AAV-Q Platform for AAV Potency
Introduction
AAV infectious Titer (AAV potency assay) are crucial for quantifying the functional viral particles in your AAV preparations. Functional titration measures the ability of AAVs to transduce cells and express the transgene, which is a more relevant parameter than AAV genomic titer and capsid titer when assessing viral potency.
The AAV infectious titer measures the number of viral particles capable of entering target cells and initiating transgene expression. Unlike genome or capsid quantification, this is a functional assessment of AAV quality based on its ability to transduce cells. In permissive cells, once AAV enters the nucleus and Rep protein is present—along with adenoviral helper functions—the AAV genome can replicate thousands of times. Only at this stage can the genome be reliably detected by real-time PCR. This principle underlies the functional titer assay, such as the Tissue Culture Infectious Dose 50% (TCID50) assay, which estimates infectious titer by identifying the viral concentration at which 50% of cell culture wells show positive replication. The result is calculated using statistical analysis of infection patterns across serial dilutions.
A modified TCID50 assay is currently used to determine the infectious titer of AAV using HeLaRC32 cells. In this system, the Rep and Cap proteins expressed by HeLaRC32 cells, along with the presence of wild-type adenovirus 5 (Ad5), enable the replication of AAV genomes within the cells. Real-time PCR is then employed to sensitively and quantitatively detect positive genome replication, providing an accurate measure of infectious titer.
At AAVnerGene, we developed AAV-Q Platform, which works for for both AAV TCID50 and rcAAV assay.
AAV-Q Platform
The AAV-Q Platform includes three specialized cell lines, two quantitative assays, and one adenoviral vector, providing a complete and standardized system for AAV infectivity and rcAAV analysis.
Three Cell Lines:
- HEK293R: AAVR overexpression based on HEK 293A cells;
- HEK293RR: Rep overexpression based on HEK 293R cells;
- HEK293Rd: Optimized for Rd-Ad5 production;
One Ad vector:
- Rd-Ad5：Replication deficient in HEK 293A, but not in HEK293Rd cells;
Two Assays:
- AAV TCID50 Assay: Measures AAV potency or infectious titer using HEK293RR cells with Rd-Ad5;
- rcAAV Assay: Detects replication-competent AAVs using HEK293R cells with Rd-Ad5;
Key Features of AAVnerGene's AAV TCID50 assay
- Ultra-sensitive: Detects a single-copy;
- Quick results: Turnaround within one week;
- Broadly compatible: works with most AAV serotypes;
- Safe design: Uses Rd-Ad5 instead of wtAd5;
- Cost-efficient: streamlined qPCR workflow;
- Highly consistent: Controlled assay conditions;
- Reliable & robust: Low variability and high reproducibility;
Note: HEK 293R only overexpresses AAVR, which is used for rcAAV testing.
Products and Services of AAV-TCID50
| Products/Services | Specification | Price | Order |
| HEK293RR | HEK293A stably expression AAV2 Rep and AAVR | License | In stock |
| Rd-Ad5 | ≥1E8 TU/ml, 100 μl, replication deficient in HEK293A cell | $1999 | In stock |
| TCID50 Service | Full scale of AAV TCID50 assay and report | $5000 | 2-3 weeks |
AAV TCID50 service at AAVnerGene
This service is for customers who need accurate TCID50 titer of the AAV virus for research and therapeutic purpose. As little as 10ul AAV at genomic titer >1.0E1+13 form the customer is sufficient for complete the assay.
(1) Thawing Frozen Cells
Retrieve the cryovial containing frozen HEK293RR cells from the shipment package or liquid nitrogen storage.
Immediately place the vial into a 37°C water bath.
Gently swirl the vial in the water bath for rapid thawing, ensuring the process takes less than 1 minute. Remove the vial when only a small ice crystal remains.
Disinfect the outside of the vial with 70% ethanol and transfer it into a laminar flow hood.
Transfer the cell suspension into a 10 cm culture dish containing 10 mL of pre-warmed DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin (PS).
After 6–12 hours, once the cells have attached, replace the medium with fresh pre-warmed DMEM containing 10% FBS and 1% PS.
Within 48 hours, the HEK293RR cells should reach confluency in the dish.
(2) Infecting HEK293RR with Ad5d-Cre-GFP adenovirus
Harvest and dissociate HEK293RR cells. Resuspend the cells in complete growth medium at a density of 6.0 × 10⁵ viable cells/mL, preparing a total volume of 6 mL.
Add 36 µL of 1.0 × 10⁸ IFU/mL Rd-Ad5d to the 6 mL cell suspension, achieving a final MOI=10.
Dispense 50 µL of the cell-virus mixture into each well of a 96-well culture plate.
