AAV6 Titration ELISA Kit | Creative Diagnostics DEIAAV6 product information

product summary

company name :

Creative Diagnostics

product type :

ELISA/assay

product name :

AAV6 Titration ELISA Kit

catalog :

DEIAAV6

quantity :

96T

more info or order :

Creative Diagnostics product webpage

product information

Catalog :

DEIAAV6

proudctName :

AAV6 Titration ELISA Kit

common :

AAV

Short Name :

AAV6 Titration ELISA Kit

Detection Method :

sELISA

Common name :

AAV

Application :

Quantitative

Sample :

cell culture supernatants, purified virus preparations

Size :

96T

Storage :

Store the test kit and components at 2-8°C. The unopened reagents are stable at 2-8°C until the indicated expiry date.

Target :

AAV6

Intended Use :

Enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of AAV serotype 6 particles in cell culture supernatants and purified virus preparations.

General Description :

Adeno-associated viruses (AAV) are non-pathogenic ssDNA viruses, which are subject of intense studies as viral vectors for gene therapy. The virus transduces a variety of dividing and non-dividing cells showing long-term gene expression with low cellular immune response. AAV has been used in several clinical trials (e.g. FIX, CFTR, Parkinson's, Canavan disease) showing no serious vector-related adverse effects. Methods for the characterization of AAV preparations currently include titration ELISA, qPCR, ddPCR, DNA dot blot, determination of transducing units, infectious center assay, SDS-PAGE or electron microscopy. Immunotitration by creative diagnostics AAV6 Titration ELISA offers a fast, sensitive and reproducible method for titration of intact AAV6 wild-type virions, AAV6 recombinant virions or assembled and intact empty AAV6 capsids.

Principle Of The Test :

The assay is based on the sandwich ELISA technique. A monoclonal antibody specific for a conformational epitope on assembled AAV6 capsids is coated onto strips of a microtiter plate and is used to capture AAV6 particles from the specimen. Captured AAV particles are detected in two steps: 1. A biotin-conjugated monoclonal antibody to AAV6 is bound to the immune complex. 2. A streptavidin peroxidase conjugate reacts with the biotin molecules. Addition of substrate solution results in a color reaction, which is proportional to the amount of specifically bound viral particles. The absorbance is measured photometrically at 450 nm (optional: reference wavelength at 620 nm). The provided Kit Control contains an AAV6 particle preparation of empty capsids. Two-fold serial dilutions of the material result in a typical titration curve. The curve allows the quantitative determination of samples of an unknown particle titer.

Reagents And Materials Provided :

1.Microtiter Plate, 12 × 8-well-strips, coated with mouse monoclonal antibody to AAV6 in aluminum bag with desiccant, 1 plate. Ready- to-use. 2.AAV6 standard, lyophilized, 3 vials. Reconstitute before use. 3.Washing Buffer 20×, 50 ml. Dilute before use. 4.Sample dilution, 50ml. Ready- to-use. 5.Anti-AAV6 Biotin Conjugate, 12ml. Ready- to-use. 6.Streptavidin Peroxidase Conjugate, 12ml. Ready- to-use. 7.TMB Substrate, 6 ml × 2. Ready-to-use. 8.Stop Solution, 7 ml. Ready-to-use.

Materials Required But Not Supplied :

1.Precision pipettes 2.Sterile pipette tips 3.Distilled water 4.Reaction tubes 5.Incubator at at room temperature (20-25°C) 6.ELISA Reader (450 nm, optional: reference wavelength at 620 nm)

Specimen Collection And Preparation :

Pre-dilute your specimen containing AAV6 particles in Sample dilution in serial dilution steps to reach a concentration within the recommended quantification range of the ELISA. It might be necessary to perform a pre-experiment to determine the approximate titer of the unknown specimen before analyzing more finetuned dilutions.

Reagent Preparation :

Prior to use, allow kit to reach room temperature (RT, 20- 25°C). Preparation and pre-dilution of components: Dilute required reagent volumes immediately before use! Washing Buffer 20× 1. Dilute 1:19 with distilled water. 2. The diluted component is named washing Buffer 1× KC (Kit Control) 1. Reconstitute with 500 μl Sample dilution. 2. Incubate for 5 min at RT and then mix by rolling for another 5 min. Avoid vortexing. 3. Find the amount of vg/ml on the label. Kit Control and Specimen Dilution We recommend to dilute the reconstituted AAV6 standard in Sample dilution in steps of 1:2: An example for dilutions is provided on the lot-specific Example Curve document. Please find the lot-specific titer of the Kit Control on the vial or on the Quality Control Certificate. Both the Example Curve document and the Quality Control Certificate are provided with the kit.

Assay Procedure :

1. Pipette 100 μL of Sample dilution (KC0), serial dilutions of KC and specimen (both in Sample dilution) in duplicates into the corresponding wells of the microtiter strips. Seal strips with adhesive foil and incubate for 1.5 h at RT. 2. Discard content of microtiter strips. For washing, fill each well with 250 μL of washing buffer 1×, incubate approximately 5 sec, discard and tap inverted plate onto absorbent paper. Carry out three washing steps in total. 3. Pipette 100 μL of Biotin into each well. Seal strips with adhesive foil and incubate for 1 h at RT. 4. Repeat washing step as described in 2. 5. Pipette 100 μL of Strep-HRP into each well. Seal strips with adhesive foil and incubate for 1 h at RT 6. Repeat washing step as described in 2. 7. Pipette 100 μL of ready-to-use TMB into each well. Seal strips with adhesive foil and incubate for 20 min at RT. 8. Stop color reaction by adding 50 μL of STOP into each well. 9. Make sure no air bubbles are in the wells. Within 30 min, measure color intensity with a photometer at a wavelength of 450 nm (optional: reference wavelength at 620 nm).

Quality Control :

The absorbance value of the undiluted AAV8 Standard should be > 1.5. The absorbance value of the Blank should be < 0.15.

Calculation :

If applicable, subtract values measured at 620 nm reference wavelength from values at 450 nm. The test is also valid if you use OD values at 450 nm only. Calculate the average absorbance values for each duplicate set of Kit Control dilutions and specimen dilutions. Create a standard curve by plotting the mean absorbance value of each Kit Control dilution (y-axis, linear scale) against the corresponding concentration (x-axis, logarithmic scale recommended). Use a best fit curve for calculating the results. We suggest using a suitable computer program for the calculation. A 4- parameter logistic fit (4PL) is recommended. Calculate the particle titer of your specimens. The kit is quantitative over the whole range of Kit Control dilutions. For highest accuracy, the OD values of unknown samples should ideally be in the recommended range for Quantification. Multiply the value obtained by the dilution factor to determine the amount of vg/ml in the sample. Please note: The standard curve needs to be determined for each experiment individually. For further orientation, please find the lot-specific Example Curve provided with the kit.

Detection Range :

0-3.00E+09vg/ml

Precautions :

1.For professional use. 2.The instruction manual is only valid in combination with the lot specific documents ( Example Curve and Quality Control Certificate), which are enclosed in each kit. Please make sure to use the instruction manual with the version number that corresponds to the number on the lot specific documents. 3.STOP (sulphuric acid) and TMB may cause skin or eye irritation. In the event of eye contact, rinse out immediately with plenty of water and consult a physician! Safety data sheet is available on request! 4.Chemicals and biological materials must be disposed of in compliance with the respective national regulations.

applicatons :

Enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of AAV serotype 6 particles in cell culture supernatants and purified virus preparations.

Contents of Kit :

Tetramethylbenzidine :