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Untersuchung des Chemokins MIP-3 β/CCL19 Measurement of CCL19 Concentrations of CCL19 in the CSF and serum were determined by sandwich ELISA (Enzyme linked immunoabsorbent assay). An ELISA Kit specific for canine MIP-3β/CCL19 (E90096Ca 96 Tests) was used according to manufacturer’s instructions (Uscn Life Science Inc., Wuhan, P.R. China). Briefly, the microtiter plate provided in the ELISA kit has been pre-coated with a monoclonal antibody specific to MIP-3β/CCL19. CSF samples were used undiluted or were tapered down to a dilution of 1:13 with the standard diluent depending on the CCL19 output concentration. Serum samples were used undiluted for the ELISA. Seven wells were prepared for the dilutions of standard, one well for blank and the rest of the 96 wells were used for the CSF and serum samples. Four CSF and serum samples of the IE group served as a control and one of these IE samples was pipetted to the plates in each experiment. As a positive control served CSF and serum samples of the neuroinflammatory group and the same sample was used in each experiment at same dilution. After a two hours incubation time at 37°C in humidified air with 5% CO2 in an incubator (SANYO Sales & Marketing Europe GmbH, Munich), the liquid of each well was removed. A biotin- conjugated polyclonal antibody preparation (Uscn Life Science Inc., Wuhan, P.R. China) specific for CCL19 was added to each well. Following a one hour incubation time the wells were washed three times with wash buffer solution (Uscn Life Science Inc., Wuhan, P.R. China). Avidin conjugated to horseradish peroxidase (Uscn Life Science Inc., Wuhan, P.R. China) was pipetted to each well and incubated for 30 minutes at 37° C. The wash process was repeated for total of five times as conducted before. Subsequently the TMB (3,3´,5,5´- Tetramethylbenzidine) substrate was pipetted to the plate. The enzyme-substrate reaction was terminated by the addition of sulphuric acid solution (Uscn Life Science Inc., Wuhan, P.R. China) and the color change measured spectrophotometrically at a wavelength of 450nm ± 10nm on a microplate reader equipped with the analysis software Gen 5 (Synergy2 HT multi-mode microplate reader, BioTek Instruments Inc., Bad Friedrichshall Germany). 38
Untersuchung des Chemokins MIP-3 β/CCL19 The detection range of this method was 15.6 - 1000 pg/ml. The standard curve was created by a lyophilized standard reagent (Uscn Life Science Inc., Wuhan, P.R. China) provided in the ELISA kit. Standard concentrations used for the ELISA were 1000 pg/ml, 500pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ ml, 31.2 pg/ml, 15.6 pg/ml. The minimum detectable dose of canine CCL19 is 6.5 pg/ml. All measurements of the CSF and serum were performed in duplicates, and OD (optical density) were converted into chemokine concentration (pg/ml) using calibration curves generated in each experiment. Chemotaxis Assay To prove chemotactic activity of measured CSF samples for mononuclear cells and to give a hint that CSF CCL19 is available in the active form, migration assays were performed by using a disposable 96-well chemotaxis chamber (Chemo TX Disposable Chemotaxis system, NeuroProbe, Gaithersburg, MD) with polycarbonate filters (5 µm pore size, 3.2 mm diameter size, 30 µl well size). CSF samples of seven patients were examined, three untreated patients diagnosed with SRMA, two untreated patients with the suspected diagnosis of MUO and two patients with IVDD. Peripheral mononuclear cells (PMCs) were separated from five milliliters of fresh EDTA blood collected via cephalic venipuncture of a healthy dog. PMCs were separated by Ficoll gradient centrifugation according to the technique described by Toth et al. 1992. PMCs were washed twice with ten milliliters Hank’s balanced salt solution (HBSS) (Fa. Sigma-Aldrich, Deisenhofen, Germany) and re-suspended in Cell-Wash fluid (Becton Dickinson, Heidelberg, Germany). The cell suspension was stained by trypanblue (0,4% in phosphate buffered saline (PBS) Fa. Sigma-Aldrich, Schnelldorf, Germany) and counted in a TÜRK cell counting chamber (Fa. Brand, Wertheim, Germany). Twenty five µl of the cell suspension (50000 cells/ 25 µl) were pipetted directly on the top side of the filter, which is coated with a hydrophobic mask around each of the test sites. The 39
