Klimabedingte Zwangsmigration: Ein Blick aus der Praxis

IHC controls. a–c, Example of control staining with secondary antibody Anti-C-Myc-Cy3 (red) alone, on cancer tissue (P757). From left to right the pictures show immunofluorescence in the blue, green and red range respectively. d–e, Example of control staining with the scFv antibody epsilon, which also was used as a negative control during screening and validation of selected antibodies. From left to right the pictures show immunofluorescence in the blue, green and red range respectively. (TIF 4987 kb

Sequences showing an error prone repeat close to primer annealing site.

<p>The sequencing data from eight clones with errors and a reference sequence showing the CDR2 and immediate downstream region. The nucleotides composing the randomized region of CDR2 are indicated at the top along with the nucleotides matching the 3’ end of the reverse primer used for the generation of the CDR2 insert. Eight of the shown clones have errors and all of the errors have occurred in the area between the randomized region of CDR2 and 3’ end of the reverse primer used in the generation of the CDR2 insert. Especially the “TATTAT” repeat seems susceptible to errors.</p

Cell staining with soluble M3-H12 Predator antibody.

<p>The cell lines HVBP (A) and HMEC-1 (B) were stained with selected M3-H12 antibody and detected with Protein A conjugated with Alexa Fluor 488 (green). The cell nuclei were stained with DAPI (blue) (scale bar, 20 µm).</p

Biophysical evaluation of isolated antibodies and affinity measurement for C3 against hen egg lysozyme.

<p>(<b>A</b>) Thermal unfolding and refolding for the lysozyme binding clones assayed by circular dichroism spectroscopy. The first melting curve for each clone is shown in blue, and the subsequent melting curve in red. After recording the first melting curve the antibodies were allowed to cool, and the experiment was repeated using the refolded antibodies. (<b>B</b>) Recorded heat change over time during successive injections of lysozyme into the cell containing C3 domain antibody. The insert shows the integrated injection heats as a function of molar ratio of the lysozyme to the antibody. Circles are the integrated signal from the larger panel, and the best fit of the 1:1 binding equation to the observation is shown as a line. The figure is representative for the three measurements performed. (<b>C</b>) Chroatogram from size exclusion chromatography of C3 antibody. The migration of two size markers is shown in the chromatogram; albumin (black triangle) with a size of 66 kDa, and TEV-protease (white triangle) with a size of 26 kDa. The single elution peak of the antibody well below the 26 kDa reference protein indicates that it exhibits a monomeric state in solution.</p

The diversity of the Predator library.

<p>The CDR2 and CDR3 of the library have been designed with specific frequencies designated to every amino acid used for all the randomized position (red boxes) in both CDR2 and CDR3 according to the different properties of the amino acids. The frequency of a given amino acid at a given position can be read from the table. The unique restriction sites used for cloning the CDR regions into the vector are shown flanking the sequences.</p

Screening ELISA from selection on lysozyme and antibody-phage ELISA dilution series on lysozyme.

<p>(<b>A</b>) The clones with the highest signal on immobilized lysozyme are shown in the figure. The signal on lysozyme is depicted as blue bars, whereas the signal on milkprotein is shown with red bars. The three clones with the lowest signal among the 96 clones screened are also shown for comparison. Clones marked with an asterisk were examined further in titered phage ELISA, ELISA using soluble domain antibodies and in western blots. (<b>B</b>) Six of the clones from the screening ELISA were tested further in a titered phage ELISA in microwells. The y-axis of the graphs depicts the signal for serial dilutions of the phages displaying the selected domain antibodies, and the x-axis depicts the phage titter in the wells of the dilution series. The binding to milk proteins is indicated with boxes, while the binding to lysozyme is indicated with circles. The error bars show one standard deviation based on three independent experiments. (<b>C</b>) The six clones were also tested in an ELISA using soluble antibody fragments. Here the x-axis depicts the concentration of antibody added to respective wells of the dilution series. The error bars show one standard deviation based on three independent experiments.</p

Trace data from sequencing of “double read” clone.

<p>The trace data from the CDR3 randomized region of one of the clones we define as having a double read. We found the trace data to be unusual as there was a perfect read both before and after the CDR3 region. Furthermore the trace data indicate that there are two very clearly defined reads mixed together in the region.</p

Screening ELISA from selection on HBVP cells and antibody-phage ELISA dilution series on HBVP.

<p>(<b>A</b>) The clones shown are the ones for which the signal on HBVP-cells was highest, and for which the signal on cell medium did not exceed 25% of the signal on cells. The three clones with the lowest signal among the 384 clones screened are shown for comparison. Blue bars depict the signal on HBVP cells, whereas signal on medium is depicted in red. The result was obtained after one round of selection on immobilized HBVP cells. Clones marked with an asterisk were examined further in titered ELISA. (<b>B</b>) Three of the clones from the screening ELISA were tested further in a titered phage ELISA in microwells. The x-axis of the graphs depicts the titer for serial dilutions of the phages displaying the selected domain antibodies, and the y-axis depicts the ELISA signal. The signal on HBVP cells is marked with boxes, while the signal on various decoy cells and on cell medium is marked with circles. The error bars show one standard deviation calculated on the basis of three independent experiments.</p