ELISA and restriction digestion
Two consecutive rounds of selection were performed with a large non-immune VHH library as described previously [4, 5] on the first 548 amino acids of the huntingtin protein [6]. From this selection, 96 individual colonies were randomly picked. To assess specific binding to the antigen of interest, ELISA was performed. In short, all 96 clones were grown first in 100 μl/well of growth medium (2xTY/Ampiciline/0,1% Glucose) for 4 hours at 37°C while shaking (220 rpm). Induction of VHH overproduction was then performed overnight after adding 20 μl of a 6 mM IPTG solution (in 2xTY/Ampiciline). A 96 well Maxisorp plate was coated with antigen overnight at 4°C. The next day; Maxisorp plates were washed twice with 1× PBS and blocked for 30 min at room temperature with 4% non-fat milk in PBS. After blocking, 50 μl of 4% non-fat milk/PBS solution was added to each well. Plates containing the VHH's were spun down at 1200 g and 4°C for 15 minutes. From each well, 50 μl of VHH-containing supernatant was added to the corresponding well of the Maxisorp plate. The Maxisorp plate was then incubated for 2 hours at room temperature while shaking (900 rpm). After incubation the plate was rinsed 3 times with PBST and 3 times with PBS. For the detection of bound VHH's, 100 μl of a 1:1000 solution of anti-myc antibody (9E10, Santa Cruz) conjugated to Horse Radish Peroxidase (HRP) in 4% non-fat milk/PBS was added to each well. The Maxisorp plate was then incubated for 2 hours at room temperature while shaking (900 rpm). After incubation the plate was rinsed 3 times with PBS containing 0.05% Tween20 and subsequently 3 times with PBS. The reaction was developed by adding an OPD-H2O2 solution to each well followed by a 35 minutes incubation at room temperature under dark conditions. Reaction was stopped by adding 50 μl/well of 1 M H2SO4. Optical densities were measured at a wavelength of 490 nm using a plate reader (Biotek, Winooski, USA). To analyse clone inserts, PCR was performed on all 96 clones on a standard block cycler (Bio-Rad, Hercules, CA) using 1 μl of overnight culture with the following primers: M13R 5'-CAGGAAACAGCTATGAC-3' and MPE25WB 5'-TTTCTGTATGGGGTTTTGCTA-3'. Amplification was performed in 1× PCR buffer, 0.7 U FastStart Taq DNA polymerase (Roche, Mannheim, Germany), 200 μM dNTPs, 1 pmol of each primer in a reaction volume of 20 μl. Cycling conditions were 5 min at 95°C followed by 35 cycles of 40 sec at 95°C, 40 sec at 55°C and 1 min at 72°C, followed by a final incubation of 5 min at 72°C. DNA fingerprint analysis was performed on 10 μl PCR product digested for 2 hr at 37°C in a total volume of 20 μl containing, 1× reaction buffer and 1 U HinfI (Fermentas, Burlington, Canada) and digests were run on a 3% agarose gel.
High resolution melt curve analysis
Amplification for HRMA was performed on 2 μl of 1:1000 dilutions of previously amplified clones using the same primers as in the first PCR reaction in a 10 μl reaction volume containing 1× LightCycler® High Resolution Melting Master (Roche), 2 mM MgCl2, and 3 pmol of each primer. All samples were amplified in duplicate in the Lightcyler®480 (Roche) and this was followed by melt curve acquisition. Initial denaturation of 10 min at 95°C was followed by 30 cycles of 10 sec at 95°C, 30 sec at 55°C and 20 sec at 72°C. After a final extension of 5 min at 72°C, melt curve acquisition started with a hold of 1 min at 95°C followed by 1 min at 40°C and ramping from 60°C to 98°C at 1°C/sec with 25 acquisitions per °C. Grouping of the clones was done using the Genescanning module of the Lightcyler®480 Software Release 1.5.0 (Roche). The sample with the highest melting temperature was selected from the normalized and temperature shifted melt curves and used as baseline for the difference plot analysis. After the software had calculated the groups, they were checked manually to ensure that samples with identical melt curves were assigned to their appropriate groups. Because the software could only assign 8 groups at once, analysis was done three times. For the second and third round of analysis, all samples assigned to groups in the previous rounds were omitted until all samples had been clustered.