Adenovirus Titration Kit

Transcription

1 Adenovirus Titration Kit Catalog # LF-RK0001(1 kit) Immunostaining method for Quantitative Detection of Adenovirus For research use only Not for diagnostic or therapeutic procedures AbFrontier Science Bldg C, 1F, 11-1, Daehyun-dong, Seodaemun-gu, Seoul , Korea Tel: Fax:

2 Contents 1. Introduction 3 2. Safety and Handling of Adenovirus 4 3. List of Components 5 4. Experimental Procedure 6 5. Troubleshooting References 11 2

3 1. Introduction Adenoviruses have been studied for many decades following the realization they were able to transform rodent cells in culture. Since then, they have served as valuable models for the exploration of the cellular pathways involved in DNA replication, regulation of gene expression and cellular proliferation. Also, adenovirus was developed as gene delivery method for gene therapy and research fields. Now, recombinant adenoviral vector is general method used in many laboratory. While most adenovirus (wild-type and recombinant) are relatively easy to grow, quantifying the number of virus particles and the infectivity of a particular inoculum is much harder. Traditionally, those wanting to accurately quantify the titer of a virus used the plaque-forming assay. However, this assay require rather lengthy incubation time (up to 14 days) and is variable. AbFrontier s Adenovirus Titration Kit uses the immunostaining method to quantifying the adenovirus stock (especially recombinant adenovirus). Dilution of recombinant adenovirus stock are used to infect adenovirus packaging cell line (HEK293). Infected cells are incubated for 48 hours to express the hexon, major capsid protein of adenovirus, reach the detectable range. This hexon-expressing cells are fixed, and detected with mouse monoclonal anti-hexon antibody and enzyme-conjugated secondary antibody. Immunostaining method for adenovirus titration has some advantage. First, this method detects the infectious virus particle. Second, this method needs relatively short experimental time rather than plaque forming assay and TCID50 assay. Third, infected cells can be confirmed with visual data. Finally, immunostaining is more reproducible than plaque forming assay. 3

4 2. Safety and Handling of Adenovirus CDC (Center of Disease Control) and NIH (Office of Biosafety, U.S. Department of Health) have described four biosafety levels based on a combination of laboratory practices and techniques, safety equipment and laboratory facilities. Different degree of personnel, environment, and product protection are offered by the various biosafety classes. According to the NIH biosafety recommendation based on risk assessment, all human adenoviruses are classified as Class II agents to be of ordinary potential harm. For more information regarding Class II practices, consult the UCSD Environmental Health and Safety Web site The virus packaged by transfected or infected HEK 293 cells with the adenoviral-based vectors described here are capable of infecting human cells. Depending on insert gene, these viral supernatants could contain potentially hazardous recombinant virus. Similar vectors have been approved for human gene therapy trials, attesting to their potential ability to express genes in vivo. For these reasons, due caution must be exercised in the production and handling of any recombinant adenovirus. The user is strongly advised not to create recombinant adenovirus capable of expressing oncogenes. The following information is a brief description of Biosafety Level 2. It is neither detailed or complete. Practices perform work in a limited access area post biohazard warning signs avoid generating aerosols decontaminate potentially infectious wastes before disposal take precautions with sharps (e.g., syringe, blades) Safety Equipment biological safety cabinet, preferably Class II protective laboratory equipments (e.g., coats, face protection, gloves) Facilities autoclave for decontamination of wastes unrecirculated exhaust air chemical disinfectants available of spills 4

5 3. List of Components Components Volume Storage temp. Mouse anti-ad5 antibody Goat anti-mouse antibody Developing Solutions Developing buffer Substrate A Substrate B 30 μl 30 μl 30 ml 100 μl 200 μl Antibodies are stable for about 1 month at 4. For extended storage, store at -20. The following materials are required but not supplied. TBS (Tris buffered saline) : 50 mm Tris-HCl (ph 7.4), 150 mm NaCl TBS-T : TBS containing 0.1% Triton X-100 Cell culture system culture medium FBS (fetal bovine serum) 12-well or 24-well culture plate Laminar flow hood CO 2 incubator Microscope Methanol Orbital shaker 5

