gene therapy

Comparison of Gator^® BLI and Progen ELISA for Quantitation of AAV Serotypes

Introduction

Determination of total capsid titer is one of the critical quality attributes for AAVs used in gene therapy.

This application note presents quantitation of AAV serotype 2, 5, 8 and 9 using Gator®Prime system and Gator® AAVX probes and compares the performance with Progen Xpress ELISA assay for the same serotypes.

ELISA was run on SpectraMax iD5, Molecular Devices. The AAV serotype standards were purchased from Virovek, Newark, CA.

GATOR^® AAVX PROBE FEATURES

Total capsid quantitation for serotypes AAV1-10
Dynamic range up to 1E+14 vp/mL (for most serotypes)
Less than 30 min analysis time
LOD ~ 1E+09 vp/mL
Crude sample tolerant
Stable over broad pH range
Cost effective
Easy to use with minimal hands-on time

QUANTITATION PRINCIPLE AND WORKFLOW

Gator® probe uses CapSelect™ AAVX nanobody as a ligand to enable direct measurement
Samples containing AAV particles are pipetted into the 96-well plate and captured on the probe
The total virus capsid concentration determined using rate of binding of the AAV serotype of interest to the probe
Different AAV serotypes bind at different rates
Gator® software calculates the binding rates from standards with known concentrations to generate a standard curve

Comparison of Gator® BLI and Progen ELISA for Quantitation of AAV Serotype 9

Method: The performance of Gator® BLI platform for quantitation of various AAV serotypes was evaluated using an established and relatively commonly used Xpress ELISA kit.

The Virovek AAV2, 5, 8 and 9 standards were at a highest concentration of 1E+11, 1E+11, 2E+11 and 2E+11 vp/mL, respectively. For ELISA, the stocks were diluted 100x, 10x, 400x and 300x, respectively, to bring into the ELISA assay.

AAV9 CONCENTRATION- GATOR® AAVX PROBES VS AAV9 ELISA

The ELISA kit performance was verified on SpectraMax iD5. As can be seen in Figure 1, the kit shows good inter-day reproducibility over 3 days. Also, as can be seen in Figure 2, the ELISA measurements were linear as specified for the kit.

VERIFICATION OF AAV9 ELISA LINEARITY

Comparison of Gator® BLI and Progen ELISA for Quantitation of AAV Serotype 2

% ERROR OF ELISA AND GATOR® FOR AAV2

Table 1: Comparison of calculated concentrations (average of three measurements) and accuracy of unknowns using ELISA and Gator®. The calibration curves were created for each method using AAV2 standards. For ELISA the stock at 1E+11 was diluted to bring it down to the specified dynamic range supported by the kit. Overall demonstrates better accuracy compared to ELISA.

AAV2 KNOWN VS ELISA VS GATOR®

CORRELATION OF ELISA AND GATOR® FOR AAV2

Comparison of Gator® BLI and Progen ELISA for Quantitation of AAV Serotype 5

% ERROR OF ELISA AND GATOR® FOR AAV5

Table 2: Comparison of calculated concentrations (average of three measurements) and accuracy of unknowns using ELISA and Gator®. The calibration curves were created for each method using AAV5 standards. For ELISA the stock at 1E+11 was diluted to bring it down to the specified dynamic range supported by the kit. Overall the Gator® demonstrates better accuracy compared to ELISA.

CORRELATION OF ELISA AND GATOR® FOR AAV5

AAV5 KNOWN VS ELISA VS GATOR®

Comparison of Gator® BLI and Progen ELISA for Quantitation of AAV Serotype 8

% ERROR OF ELISA AND GATOR® FOR AAV8

Table 3: Comparison of calculated concentrations (average of three measurements) and accuracy of unknowns using ELISA and Gator®. The calibration curves were created for each method using AAV8 standards. For ELISA the stock at 2E+11 was diluted to bring it down to the specified dynamic range supported by the kit. Overall the Gator® demonstrates better accuracy compared to ELISA.

CORRELATION OF ELISA AND GATOR® FOR AAV8

AAV8 KNOWN VS ELISA VS GATOR®