Gene Therapy Strategies: Constructing an AAV Trojan Horse - Genomics: Essential Methods - page 297

(d) Wash buffer: dilute 20 × wash buffer with distilled water to appropriate volume of
1 × wash buffer.
(e) Biotin conjugate (anti-AAV 2): reconstitute the lyophilized biotin conjugate with
750 μ l of distilled water, forming 20 × biotin conjugate at first time, then dilute
20 × biotin conjugate with 1 × wash buffer t to appropriate volume of 1 × biotin

conjugate: B*.
(f) Streptavidin peroxidase conjugate: dilute 20 × streptavidin peroxidase conjugate
with 1 × wash buffer u

to appropriate volume of 1 × streptavidin peroxidase

conjugate: C*.
(g) Substrate: dilute 20 × substrate with distilled water to appropriate volume of 1 ×
substrate: S*.
2 Take appropriate numbers of eight-well microtiter strips coated with mouse monoclonal
antibody and place on the microtiter plate.
3 Pipette 100 μ lperwellof1 × sample buffer (as blank), serial dilution of standard
samples and tested samples into the wells of the microtiter strips, seal strips with
adhesion foil and incubate at 37 ◦ Cfor1h.
4 Wash the wells with 200 μ lof1 × wash buffer/per well (incubate for 5-10 s)
twice.
5 Pipette 100 μ lperwellof1 × biotin conjugate (B*), seal strips with adhesion foil and
incubate at 37 ◦ Cfor1h.
6 Repeat washing step in 4.
7 Pipette 100 μ lperwellof1 × streptavidin peroxidase conjugate (C*), seal strips with
adhesion foil and incubate at 37 ◦ Cfor1h.
8 Repeat washing step in 4.
9 Pipette 100 μ lperwellof1 × substrate (S*) and incubate at room temperature for
10-15min.
10 Add 100 μ l per well of stop solution to stop color reaction.
11 Measure intensity of color reaction in plate reader at 450 nm within 30 min.
12 Calculate the titers of rAAV virus according to the intensities and the standard
curve.

Notes
s Prepare serial dilutions of standard and diluted tested rAAV samples with 1 × sample buffer.
t Dilute 20 × biotin conjugate into 1 × biotin conjugate with 1 × wash buffer.
u Dilute 20 × streptavidin peroxidase conjugate into 1 × streptavidin peroxidase conjugate with
1 × wash buffer also.