Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 42

Decant the gel fixing solution. Add ultrapure water to remove
residual solution from the gel. Agitate for 5 min.
Carefully decant the water. Immerse the gel in 1× Krypton™
Infrared Protein Stain. Cover the tray with aluminum foil
to minimize exposure to light. Agitate for 1 h to overnight
at room temperature.
Decant the stain solution. Add the destaining solution (5 % acetic
acid and 0.1 % Tween-20), cover the tray and agitate for 5 min.
Remove the destaining solution and replace with an equal volume
of ultrapure water. Agitate for 10 min.
Decant water and repeat washing step.
Image the gel with the LI-COR/Odyssey infrared imaging system
at 680-nm excitation and 720-nm emission.
(b) Endotoxin assay .
Since endotoxin contamination may induce an acute toxicity
response in animals, the level of gram-negative bacterial endo-
toxin is quantitatively determined using the Limulus
Amebocyte Lysate (LAL) test as briefly described below.
Perform four dilutions of the endotoxin positive control in LAL
reagent water as per manufacturer's instructions.
For viral samples, take 1.5 μl of virus in 148.5 μl LAL reagent water.
Prewarm the microplate at 37 °C in a dry block heater. Leave the
microplate within the dry block heater during all the
procedure.
Add 50 μl of diluted viral sample and standard serial dilutions to
each well of the microplate. Include a negative control (LAL
reagent water). Perform samples and controls in duplicate.
Add 50 μl of LAL to each well. Incubate for 10 min.
Add 100 μl of prewarmed substrate solution. Incubate for 6 min.
Add 100 μl of stop reagent.
Measure optical density at 405-410 nm on a 96-well plate
reader.
(c) Replication-competent AAV detection
Vector preparations may be contaminated with wtAAV and
replication-competent AAV (rcAAV) as a result of nonho-
mologous recombination during vector production
between the AAV vector and helper plasmids [ 65 ]. Vectors
stocks are therefore tested for contaminated wtAAV or
rcAAV using a modified infectious center assay. In this
assay, HeLa or HEK_293 cells are coinfected with rAAV
and adenovirus. Since no wtAAV (i.e., no Rep) is provided,
only particles in the vector preparation that have packaged
wtAAV or pseudo AAV genome are capable of replication