Horseradish peroxidase conjugated anti mouse antibody

Peptide SPOT synthesis [43] (link) was performed essentially as described in [44] (link). Briefly, cellulose membrane-bound peptides were prepared in an automated Spot synthesizer (MultiPep, Intavis AG Bioanalytical Instruments, Köln, Germany) using Fmoc derivatives of amino-acids (Novabiochem, Darmstadt, Germany). GST-nTRIP6 was expressed in Escherichia coli BL21 and purified using glutathione Sepharose 4B beads (GE Healthcare, Freiburg, Germany). After activation of the membranes with methanol the membrane-bound peptide arrays were blocked for 3 h in blocking buffer (2% milk powder and 5% sucrose in Tris-buffered saline (TBS), pH 8.0) and then incubated overnight at 4°C with 10 µg/ml purified GST-nTRIP6 in blocking buffer, which was then detected with an anti-GST antibody (G1160; Sigma-Aldrich, München, Germany), revealed by a horseradish peroxidase conjugated anti-mouse antibody (Sigma-Aldrich) and ECL. The QRALAKDLIVPRRP peptide, recognized by the anti-GST antibody, was used as a positive control.

Anti-phospho-Akt, anti-Akt, anti-phospho-p70S6K and anti-hemagglutinin (HA) antibodies were purchased from Cell Signaling Technology, Inc., and used at a dilution of 1:1,000, according to the manufacturer’s instructions. The JC12 anti-FOXP1 antibody was provided by Professor Alison H. Banham and used at a dilution of 1:30. β-actin mouse monoclonal antibody (dilution, 1:5,000) and horseradish peroxidase-conjugated anti-mouse antibody (dilution, 1:2,500) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Agilent Technologies, Inc. (Santa Clara, CA, USA), respectively.

For the competitive ELISA, 96-well microplates were coated with preS1 peptide p20-47 using 100 μL of peptide solution (10 μg/mL) per well. After incubation overnight at 4 °C, the plates were blocked with 1 % BSA in PBS for 30 min at 37 °C. Subsequently, 50-μL aliquots of serial dilutions of competing protein and 50 μL of the monoclonal antibody MA18/7 [33 (link)] were simultaneously added to the wells; to this end, the MA18/7 dilution was used, which resulted in an OD492 value within the range of 0.5–0.6 for the control sample (without competing protein). After incubation for 1 h at 37 °C, the plates were washed three times with PBS containing 0.05 % of Tween-20. Thereafter, 100 μL of horseradish peroxidase-conjugated anti-mouse antibody (Sigma–Aldrich St. Louis, MO, USA) was added at a 1:10,000 dilution. After incubation for 1 h at 37 °C, the plates were washed as before, and OPD substrate (Sigma–Aldrich St. Louis, MO, USA) was added to develop the colour. The per cent inhibition (I %) of antibody binding by the competing protein was calculated as follows: $$I\%=\left[\left(\text{OD}492\text{test}\text{sample}-\text{OD}492\text{negative}\text{control}\right)/\left(\text{OD}492\text{positive}\text{control}-OD492\text{negative}\text{control}\right)\right]\times 100.$$ The molar amounts of the proteins necessary for 50 % inhibition (I₅₀) were calculated.

For the first assay (Fig. 6A), 10⁸ vg of indicated AAV vectors were adjusted to the “pH buffer” (citric acid/Na₂HPO₄) at pH 7.2 and pH 5.2, for 15 min. at room temperature. Then samples were exposed to indicated temperatures (50, 55, 60, 65, 70, 75, 80 °C) for 15 min and then diluted in PBS. For the second assay (Fig. 6B), wells of a qPCR plate were loaded with 5 × 10⁸ vg of indicated AAV vectors diluted in PBS. The temperature gradient was generated by a LightCycler® 96 System (Roche Life Science) using a self-designed program (Supplementary Table S3). After the run, PBS was used to dilute samples. In both experiments, after PBS dilution, samples are transferred to a nitrocellulose membrane using a vacuum blotter for a native dot blot assay. After saturation, the membranes were incubated overnight at 4 °C with A20, or B1 antibodies^{63 (link)}. A horseradish peroxidase-conjugated anti-mouse antibody (Sigma,1/10,000 dilution) was then applied for 1 h at room temperature. Finally, the membranes were incubated with an enhanced chemiluminescence reagent (West Dura; Pierce) and analyzed by autoradiography film exposure or FusionFX device (Peqlab).

A 96‐well qPCR plate was loaded with 2E9 vector particles/well diluted in PBS and subjected to a temperature gradient using the LightCycler 96 System (Roche Life Science) as previously described (Rossi et al, 2019). Subsequently, samples were diluted in PBS and transferred to a nitrocellulose membrane using a vacuum blotter. Membrane was probed with B1 (1:5,000, 16048, Abcam) antibody recognising the C’‐terminus of all AAV2 capsid proteins and with A20 (1:50) antibody (Wistuba et al, 1997) binding to intact capsids. B1 and A20 were kindly provided by Martin Müller (DKFZ, Heidelberg, Germany). As secondary antibody, a horseradish peroxidase‐conjugated anti‐mouse antibody (1:10,000, Sigma‐Aldrich) was applied. Finally, the membranes were treated with an enhanced chemiluminescence reagent (West Dura, Pierce) and analysed by FusionFX device (Peqlab).

For the competitive ELISA, 96-well microplates were coated with 100 µL of preS1(20–47) peptide solution (10 µg/mL) in 50 mM sodium carbonate buffer, pH 9.6 per well and incubated overnight at 4 °C. After blocking with phosphate-buffered saline (PBS) containing 1% BSA for 1 h at RT, 50 µL aliquots of serial dilutions of test proteins and 50 µL of the anti-preS1 mAb MA18/7 (dilution 1:500) [9 (link)] were added to the wells simultaneously. Plates were incubated at 37 °C for 1 h, then washed four times with Tween-20 containing (0.05%) PBS. Thereafter, 100 μL of horseradish peroxidase-conjugated anti-mouse antibody (Sigma-Aldrich St. Louis, MO, USA) was added to wells at a 1:10,000 dilution and incubated at 37 °C for 1 h. After washing the plates four times as before, OPD substrate (Sigma–Aldrich St. Louis, MO, USA) was added to develop the color. The percent inhibition (I%) of antibody binding by the competing protein was calculated as follows: I% = [(OD492 test sample—OD492 of negative control)/(OD492 of positive control—OD492 negative control)] × 100. The molar amount of the protein necessary for 50% inhibition (I50) was calculated.