Page 1 and 2: Tierärztliche Hochschule Hannover
Page 3 and 4: Meinen Eltern
Page 5 and 6: Inhaltsverzeichnis INHALTSVERZEICHN
Page 7 and 8: Einleitung Kapitel 1 Einleitung Ent
Page 9 and 10: Literaturübersicht Kapitel 2 Liter
Page 11 and 12: Literaturübersicht erhöhtem Gesam
Page 13 and 14: Literaturübersicht der Entwicklung
Page 15 and 16: Literaturübersicht Häufig wird ei
Page 17 and 18: Literaturübersicht besitzen Patien
Page 19 and 20: Literaturübersicht Sie können Obe
Page 21 and 22: Material und Methoden Kapitel 3 Mat
Page 23 and 24: Material und Methoden - Tischzentri
Page 25 and 26: Material und Methoden 3.3. Methode
Page 27 and 28: Material und Methoden Abb. 1: Prinz
Page 29 and 30: Material und Methoden wurde jeweils
Page 31 and 32: Untersuchung des Chemokins MIP-3 β
Page 37: Untersuchung des Chemokins MIP-3 β
Page 59 and 60: Falldarstellung Kapitel 5 Falldarst
Page 61 and 62: Falldarstellung the central nervous
Page 63 and 64: Falldarstellung set at a rate of 49
Page 65 and 66: Falldarstellung Fig. 3 Fig. 1, Fig.
Page 67 and 68: Falldarstellung present, the evalua
Page 69 and 70: Falldarstellung bacterial growth (D
Page 71 and 72: Falldarstellung high risk of brain
Page 73 and 74: Falldarstellung However, at the ini
Page 75 and 76: Falldarstellung Adjunctive glucocor
Page 77 and 78: Falldarstellung Versed®; Roche Eto
Page 79 and 80: Falldarstellung in: A. LAJTHA, A. G
Page 81 and 82: Falldarstellung KING, N. (2010): Br
Page 83 and 84: Falldarstellung PLATT, S.R., u. N.
Page 85 and 86: Falldarstellung VITE, C.H. (2005):
Page 87 and 88: Übergreifende Diskussion Organismu
Page 89 and 90:
Übergreifende Diskussion Neutrophi
Page 91 and 92:
Übergreifende Diskussion Rückenma
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Zusammenfassung Kapitel 7 Zusammenf
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Zusammenfassung In dieser Studie ko
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Summary Increased intrathecal chemo
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Abkürzungsverzeichnis Kapitel 9 Ab
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Abkürzungsverzeichnis SRMA Std. T
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Literaturverzeichnis BAUMGÄRTNER,
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Literaturverzeichnis CHERUBINI, G.
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Literaturverzeichnis DE LAHUNTA, A.
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Literaturverzeichnis FUCHS, C., S.
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Literaturverzeichnis JEFFERY, N.D.
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Literaturverzeichnis KLOPP, L.S., J
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Literaturverzeichnis MADDISON, J.E.
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Literaturverzeichnis PASHENKOV, M.,
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Literaturverzeichnis SCHATZBERG, S.
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Literaturverzeichnis SERAFINI, B.,
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Literaturverzeichnis TALARICO, L.R.
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Literaturverzeichnis UCHIDA, K., T.
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Anhang Kapitel 12 Anhang Materialvo
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Anhang E: TMB Substrat: Das benöt
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Anhang 9. Die Flüssigkeit, welche
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Anhang 4. Anschließend wird das ve
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Anhang 1. Anzahl der Hunde, Diagnos
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Anhang 2. Anzahl der Hunde, Diagnos
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Anhang Lfd. Nr. Diagnose Grad Vorst
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Danksagung Kapitel 13 Danksagung Me
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