Hrp conjugated anti mouse igg antibody

Protein extracts were obtained from 1 × 10⁶ cells after sonication in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Roche Applied Science). Total protein was determined by using Total protein concentration BCA protein assay kit.
(Pierce). For MBNL1 immunodetection 20 mg of total protein was heated 5 min at 100 ºC, electrophoresed on 12% SDS-PAGE gels, and transferred using a semi-dry system (Bio-Rad) onto nitrocellulose membranes (GE Healthcare). Then, membranes were blocked for 1 h at RT in PBS-T (0.05% Tween 20 [pH 7.4]) supplemented with 5% non-fat dried milk and incubated ON at 4 ºC with antibody mouse anti-MBNL1 (1:200, cloneMB1a, The Wolfson Centre for Inherited Neuromuscular Disease, UK) on blocking solution. After three washes with PBS-T membranes were incubated 1 h RT with secondary antibody in blocking solution (anti-mouse-IgG goat horseradish peroxidase (HRP)-conjugated 1:5000, Sigma-Aldrich). β-ACTIN was detected with a primary mouse anti-β-Actin antibody (1 h, 1:5000, Sigma-Aldrich) followed by HRP-conjugated anti-mouse-IgG antibody (1 h, 1:5000, Sigma-Aldrich). Bands were detected using ECL western blotting substrate (Pierce). Images were acquired using ImageQuant LAS 4000 (GE Healthcare).

Membranes were saturated with 5% non-fat dried milk. Western blotting was carried out with total sera (1/100). One membrane per patient’s serum has to be used in order to analyze the related antigenic map. For each study, one membrane is dedicated for the landmark map using a pool of commercial mAbs (anti-HSP71, 1/1,500; anti-ENOA, 1/5,000;anti-ACTB, 1/750; anti-G3P, 1/750; anti-TPIS, 1/200,000). Sera and commercial mAbs were diluted in Tris buffer saline (TBS: 100 mM Tris, pH 8.0; 0.3 M NaCl) containing 0.5% Tween20 (w/v) and 5% non-fat dried milk. After incubation overnight at 4°C, each antigenic map was revealed with an anti-human IgG horseradish peroxidase (HRP)-conjugated antibody 1/5,000 (Sigma Aldrich) and the landmark was revealed by HRP-conjugated anti-mouse IgG antibody 1/5,000 (Sigma Aldrich). All fluorograms were prepared using an enhanced chemiluminescence kit (GE Healthcare).

Exosomes were directly coated on the surface of a 96-well plate (Greiner). Fifty microliters/well of purified exosome solution was added and incubated overnight at 4 °C. The plate was washed with D-PBS and blocked with 100 μl/well of blocking solution (D-PBS containing 5 % BSA) at room temperature for 1.5 h. Following three washes of D-PBS, 50 μl/well of primary antibodies at 2 μg/ml in blocking solution were added for 1 h at room temperature (RT). After three washes of D-PBS, the plate was incubated with 50 μl/well of HRP-conjugated anti-mouse IgG antibody (Sigma, 1:10,000 dilution) in blocking solution for 1 h at RT. The wells were finally washed three times. HRP-bound antibodies were detected by adding 100 μl of 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma) solution, and the reaction was stopped with 100 μl 2 M H₂SO₄ after 30 min. The optical densities were recorded at 450 nm using the multiwell plate reader SpectraMax 360 (Molecular Devices).

The assessment of total serum IgG levels (anti-Toxocara) was performed by indirect immunoenzymatic assay (ELISA) using TES as the antigen at a concentration of 1 μg/ml and serum dilution of the primary antibody (1:50), as described by Avila et al. (2012) (link). For the secondary serum, horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody produced in goat (Sigma-Aldrich, St. Louis, Missouri, USA), a 1:5000 dilution in PBS-T, was used. Serum samples were examined individually and in duplicate, and readings were performed at a wavelength of 492 nm. The evaluated isotypes were IgG1, IgG2a, IgG2b and IgG3, following the recommendations of Monoclonal Mouse Antibody Isotyping Reagents (Sigma-Aldrich, St. Louis, Missouri, USA). The concentration of antigen used in sensitization and the dilution of the primary serum were the same as those used for the evaluation of total IgG. Each sample (pool of sera) was examined in triplicate to obtain the mean absorbance. The secondary antibodies anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Sigma-Aldrich, St. Louis, Missouri, USA) produced in goat were diluted 1:2000 in PBS-T and applied to the plates. After, the peroxidase-conjugated anti-goat antibody produced in rabbit (Sigma-Aldrich, St. Louis, Missouri, USA) was diluted 1:4000 in PBS-T was added.

The ELISA procedure was based on a previously described method (23 (link), 24 (link)) with minor modifications. Briefly, Maxisorb ELISA plates (Nunc) were coated at 4°C for 16 h (ON) with 100 μl/well of FMDV 3ABC protein at a concentration of 60 ng/ml suspended in PBS. Following incubation plates were washed three times with washing buffer [PBS containing 0.05 % Tween 20 (PBS-T)] and then blocked for 1 h at RT with 5 % skimmed milk (Sigma). The same washing procedure was performed after each incubation step. Next, serial dilutions of tested Nbs in concentrations from 4 μg/ml to 60 ng/ml were added at a volume of 100 μl/well and incubated for 1 h at RT. Following incubation plates were washed and 100 μl/well of 1 μg/ml anti-HA (Sigma) was added and incubated for 1 h at RT. Afterward, plates were washed and 100 μl/well of 1 μg/ml HRP-conjugated anti-mouse IgG antibody (Sigma) was added and incubated for an additional 1 h at RT. Finally, plates were washed four times and 90 μl/well of TMB One solution (SouthernBiotech) was added to develop the color reaction. The color reaction was stopped after 15 min with 1 M Sulfuric acid and readouts were obtained with absorbance read at 470 nm using a standard luminometer (Thermolabsystems-Luminoskan Ascent).

Serum was collected one week after the second vaccination by retro-orbital bleeding. Sera (1:50 dilution) were incubated on microplates previously coated with recombinant mouse xCT protein (Cloud-Clone-Corp., Katy, TX, USA; 40 ng/well) and binding was detected with HRP-conjugated-anti-mouse-IgG antibody (Sigma-Aldrich) using a 680XR microplate reader (Bio-Rad), as previously described [34 (link)].

The detection of LCMV-glycoprotein GP-1-specific IgG by ELISA has been previously described59 (link). In short, 96-well flat-bottom Nunc Immuno Plates (Thermo Scientific) were coated with anti-human IgG (Jackson ImmunoResearch Laboratories, Inc.) in coating buffer (0.1 M Na₂CO₃+0.1 M NaHCO₃; pH 9.6) overnight at 4 °C. On the next day, plates were washed with washing buffer (PBS with 0.05% Tween-20), and unspecific binding was blocked with 2% FCS in PBS for 2 h. Plates were incubated with LCMV Gp-Fc supernatant (made in-house) for 3 h at room temperature. Plates were washed and titrated with pre-diluted (1: 20) serum over 12 wells with 1: 3 dilutions in successive wells. After a 90-min incubation, plates were incubated with HRP-conjugated anti-mouse-IgG antibody (Sigma). After a 1-h incubation, plates were developed as described below.