rev: January 25, 2005
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AAV TITRATION ELISA-For in vitro research use only-not for use in diagnostic procedures
Please call or EMAIL us with your address and we will gladly send you the full kit inserts . Information below is for illustartive purposes only-see actual kit insert inside each kit for batch specific information. copyright by owner
Enzyme Immunoassay for the Quantitative Determination of AAV Particles in Cell Culture Supernatants and Purified Virus Preparations
cat#RDI-PROPRATV 96 wells $1000.00/kit $969.00/kit 2 -9 $938.00/kit 10+ or more Bulk on request
Size of the Kit: 12 x 8 Determinations
Storage: 2 - 8° C
In vitro RESEARCH USE ONLY
-AAV2 and AAV3 serotypes can be titrated with Cat. #RDI-PROPRATV.We are presently developing a titration test for AAV5, available in about 2 months time. Other AAV serotype tests will follow soon after.
Adeno-associated virus (AAV) has been a topic of intense study in gene therapy because it is a naturally viral gene delivery system which never has been shown to have any pathogenic effects in humans despite the fact that most of the population has been exposed to AAV early in life. Beside its safety, its efficacy of delivery and simplicity of its components, its long persistence in infected cells makes it a very promising tool for therapeutic gene transfer. Characetrization of AAV preparations currently includes the determination of rnasducing units, an infectious center assay, a protein assay or protein gel, a DNA dot blot or electron microscopy.
Immunotitration with the AAV Titration ELISA test is a fast, sensitive and reproducible method for the titration of AAV virions, AAV gene shuttles or assembled and AAV intact empty particles.
The assay is based on a sandwich ELISA technique. A monoclonal antibody specific for AAV assembled capsids is coated onto microplate strips and is used to capture AAV particles from the specimen. Captured AAV particles are detected in two steps. First a biotin conjugated monoclonal antibody to AAV is bound to the immune complex. In the second step streptavidin peroxidase conjugate reacts with the biotin molecules. Addition of substrate solution results in a color reaction which is proportional to specifically bound virus particles. The absorbance is measured photometrically at 450 nm.
The kit control provided contains an AAV particle preparation of empty capsids. It shows a typical titration curve whenused in dilutions of steps of two. It allows the quantitative determination of samples of an unkown particle titer (immunological titer) and the calibration of an inhouse AAV preparation(e.g. infectious titer, transducing units, DNA dot blot titer).
· Precision pipettes
· Sterile pipette tips
· Distilled water
· Vials for specimen dilutions
· Incubator for 37°C
· microplate Plate Spectrophotometer (450 nm)
6 x 8-well microplate strips, coated with mouse monoclonal anti-AAV
KC Kit control (empty AAV cpasids). lyophilized
SB Sample Buffer (20x), 20 ml
WB Wash Buffer (20x), 20 ml
B Anti-AAV, Biotin Conjugate (20x), lyophilized
C Streptavidin Peroxidase Conjugate (20x), lyophilized
S TMB Substrate Concentrate (20x), 750ul
SS Stop Solution (ready to use), 13 ml
All components contain Thimerosal as preservative reagent!
Allow kit to reach RT. Buffer concentrates may contain salt cristals which dissolve quickly at 37°C. Dilute required volumes of reagents directly before use!
Example for 1x 8-well microplate strip:
SB Dilute 1:20 (1 ml + 19 ml distilled water
WB Dilute 1:20 (1 ml + 19 ml distilled water
KC Reconstitute with 500ul distilled water
Contains 5 X 10 E09 particles/ml ((see vial label for exact concentration)
B * Reconstitute with 750ul distilled water. Dilute 1:20 with ready-to-use wash buffer for ready-to-use AAV biotin conjugate
C * Reconstitute with 750ul distilled water. Dilute 1:20 with ready to use wash buffer for ready to use strepatvidin peroxidase conjugate
S * Dilute 1:20 with distilled water for ready to use substrate
CAUTION: dilute substrate in glass ir polypropylene tubes only!
* dilute immediatley before use
The linear range of the ELISA covers 5 X 10 E07 -1 X 10 E09 particles. The kit ocntrol (KC) once reconstituted in water canbe kept at 4 DEG C and should be used within 4 months after reconstitution. Dilute specimen containing AAV particles to reac a concentration within the linear range of the ELISA using read-to-use sample buffer. DILUTE the reconstituted Kit control also in ready-to-use sample buffer
Sample:Dilute specimen (cell culture supernatants containing AAV particles) in steps of 10 in ready-to-use sample buffer. According to an expected value the dilution may be chosen as follows:
expected OD range
|1||infectious titer||1 X 108/ml||1012P/ml||1:1000||2.0|
|2||DNA dot blot||1 X 10 10/ml||1 X 1011P/ml||1:100||2.0|
For measurement dilute further in steps of 1:5. A minimum of 2-3 different dilutions should be tested.
1. Pipette 100 µl of ready-to-use sample buffer (blank), serial dilutions of your Inhouse Standard, Kit control, and dilutions of specimen in to the wells of the microtiter strips. Seal strips with adhesion foil and incubate for 1h at 37°C.
2. Empty contents of microtiter strips.
Fill wells with 200 µl each of ready-to-use wash buffer, let react approx. 5 sec, aspirate and tap inverted plate. Repeat washing step 2x.
3. Pipette 100 µl per well of ready-to-use biotin conjugate. Seal strips with adhesion foil and incubate for 1h at 37°C.
4. Repeat washing step as described in 2.
5. Pipette 100 µl per well of ready-to-use streptavidin conjugate. Seal strips with adhesion foil and incubate for 1h at 37°C.
6. Repeat washing step as described in 2.
7. Pipette 100 µl per well of substrate. Incubate for 10-15 min at RT.
8. Stop color reaction by adding 100 µl of stop solution into each well.
9. Measure intensity of color reaction with a photometer at 450 nm within 30 min.
The results are calculated by using an ELISA reader software or by means of a standard curve. Create a standard curve by plotting the absorbance values of the standards (y-axis) versus the serial dilution of your calibrated Inhouse Standard (x-axis). Use this standard curve for the calculation of the particle titer of unkown specimens.
SAMPLE DATA- DO NOT USE FOR CALCULATION
|undiluted (1:1)||25 X 108||2.120|
|1:2||12.5 X 108||1.850|
|1:4||6.25 X 108||1.422|
|1:8||3.15 X 108||0.841|
|1:16||1.56 X 108||0.478|
|1:32||0.78 X 108||0.226|
|1:64||0.39 X 108||0.134|
Kit control (undiluted) OD >2.0
Wistuba A.,Weger S., Kern A., and Kleinschmidt J. (1995) Intermediates of adeno-associated virus type 2 assembly: Identification of soluble complexes containing Rep and Cap proteins. J. Virol. 69, 5311-5319.
Wistuba A., Kern A., Weger S., Grimm D., and Kleinschmidt J. (1997) Subcellular compartmentalization of adeno-associated virus type 2 assembly. J. Virol. 71, 1341-1352.
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