Incubate the plate at 37°C with 5% CO₂ for 24 hours.
Check EGFP expression (from Rd-Ad5) under microscope.
(3) Serial Dilution Setup for rAAV Titration
(Example using AAV2 with a genome titer of ~1.0 × 10¹³ gc/mL)
- Label 10 × 1.5 mL Eppendorf tubes (D1 to D10) and
- add 90 µL of complete cell culture medium to D1
- add 900 µL of complete cell culture medium to D2-D10.
D1 (1×10¹): Add 10 µL of the original AAV stock to the first dilution tube (already containing 90 µL medium).
D2 (1×10²): Transfer 100 µL from D1 to D2, mix well.
D3 (1×10³): Transfer 100 µL from D2 to D3, mix well.
D4 (1×10⁴): Transfer 100 µL from D3 to D4, mix well.
D5 (1×10⁵): Transfer 100 µL from D4 to D5, mix well.
D6 (1×10⁶): Transfer 100 µL from D5 to D6, mix well.
D7 (1×10⁷): Transfer 100 µL from D6 to D7, mix well.
D8 (1×10⁸): Transfer 100 µL from D7 to D8, mix well.
D9 (1×10⁹): Transfer 100 µL from D8 to D9, mix well.
D10 (1×10¹⁰): Transfer 100 µL from D9 to D10, mix well.
(4) AAV TCID₅₀ Assay Setup
(Example using rAAV2 with ~1.0 × 10¹³ gc/mL genome titer)
- Confirm Adenoviral Infection
- Inspect HEK293RR cells in the 96-well plate under a fluorescence microscope.
Expected outcome: Uniform and strong GFP expression across all wells, with no signs of cell death or detachment. This confirms effective Rd-Ad5 infection.
- Add Serial Dilutions of Test AAV2
Add 50 µL of AAV2 dilutions (D7 to D10, corresponding to 1×10⁷ to 1×10¹⁰ dilution) to designated wells.
For each dilution, use one full row with 10 replicate wells per row (columns 1–10).
- Set Up Controls
Add 50 µL of complete medium (no AAV) to wells 11 and 12 in each row as the adenovirus-only negative control.
- Incubation
Return the plate to a 37°C, 5% CO₂ incubator.
Incubate for 72 hours to allow sufficient time for AAV replication and transgene expression.
(5) Genomic DNA Extraction and Real-Time PCR Assay
- Confirm Adenoviral Infection
Examine the HEK293RR cells in the 96-well plate under a fluorescence microscope.
Expected result: Uniform and robust GFP expression across all wells, with no signs of cell death or cell detachment.
- Cell Lysis and Protein Digestion
Add 20 µL of 10× Proteinase K digestion buffer (1% SDS, 10 mM EDTA, 10 mM PBS, pH 8.0) to each well.
Add Proteinase K to a final amount of 25 µg per well.
- Incubation for Lysis
Incubate the plate at 37°C for 1 hour to allow complete digestion.
- Confirm Cell Lysis
Re-examine the plate under a microscope.
Expected result: Complete cell lysis in all wells, with no visible cell patches remaining.
- Inactivate Proteinase K
Transfer the entire lysate (~120 µL per well) to a 96-well PCR plate.
Heat at 95°C for 10 minutes to inactivate Proteinase K.
- Dilution for PCR
Prepare a fresh 96-well PCR plate by adding 90 µL of nuclease-free water to each well.
Transfer 10 µL of the heat-inactivated lysate from each corresponding well into the new plate.
This results in a 10× dilution, suitable for downstream real-time PCR analysis.
(6) Setup real-time PCR Assay and analyze the data
Perform real-time PCR using your preferred system. Use 2 µL of diluted lysate from each well as the template.
After the run, calculate the negative threshold using the following formula:
Threshold Ct = Average Ct of all negative control wells − (3 × standard deviation)For each sample well, if the Ct value is lower than the calculated threshold, the sample is considered positive for AAV infection.
Analysis the data with Improved Ka¨rber Method.
This method gives the 50% endpoint as:
logID50= -log (highest dilution giving 100% positive)-log (dilution factor)*(sum of infection rate at each dilution-0.5)
- Highest dilution giving 100% positive results would be: 1e7
- Dilution factor: 10
- Sum of infection rate at each dilution: 10/10+7/10+1/10=1.8
logID50=-7-1*(1.8-0.5)= -8.3
The final infection titer of AAV would be: 1E8.3*120/2/50*1000=1.995E11 IU/mL
- 120 µL is entire lysate volume
- 2 µLis the volume added for qPCR
- 50 µL is the diluted AAV samples used to infect