Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography - A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography. - Bio

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Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography
A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography.


BioPharm International


Mice Immunization and Antibody Detection

BALB/c (H-2d) female mice, 6 to 8 weeks old and weighing 18–20 g were purchased from CENPALAB (Havana, Cuba) and used for all in vivo studies. Groups of 22 animals were immunized with the mixture of Co.120 (10 μg) plus plasmid pIDKE2 (100 μg), obtained using the method described by Horn9 or the method described here. Mice were injected intramuscularly at 0, 3, 7, 12, and 16 weeks in the quadriceps muscle with 100-μL final volume of 0.9% NaCl-based solution. Normal saline was administered to negative control group under similar conditions. Blood samples were collected 17 weeks after primary immunization from retro-orbital sinus and the sera were analyzed for antibodies (Ab) to HCV Co.120, E1.340, and E2.680 proteins.

To detect murine anti-HCV antibodies, 96-well microtiter plates (Costar, Cambridge, MA) were coated with 100 μL of recombinant proteins Co.120, E1.340, or E2.680 (10 μg/mL) diluted in coating buffer (50 mM carbonate buffer, pH 9.6) and incubated for 12 h at 4 °C. The wells were washed three times with 0.05% v/v Tween 20 in phosphate buffered saline pH 7.5 (PBST) and blocked with 200 μl of PBST containing 1% w/v skim milk (Oxoid Ltd, England) for 1 h at 28 °C. After three washes with PBST, 100 μL of serial double dilutions of individual mouse sera in PBST were added and incubated at 37 °C for 1 h. The plates were washed three times with PBST, and 100 μL of horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham, UK) diluted 1: 25,000 was added at 37 °C for 1 h. Positive reactions were visualized with o-phenylenediamine (Sigma) in 0.1 M citric acid, 0.2 M NaH2PO4 pH 5.0, and 0.015% v/v hydrogen peroxide as substrate, and the reaction was stopped with 50 μL of 2.5 M H2SO4. Optical density (OD) was determined at 492 nm using a plate reader (SensIdent Scan, Merck, Ger-many).


Figure 1
The cut-off value to consider a positive antibody response was established as twice the mean OD 492 nm of the negative control sera.


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