capture matrix was studied using the biosensor and GFP-E. coli O157:H7. The amount of cells captured was less than one percent of the sample concentration. This limit of detection and capture efficiency was comparable to the streptavidin-biotin capture matrix.

West Nile virus (WNV) and St. Louis Encephalitis (SLEV) are arthropod-borne viruses belonging to the genus Flavivirus and are classified as significant human pathogens of global epidemiological importance. Since its introduction into the United States in 1999, WNV has spread throughout most of the country and has caused major epidemics of neuroinvasive disease (Hayes and Gubler, 2005). SLEV is endemic to the United States and is maintained in an enzootic transmission cycle in Florida.The Florida Sentinel Chicken Arboviral Surveillance Network was established in 1978 following a widespread rural epidemic of SLEV in central Florida to monitor the activity of arboviruses (Day and Stark, 1996). This program ultimately impacts vector control strategies and may warrant medical alerts to warn the population. Current serological detection methods for sentinel chickens include hemagglutination inhibition antibody test (HAI), IgM antibody capture enzyme-linkedimmunosorbent assay (MAC-ELISA), and Plaque Reduction Neutralization Test (PRNT).These serological assays may take over three weeks to generate a final result. A more rapid and equally sensitive test to replace these current serological methods would be of benefit. Microsphere-based immunoassays (MIAs) are a more rapid serological option for laboratory diagnosis of many diseases (Kellar et al, 2001). The objective of this study was to develop and validate a protocol for a MIA to detect antibodies to WNV and SLEV in sentinel chicken sera. A total of 385 sentinel chicken sera from 2005 were assayed using the MIA for WNV and 424 sera from multiple years were assayed for SLEV. The capability of the MIA to multiplex allowed for simultaneous detection of antibodies to WNV and SLEV in sentinel chicken sera. The MIA was found to be more sensitive and specific than both the HAI and MAC-ELISA for the detection of antibodies to WNV, and just as sensitive and specific as the MAC-ELISA for the detection of antibodies to SLEV in sentinel chicken sera. These results indicate that there is a potential of the MIA to decrease turn-around time and allow for earlier detection and improvement to the current surveillance system.