Adenovirus ELISA 试剂盒

1. Anti-Hexon Antibody : One tube - 30 μL.
2. Secondary Antibody, HRP Conjugate : One tube - 50 μL.
3. TMB Substrate : One amber bottle - 20 mL.
4. Stop Solution : One bottle - 20 mL.

Box 2 (shipped on blue ice packs)

1. Ad-β gal Positive Control : One tube - 50 μL at 1.0 x 109 ifu/mL.

1. Recombinant adenovirus of interest
2. HEK 293 cells and cell culture growth medium
3. Methanol
4. 1 % BSA/PBS
5. Wash Buffer such as PBS
6. Microtiter plate reader

• More accurate adenoviral titer than traditional plaque-forming unit assays
• Faster results: 2.5 days vs. 10 days
• No agar overlay steps

• 1X Anti-Hexon antibody solution: Prepare a 1X anti-hexon antibody solution by diluting the provided Anti-Hexon antibody stock 1:1000 in 1 % BSA/PBS. Store the diluted solution on ice.
• 1X Secondary antibody solution: Prepare a 1X Secondary antibody solution by diluting the provided stock 1:2000 in 1 % BSA/PBS. Store the diluted solution on ice.

• For unknown viral samples, create 10-fold serial dilutions with culture medium. For example, start with a 1:100 dilution of the original viral samples by adding 10 μL of viral sample to a sterile tube containing 990 μL of culture medium. Transfer 100 μL of the mixture to the next tube containing 900 μL of culture medium. Repeat this step several times. For accurate assessment of viral titer, one of the dilutions for your unknown viral sample should be within the range of the Ad-β gal standard curve (4.0 x 103 ifu/mL to 2.5 x 105 ifu/mL).

I. Virus Infection

1. Harvest HEK 293 cells and resuspend cells in culture medium at 5 x 105 cells/mL. Seed 100 μL in each well of a 96-well plate and incubate at 37 °C, 5 % CO2 for 1 hr. Note: Adenovirus titer assay is critically dependent on the firm attachment of cells. If the cells look thin and easy to come off during immunostaining steps, you won't get consistent results. Only use low passage 293 cells with flattened morphology or 293AD (Cat. # AD-100), a selected 293 cell line for plasmid transfection, adenovirus amplification and titering. To improve cell adhesion, you can also precoat the plate with polylysine or extracellular matrix.
2. Prepare serial dilutions of the Ad-β gal positive control and your viral sample in culture medium. Dropwise add 50 μL of diluted viral sample to each well of the 96-well assay plate (note: a negative control should be performed simultaneously). To ensure accuracy, perform each sample in duplicate.
3. Incubate infected cells at 37 °C, 5 % CO2 for 2 days.

II. Immunoassay

1. Slowly remove medium from the wells by tilting the plate and aspirating from the edge, then fix infected 293 cells by gently adding 100 μL of cold methanol down the side of each well of the 96-well assay plate, taking care not to dislodge the cells. Incubate 20 minutes at -20 °C.
2. Gently wash the fixed cells three times with 1X PBS, five minutes each wash.
3. Block for 1 hr with 200 μL of 1 % BSA in PBS per well at room temperature on an orbital shaker.
4. Add 100 μL of diluted 1X anti-Hexon antibody solution to each well and incubate for 1 hr at room temperature on an orbital shaker.
5. Gently wash the fixed cells three times with 1X PBS, five minutes each wash.
6. Add 100 μL of diluted 1X Secondary antibody solution (HRP-conjugated) to each well and incubate for 1 hr at room temperature on an orbital shaker. 4
7. Gently wash the fixed cells five times with 1X PBS, five minutes each wash.
8. Warm TMB Substrate to room temperature. Add 100 μL of TMB Substrate solution and incubate at room temperature for 5 to 10 minutes. Stop reaction by adding 100 μL of Stop Solution to each well.
9. Measure Optical Density at 450nm on a 96-well plate reader. Calculate the viral titer based on the standard curve from Ad-β gal positive control titrations.