6 4. Experimental Procedure To ensure accuracy, each experiment should be assayed in duplicated. A. Infection of Adenovirus 1. Before 24 hour infection, seed cells (healthy HEK 293) in each wells of a 12-well culture plate with standard growth medium (e.g., DMEM + 10% FBS + antibiotics). Note: 5% FBS containing medium also available. 2. Next day, prepare 10-fold serial dilutions of your viral sample (from 10-3 to 10-8 ) with serum-free growth medium (e.g., DMEM). Note : with this dilution, you can determine up to IFU/ ml viral stock. If your viral stock is over than this titer, use up to dilution. 3. Make the infection solution with 0.1 ml of diluted viral stock and 0.9 ml of complete growth medium containing FBS and antibiotics. For example, you can make 10-7 dilution by mixing 0.1 ml of 10-6 viral stock and 0.9 ml of complete growth medium. To improve accuracy, you may need to adjust dilutions to , , , etc., depending on the expected viral titer. 4. Replace the culture medium in 293 cell seeded 12-well culture plate with previous infection solution. 5. Incubate cells at 37 in 5% CO 2 for 48 hr. B. Immunostaining of Infected 293 cells 1. After 48 hr infection, aspirate the culture medium and allow cells to dry in hood for 5 min. Note: Cell dry step is critical to avoid disruption of cell monolayer 2. Fix cells by adding 1 ml of ice-cold 100% methanol to each wells. Note: Add methanol very gently. The monolayer can easily dislodge until cells are fixed. 3. Incubate the plate at -20 for 10 min. 4. Aspirate methanol, and gently rinse the wells with 1 ml of TBS-T for 3 times. 6

7 5. Aspirate final rinse from the wells, and add 0.5 ml of anti-ad5 antibody in TBS-T (2% BSA) Note: Dilute mouse anti-ad5 antibody 1:1,000 in TBS-T (2% BSA). 6. Incubate 1 hr at 37 or room temperature. Note: Orbital shaker is optional in experimental procedure. 7. Aspirate anti-ad5 antibody, and gently rinse wells 3 times with 1 ml of TBS-T. 8. Aspirate final rinse from the wells, and add 0.5 ml of anti-mouse antibody in TBS-T (2% BSA). Note: Dilute mouse anti-mouse antibody 1:1,000 in TBS-T (2% BSA). 9. Aspirate anti-mouse antibody, and gently rinse each wells 3 times with 1 ml of TBS-T. 10. After removing the final TBS-T rinse, add 500 μl of Staining Solution to each well. Note: Prepare the Staining Solution by mixing like belows; 10 ml of Development Buffer 33 μl of Substrate A 66 μl of Substrate B 11. Incubate at room temperature for 10 min. Then aspirate the Staining Solution and add 1 ml of PBS to each well. C. Calculation of Titer 1. Count the brown or black positive cells using a microscope with a 20 objective (minimum of 3 fields; random selection), and calculate the mean number of positive cells in each well. Note: Ideal field should contain 10 to 50 positive cells. 2. Calculate infectious units (IFU/ ml ) for each wells as follows; [ (infected cells / field) (fields/well) ] / [ (volume of virus) (dilution factor) ] # Example Calculation Mean positive cells / field = 10 cells fields/well (20 objective) = 466 Amount dilution added = 1.0 ml 7

8 dilution factor (counted dilution) = 10-6 dilution viral titer = [(10 cells/field) (466 fields/well)] / [(1.0 ml ) (10-6/ ml )] = IFU/ ml Note: number of fields/well is variable according to plate. Derivation of Area counted in Fields/Well Since radius of a standard 20 objective = 0.5-mm then area of cells counted per field = mm 2 Area of a well (for average TPP 12-well plate) = 3.66 cm 2 therefore, fields/well = 466 fields 8

9 Example of Immunostaining (GFP Ad) GFP Expression Immunostaining 10-5 Dilution 10-6 Dilution 10-7 Dilution Figure 1. Titration of GFP Ad with Adenovirus Titration Kit After 48 hours post-infection, GFP expression was detected with fluorescence microscopy (10 objectives). This cells was used for immunostaining with Adenovirus Titration Kit. Infected cells (black purple spots) was detected with 10 objectives. 9

10 5. Troubleshooting A. Disruption of Cell Monolayer 1. Perform the addition of methanol and TBS-T very gently 2. Use collagen coated plate B. No Positive Cells 1. Problems of antibody ; check the addition, dilution, and storage condition of antibody 2. Check the dilution of adenoviral stock 3. Incubation time. ; As a result of short incubation, expression of hexon protein did not reach detection threshold C. All Positive Cells 1. Check the dilution of adenovirus stock 2. Incorrect dilution of first and second antibody 3. Check the rinse step, and rinse 4~5 times. 4. Check development solution. Incorrect preparation of development cause high background signals. 10

11 6. References 1. Aiello, L., et al., (1979) Adenovirus 5 DNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad5 or 293). Virology 94: Gregory, J. P, et al., (2000) Quantifying adenoviral titers by spectrophotometry, BioTechniques 28: Eykholt, R. L., et al., (2000) Accelerated titering of adenovirus. BioTechniques 28: Graham, F. L., et al., (1997) Characterization of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36: Price, J., et al., (1987) Lineage analysis in the vertebrate nervous system by retrovirusmediated gene transfer. Proc. Natl. Acad. Sci. USA 84:

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