Melon gel igg purification kit

New Zealand white rabbits 4–6 weeks old were immunized subcutaneously with 30 µg of rIxofin3D-PF or ovalbumin in complete Freund's adjuvant and boosted twice with 30 µg of rIxofin3D-PF or ovalbumin once every 3 weeks in incomplete Freund's adjuvant. Test bleeds were obtained from ear veins 2 weeks after the final boost and reactivity to recombinant rIxofin3D-PF and ovalbumin assessed by western blot. Rabbits were euthanized and serum was obtained by cardiac puncture. Polyclonal IgG was purified from the sera using the Melon Gel IgG purification kit (Thermoscientific, IL). For passive immunization, mice were passively immunized 24 h prior to tick placement by intraperitoneal inoculation with 100 µg of purified rabbit IgG against rIxofin3D-PF or ovalbumin. For active immunization, mice were immunized with 10 µg of rIxofin3D-PF or ovalbumin as described for rabbits. To address the role of rIxofin3D-PF in B. burgdorferi transmission, four B. burgdorferi N40 infected nymphs were placed on each immunized mouse. Nymphs were allowed to feed to repletion. Salivary glands and guts were dissected and combined in pools of 2–3 ticks for quantitative RT-PCR as described above. DNA was isolated from skin punch-biopsies at 7, 14 and 21 days and from heart and joints 21 days post tick-detachment and Borrelia burden assessed by quantitative PCR as described [35] (link).

ADCC capacity was evaluated using pooled IgG isolated from the HIV-1 infected individuals included in the study. IgG was isolated from plasma using the Melon Gel IgG Purification kit according to the manufacturer's instructions (Thermo Scientific) and quantitated using the Bicinchoninic acid (BCA) assay. HIV immune globulin (HIVIG; NIH AIDS reagent program) was used as a positive control.

As described before [18 ], the plasma samples were sonicated, centrifuged and their protein concentrations were measured. All plasma samples and HSA standards were diluted to 5 μg/mL and analyzed in triplicates by an indirect Enzyme-Linked Immunosorbent Assay (ELISA). The isoAsp levels in each blood sample were calculated according to the standard curve of HSA constructed from standards on each plate. The results were normalized by the average value in the control group.
The IgG antibodies were purified from plasma using the Melon Gel IgG Purification Kit (Thermo Fisher Scientific, San Diego, CA, USA). To determine in each sample the amount of IgG against aged HSA (aHSA, ≈60% isoAsp), the purified IgG antibodies were used as the primary antibody in the indirect ELISA [17 ], while the secondary antibody was Goat anti-Human IgG (H + L) Secondary Antibody conjugated with HRP (Thermo Fisher Scientific, San Diego, CA, USA). To eliminate the effect of sample location on the plate, the data were normalized by the average reading in each row and column.

IgG purification from plasma was performed with the Melon Gel IgG purification kit (Thermo Fisher) according to the manufacturer’s instructions. ELISAs against recombinant proteins YU2 gp120 (provided by Dr. John Mascola) and DIII gp41 (Abcam) were run as previously described (31 (link)), with the following modifications. IgG was diluted to 50μg/mL and serially diluted 5-fold to 0.0032μg/mL in B3T buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 3.3% fetal bovine serum, 2% bovine albumin, 0.07% Tween 20). Briefly, 96-well ELISA plates were coated with 2 μg/ml of the specified recombinant protein in phosphate-buffered saline (PBS) overnight at 4°C. The following day, the plates were blocked with B3T buffer. IgGs were detected using peroxidase-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch). All incubations were for 1 h at 37°C, and all volumes were 100 μl, except for the blocking step, which was 200 μl. The plates were washed between incubations with 0.1% Tween 20 in PBS, detected using SureBlue TMB substrate (SeraCare, VWR), and subsequently read at 450 nm.

The abrogation of the immunodominant Ab response to the selected epitopes in vaccinated pigs was assessed by surface plasmon resonance on a Reichert SR7500DC instrument (Reichert Technologies, Buffalo, NY, USA). Biotinylated peptides encoding the wildtype peptide sequences of epitopes A and B described above were commercially synthesized (Biomatik, Wilmington, DE, USA). Pooled sera collected at DPV 28 from the three treatment groups and from archived sera collected from PCV2b infected pigs [17 (link)] (provided by X. J. Meng, Virginia Tech, Blacksburg, VA, USA) were used to purify IgG using a commercial kit (Melon gel IgG purification kit, Thermo Fisher, Waltham, MA, USA). The biotinylated peptides were immobilized on streptavidin coated carboxymethyl dextran sensor chips (Reichert Technologies, USA) by injecting 0.16 μg/μL peptide solution over the sensor chip at a flow rate of 25 μL/min. After an increase of about 300 µRU was observed, indicating immobilization of each peptide had occurred, the purified IgGs for the experimental groups were injected over the flow cells at a concentration of 20 µM in phosphate buffered saline with 0.005% Tween 20 (PBST), at a flow rate of 25 μL/min for 240 s. Binding of the IgGs to the peptides was assessed by the response in µ response units (µRU).