Rabbit anti human igg

Maxisorp micro titer plates (Nunc, Maxisorp) were coated with the individual recombinant antigens in a concentration of 0.5 μg/ml and antibody levels in serum/plasma samples were determined by ELISA as previously described21 (link). In brief, free binding sites were blocked with 3% skimmed milk (w/v) and plates were incubated with individual plasma samples in 10-fold serial dilutions starting with a 1:10 dilution. Antigen-specific IgG was detected with HRP-conjugated secondary antibodies which for human samples was polyclonal rabbit anti-human IgG (Dako) diluted 1:6000 or rabbit anti-mouse IgG (Zymed) diluted 1∶5000 for murine samples. Substrate was TMB-PLUS (Kem-En-TEC). Reciprocal serum dilutions corresponding to 50% maximal binding (EC₅₀) were computed using the GraphPad Prism 6.04.

Anti-PTM antibodies were detected using an in-house enzyme-linked immunosorbent assay (ELISA), based on modified fetal calf serum (FCS) as described previously (10 (link)). Briefly, modified and non-modified FCS were coated to a Nunc Maxisorp ELISA plate (430,341, Thermofisher). In between each sequential step plates were washed three times using Phosphate Buffered Saline (PBS)/0.05%Tween (Sigma, P1379). After blocking [PBS/1% Bovine Serum Albumin (BSA)] for 6 h at 4°C, plates were incubated overnight at 4°C with 1/50, 1/100 or 1/1000 diluted serum. Each plate contained a standard of anti-PTM antibody positive serum to calculate arbitrary units. After incubation, IgG levels were detected using horseradish peroxidase (HRP) labeled Rabbit-anti-Human IgG (Dako, P0214). Plates were developed by incubating with 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS)/0.015% H₂O₂ (A1888 and 7,722-84-1, both from Merck) and absorbance at 415 nm was measured using a microplate reader (Bio-Rad iMark). The cut-off for positivity was set as the mean arbitrary units plus two times the standard deviation of 100 HCs, excluding values higher than 10x the mean.

Immunostaining for CCN-2 was performed on 3 μm formalin-fixed paraffin-embedded tissue sections. After deparaffinization, antigen retrieval was performed by predigestion with Protease XXIV (0.2 M phosphate; Sigma) followed by blocking of endogenous peroxidase activity (1 % H₂O₂ in phosphate/citrate buffer). Sections were incubated with CCN-2-specific human monoclonal antibody (FibroGen) (24 μg/ml in PBS/1 % BSA) for 1 h followed by incubation with rabbit anti-human IgG (Dako, Glostrup, Denmark) (1:100, 30 min). Amplification was performed with Powervision poly-peroxidase goat anti-rabbit IgG (Klinipath, Duiven, The Netherlands) (30 min) and FITC Tyramide Amplification Reagent (PerkinElmer, Boston, MA) (1:50, 10 min). TO-PRO 3 Iodide (Molecular Probes, Eugene, OR) was used for nuclear counterstaining. Slides were mounted in Vectashield (Vector Laboratories, Ontario, Canada), and visualized by fluorescence and confocal laser scanning microscopy. For all stainings, incubation with secondary antibody alone served as a negative control.

Total cell lysates were prepared and then separated using SDS-PAGE and transferred to a PVDF membrane (Millipore). The membranes were then blocked with 5% skim milk at room temperature for 1 h. The membranes were washed and incubated with primary antibody at 37 °C for 2 h and secondary antibody at 37 °C for 1 h. The primary antibodies used were: rabbit anti-human IgG (Dako) and horseradish peroxidase-conjugated anti-GAPDH (KangChen Bio-tech). The secondary antibodies used were: horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG (Dako). After washing, the proteins were visualized using the SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce Biotechnology) according to the manufacturer’s instructions. Densitometry analysis was performed using AlphaImager 2200 analysis software.

Coating was with polyclonal Rabbit anti-Human IgG (DakoCytomation, 1/2000 in 50 µl PBS). After blocking with skimmed milk (4% w/v Marvel in PBS), incubation was with EGFR ectodomain (EGFR-ect) containing an Fc-tail (85 ng in 50 µl 2% Marvel) and next incubated with biheads at indicated concentrations in 1% Marvel. In FLISA results were analyzed by the Odyssey Infrared Imaging System (Li-Cor Biosciences). For ELISA αMyc (1/2000), αMouse-Ig-peroxidase (1/5000) and o- Phenylenediamine (OPD) were used.

Ninety six-well flat-bottom plates were coated with T. cruzi Brazil strain lysate (5 µg/ml) or rabbit anti-human IgG (10 µg/ml; Dako, Denmark) overnight at 4°C. Plates were washed with PBS-Tween-20 and blocked with 5% dry, non-fat milk-PBS for 1 h at room temperature, followed by incubation with undiluted culture supernatants in duplicate wells for 45 minutes at 37°C. Plates were then incubated with 100 µl/well of HPR-labeled rabbit anti-human IgG (1∶1000; Dako) for 1 hour at 37°C. Color development was obtained by the addition of O-phenylenediamine (OPD, Sigma-Aldrich, Saint Louis, MO, USA) and read at 490 nm in an automatic ELISA reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Titers were calculated by subtracting the values obtained for the wells incubated with supernatants of unstimulated cultures from the values of wells incubated with supernatants of T. cruzi lysate- or cytokine cocktail- stimulated cultures. Selection of the cut-off point for T. cruzi-specific responses was based on the mean +3 SD of IgG titers obtained for lysate or cytokine cocktail stimulated supernatants in the control group, and responses were considered to be positive if the titer was higher than the cut-off value.