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Med hjälp av immunohistokemi kan antigen och antikroppar som är bundna till vävnaden detekteras. Autoimmuna hudsjukdomar är exempel på sjukdomar som diagnosticeras med immunohistokemi. På Falu lasarett användes immunofluorescens för diagnostik av autoimmuna hudsjukdomar. Syftet med denna studie var att optimera enzym-baserad immunohistokemi för epitoperna IgA, IgG, IgM och C3 och jämföra med immunofluorescens vad gäller specificitet, signalstyrka och upplösning. Vävnader som analyserades var tonsill, lever, tarmslemhinna och hudbiopsier. Fixering gjordes i 4% formaldehyd av vävnaderna som infärgades med ultraView DAB och ultraView DAB med FITC. Vävnad som infärgades med DIF med FITC sköljdes enbart i Reaction Buffer. Vävnaderna färgades enligt protokollen ultraView DAB, ultraView DAB med FITC och DIF med FITC. En manuell infärgning med aktiverat DAB utfördes. Resultatet visade bakgrundsinfärgning för samtliga infärgningar. DIF med FITC var tydligare infärgad och lättare att skilja mellan specifik och ospecifik infärgning. Det är svårt att optimera enzym-baserad IHC och epitopen IgA hade generellt starkare infärgning jämfört med epitopen IgM. För att erhålla tillförlitliga resultat krävs det att flera vävnadsprover analyseras. / Using immunohistochemistry, antigens and antibodies bound to the tissue can be detected. Autoimmune skin diseases are examples of diseases that are diagnosed with immunohistochemistry. At Falu Hospital, immunofluorescence was used to diagnose autoimmune skin diseases. The aim of this study was to optimize enzyme-based immunohistochemistry for the epitopes IgA, IgG, IgM and C3 and to compare with immunofluorescence in terms of specificity, signal strength and resolution. Tissues analyzed were tonsil, liver, intestinal mucosa and skin biopsies. Fixation was done in 4% formaldehyde of the tissues stained with ultraView DAB and ultraView DAB with FITC. Tissue stained with DIF with FITC was rinsed in Reaction Buffer only. A manual staining with activated DAB was performed. The result showed background staining for all stainings. DIF with FITC was more clearly stained and easier to distinguish between specific and nonspecific staining. It is difficult to optimize enzyme-based IHC and the epitope IgA generally had stronger staining compared to the epitope IgM. To obtain reliable results, several tissue samples must be analyzed.

In this thesis, improved techniques for biomolecule analysis using capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and hyphenations between those have been presented.A pre-concentration method which is possible to apply in both techniques, has also been investigated. In this work the off-line MS mode has been used either in the form of fractionation (Paper I) or by incorporating the MALDI target in the CE separation system (Paper II).In Paper I, a protocol for CE-MALDI analysis of cyanogen bromide digested bacteriorhodopsin (BR) peptides as model integral membrane protein peptides were established. Also, an improved protocol for partially automated manufacturing of a concentration MALDI-target plate is presented. The design of the targets was suitable for the fractions from the CE. A novel technique for the integration of CE to MALDI-MS using a closed-open-closed system is presented in Paper II, where the open part is a micro canal functioning as a MALDI target window. A protein separation was obtained and detected with MALDI-MS analysis in the micro canal. A method has been developed for detection of monosaccharides originating from hydrolysis of a single wood fiber performed in a micro channel, with an incorporated electromigration pre-concentration step preceding CE analysis in Paper III. The pre-concentration showed to be highly complex due to the fact that several parameters are included that affecting each other. In Paper IV a protocol using enzymatic digestion, MALDI-TOF-MS and CE with laser induced fluorescence (LIF) detection for the investigation of the degree of substitution of fluorescein isothiocyanate (FITC) to bovine serum albumin (BSA), as a contact allergen model system for protein-hapten binding in the skin, is presented. The intention of a further CE-MALDI hyphenation has been considered during the work. In Paper V 2,6-dihydroxyacetophenone (DHAP) was investigated, showing promising MALDI-MS matrix properties for hydrophobic proteins and peptides. 2,5-dihydroxybenzoic acid (DHB) was undoubtedly the better matrix for the hydrophilic proteins, but its performance for the larger and hydrophobic peptides was not optimal. Consequently, DHAP can be used as a compliment matrix for improved analysis of hydrophobic analytes. / QC 20101214