Anti mouse hrp conjugate

For monoclonal phage ELISA, individual colonies were picked after the second and third rounds of selection and grown into 2xTY medium containing 100 µg/ml ampicillin and 4% glucose in a 96-well plate and incubated at 37 °C, 250 rpm for 12 hours. Then, the overnight culture for each clone diluted 100-fold into 200 µl 2xTY medium (100 µg/ml ampicillin and 0.1% glucose) and incubated at 37 °C, 250 rpm until OD600 = 0.4. The culture was infected with 4 × 10⁸ KM13 helper phages for 30 min at 37 °C, the bacteria pelleted by centrifugation and resuspended in 150 µl of 2 × YT containing ampicillin (100 µg/ml) and kanamycin (50 µg/ml) before growth overnight at 25 °C, 250 rpm for 16 hours. Then, the plate was centrifuged and 100 µl of culture supernatant of each well was used for ELISA plates that pre-coated with epsilon toxoid (100 µg/ml) and blocked with 4% MPBS buffer. Phage binding was detected with a monoclonal anti-c-Myc antibody (Biolegend) and anti-mouse HRP conjugate (Sigma).

The western blot analysis for the HTT protein (17/68Q tract) was performed as previously described (Fiszer et al., 2011 (link)). Briefly, 30 μg of total protein was run on a Tris-acetate sodium dodecyl sulfate (SDS)-polyacrylamide gel (1.5 cm, 4% stacking gel/4.5 cm, 5% resolving gel, acrylamide:bis-acrylamide ratio of 49:1) in XT Tricine buffer (Bio-Rad, Hercules, CA, USA) at 130 V in an ice-water bath. Subsequently, the proteins were wet-transferred to a nitrocellulose membrane (Sigma–Aldrich). All of the immunodetection steps were performed using the SNAPid system (Millipore). The primary antibodies anti-huntingtin (1:1000, MAB2166, Millipore) and anti-plectin (1:1000, ab83497, Abcam, Cambridge, UK) and secondary antibodies anti-mouse HRP-conjugate (1:2000, A9917, Sigma–Aldrich) and anti-rabbit HRP-conjugate (1:2000, 711-035-152, Jackson ImmunoResearch) were used in a PBS/0.1% Tween-20 buffer containing 0.25% non-fat milk. The immunoreaction was detected using WesternBright Quantum HRP Substrate (Advansta, Menlo Park, CA, USA). The protein bands were scanned directly from the membrane using a camera and were quantified using Gel-Pro Analyzer.

Acetylshikonin (CAS Number: 24502-78-1) and simvastatin (CAS Number: 79902-63-9) were purchased from Chem Face China Company. Temozolomide (CAS Number: 85622-93-1), propidium iodide (CAS Number: 25535-16-4), 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) (CAS Number: 298-93-1), acridine orange (CAS Number: 65-61-2), bafilomycin A1 (Cat#B1793-10UG) and anti-rabbit LC3β antibody (Cat#L7543—100UL) were purchased from Sigma-Aldrich Co. Anti-rabbit p62 antibody (Cat#39749), anti-rabbit Beclin-1 antibody (Cat#3738), anti-mouse Bcl2 antibody (Cat#15071), anti-rabbit Mcl-1 antibody (Cat#4572), anti-rabbit Bcl-XL antibody (Cat#2762), and anti-rabbit GAPDH antibody (Cat#2118) were purchased from Cell Signaling Company. The secondary antibodies, anti-rabbit HRP-conjugate and anti-mouse HRP-conjugate, were purchased from Sigma-Aldrich (Oakville, ON, Canada) as well. The enhanced chemiluminescence (ECL) (CAS Number: 12630) was acquired from Cell Signaling Technology Co. (Beverly, MA, USA). The bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Fisher Scientific (Winnipeg, MB, Canada).

The rP32Trx antigen (0.6 mg/mL in carbonate-bicarbonate buffer; pH 9.6) was immobilized onto a 96-well plate. The unbound proteins were removed using PBS-T. Blocking was performed by adding 3% skimmed milk and incubating for 40 min at 37°C. The sera of the mice were added at a dilution of 1:100, followed by a double titration. Next, anti-mouse HRP conjugate (Sigma-Aldrich) was added at a dilution of 1:20,000. Further, o-phenylenediamine dihydrochloride substrate was added to develop color, and the reaction was stopped by adding 0.2 M sulfuric acid. The optical density was measured at a wavelength of 490 nm using a plate spectrophotometer 680 (BioRad, Italy).

ELISA was carried out using commercially available anti-TTR monoclonal antibody (ProSci Antibody, USA). For comparison between patient and control group, 60 plasma samples from each group were taken. Individual plasma samples from the enrolled participants were diluted (1 μl/200 μl) in a coating buffer (0.01 M Na₂CO₃ and 0.035 M NaHCO₃, pH 9.6), coated on 96 well plates (Thermo Scientific, Nunc, USA) and incubated overnight at 4°C. Next day the plates were washed three times with 100 μl PBST and incubated with 100 μl blocking buffer (1% BSA in 1X PBS) for 1 h at RT. The blocking buffer was extracted, plates were washed and incubated with 100 μl of anti-TTR as a primary antibody (1∶2000 dilution) for 2 h at RT. The plates were then washed three times and incubated with 100 μl of anti-mouse HRP conjugate (1∶1000 dilution) for 1 h and developed with ortho phenylene diamine (1 mg/ml in 0.05 M citrate buffer and 5 μl/ml H₂O₂, Sigma-Aldrich, USA). The reactions were stopped by adding 2N H₂SO₄ in each well and binding efficiency was checked by reading absorbance at 492 nm in an ELISA reader (Spectramax Plus; Molecular Devices, USA) [20] (link). Plasma levels of TTR in RA patients was also compared from patients of different diseases.