3.5.2.1 Cell-based assay by flow cytometry
Cell-bound recombinant protein antigens in native conformation were used to probe for reac- tive autoantibodies in human serum. For each serum sample, TE671 cells expressing human NF155 and human NF186, and also cells transfected with the empty vector were stained. The 3 cell lines (TE-RSV0, TE-NF155, TE-NF186) were plated at 200 000 cells per well. After pelleting by centrifugation, the cells were stained with antibodies.
Initial experiments to set up a staining protocol utilized pan-NF mAbs (A12/18.1, A4/3.4) and secondary detection antibodies conjugated to fluorescent molecules. The initial staining proto- col was the following: 100 µL of 1:100 diluted primary antibody or 1:100 diluted human serum was used to resuspend the cell pellet and this was incubated on ice for 1 h. Following 2 washes with 200 µL PBS/1% FCS, 100 µL secondary detection antibody solution (1:200 anti-mouse- Ig-PE (Dako, Eching, Germany) or 1:100 anti-human-IgG-DyLight488 (JacksonImmuno, New- market, UK)) was applied and the cells were incubated on ice for 45 min. After 3 washes, the cells were resuspended in 100 µL PBS with TO-PRO3 (1:4000; Invitrogen). The stained cells were acquired on FACS Calibur (BD Biosciences), and the data was analyzed on FlowJo (Tree Star Inc., Ashland, USA). The fluorescence intensity measured on the NF-expressing cells was divided by the intensity measured on cells with the empty vector to normalize for background staining.
The optimized protocol contained the following modifications: serum samples were diluted 1:50 in PBS/1% FCS and 100 µL was used to stain cells in each well, this was incubated for 1 h on ice; after 2 washes with 200 µL PBS/1% FCS, 100 µL secondary antibody solution (1:150 anti-human-IgG-PE or 1:150 anti-human-Ig-PE, both from JacksonImmuno) was added and incubated on ice for 35 min. Following 3 washes, the cells were again resuspended in PBS with TO-PRO3 iodide for analysis as in the previous protocol. It was found to be important to keep the cells cold during the entire procedure.
3.5.2.2 ELISA
Soluble recombinant human NF155 and NF186 were immobilized on a solid surface to be displayed in random orientation for antibody detection. The optimized protocol was the fol-
CHAPTER 3. MATERIAL AND METHODS 43
lowing: 5 µg/mL of human NF155 and NF186, rat NF155 (R&D Systems, Abingdon, UK), and BSA (Serva) were used for coating in 0.1 M carbonate buffer at pH 9.5 (maxisorp) or 1 x PBS (Ni-NTA) at 4◦C overnight. After washing with PBS, the plate was blocked with 200 µL of PBS/0.05% Tween-20 (PBST) with 3% BSA (Serva) at 37◦Cfor 1 h. The plate was then washed with PBST. To each well, 100 µL of sera diluted 1:100 or 0.2 µg/mL mAb (A4/3.4) in blocking buffer was applied to wells coated with BSA, human NF155 and NF186 and rat NF155; this was incubated for 1 h at 37◦C. After washing with PBST, 100 µL of anti-human-Ig-horseradish peroxidase (HRP; 1:7000; JacksonImmuno) or anti-mouse-IgM-HRP (JacksonImmuno) was added into each well and the plate was incubated at 37◦C for 30 min. After washing with PBST, 100 µL of TMB solution (eBioscience) was added to each well and 50 µL of 2 N sulfuric acid was used to stop the reaction once sufficient colour change was observed. The optical density was measured at 450 nm, and the data was analyzed using Prism Graphpad (Graphpad software, La Jolla, USA). For antigen crossreactivity experiments, human contactin-2 and hu- man oligodendrocyte myelin glycoprotein (OMgp) (both from R&D systems) were incorporated to the panel of test antigens used for screening.
3.5.2.3 Western blot
To screen human serum for NF reactivity by Western blot, we blotted NF155 and NF186 onto polyvinylidene fluoride (PVDF) membrane, and stained each blot with a diluted serum sample. In detail: 1 µg of each neurofascin and 3 µL of Protein Rainbow Marker (GE Healthcare, Munich, Germany) were loaded for every 3 lanes on a 4-12% BIS-TRIS gel (Invitrogen). The gel was subjected to electrophoresis at 200 V for 1 h. Hybond-P PVDF-membrane (GE Healthcare) was cut to the size of the gel,activated by methanol, washed with water and then incubated in cold transfer buffer (20x NuPAGE transfer buffer, Invitrogen). When the gel electrophoresis was completed, the ’blot sandwich’ was built and placed between the electrode-plates of a semi-dry transfer apparatus. The ’blot sandwich’ from cathode to anode consisted of 3 sheets of 3 mm Whatman filter paper pre-soaked in transfer buffer, the gel on top of the membrane, and then another 3 sheets of pre-soaked filter paper. The semi-dry transfer set at 0.8 mA/cm2 for 90 min. Afterwards, the membrane was cut into strips where each strip contain the protein marker, NF155, and NF186. These strips were blocked in 5% skim milk in PBST for 1 h at room temperature. After washing with PBST, each strip was incubated in diluted human serum (1:400 in blocking solution) overnight at 4◦Con a shaker. These strips were washed with PBST for 1 h
CHAPTER 3. MATERIAL AND METHODS 44
at room temperature, and anti-human-Ig-HRP (1:7000; JacksonImmuno) was applied for 1 h at room temperature on a shaker. After washing with PBST, enzymatic chemiluminescence (ECL) solution was applied for 1 min. The ECL solution was a mixture of 10 mL ECL-A (0.25% (w/v) luminol in 1M Tris/HCl), 100 µL ECL-B (0.11% (w/v) para-hydroxycoumarin acid in DMSO), and 3.1 µL of 30% H2O2. Then the strip was exposed onto autoradiography film (GE Healthcare)
for 3 sec to 1 min. Visual analysis was done on a lightbox for presence of bands where each neurofascin isoform was blotted.