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In AAV Research & Gene Therapy

Progen

STANDARDIZED RESEARCH WITH AAV TITRATION ELISA

Reliable determination of AAV titers

The increasing interest in rAAV for clinical applications demands a dependable and reproducible quantification of accurate rAAV titers to ensure a safe and reliable gene transfer. In view of the scientific
and clinical significance, PROGEN has established a line of AAV quantification ELISAs for different AAV serotypes (1, 2, 3, 5, 6, 8, 9, and rh10), utilizing its portfolio of capsid-specific AAV antibodies.
The assays for the determination of rAAV2 and rAAV8 titers have been validated in international studies8, 1 and have been classified as superior method for a reliable, standardized rAAV particle titer quantification.

Serotype AAV1 AAV2 AAV2 AAV3
Cat. No. PRAAV1 PRATV PRAAV2R PRAAV3R
Serotype AAV5 AAV6 AAV8 AAV9 AAVrh10
Cat. No. PRAAV5 PRAAV6 PRAAV8 PRAAV9 PRAAV10

Development of internal AAV standards for ELISA kit calibration

The development of standardized AAV capsid material is an essential requirement for the calibration of all of PROGEN's AAV titration ELISA assays to ensure the reliable quantification of intact capsids in rAAV vector preparations. The alignment of the AAV2 and AAV8 Kit Controls for PROGEN's titration ELISAs is based on the ATCC standard material8, 9. For the remaining Kit Controls of the AAV1, 3, 5, 6 and 9 titration ELISAs PROGEN utilizes electron microscopy in combination with qPCR and ddPCR for the development of internal AAV gold standards (data for the development of the AAV5 standard shown below).


A) + B) Comparison of AAV5 sample and reference AAV8 (WL217S) capsids & distinction of full and empty capsids by EM: A) AAV5 micrograph.
B) AAV8 (WL217S) micrograph, which was used to characterize the current AAV8 RSM material from ATCC8. Samples were stained with uranyl acetate.
C) Comparison of AAV5 qPCR titers with ATCC standards for AAV2 and AAV8: A: 3 independent labs provided qPCR / ddPCR data for the AAV5 standard material. The mean titer of viral genome copies and CV was calculated. B: Published qPCR data for ATCC standard material AAV29. C: Published qPCR data for ATCC standard material AAV88.
D) Alignment of the Kit Control with the established gold standard in the AAV5 Titration ELISA: Aligned curves of gold standard material and Kit Control, measured with the AAV5 Titration ELISA (OD vs. concentration). Black: Curve of the gold standard material, Red: Curve of the Kit Control.

1 Naldini L, 2015, Nature, 526:351–360 (Review)
2 Bennett AD et al., 2018, Virology, 518:369–376
3 Tseng YS et al., 2015, J Virol, 89(3):1794–1808
4 Gurda BL et al., 2012, J Virol 86:7739–7751
5 Sonntag F et al., 2011, J Virol 85:12686–12697
6 Moskalenko M et al., 2000, J Virol, 74(4):1761–1766
7 Wobus CE et al., 2000, J Virol, 74(19):9281–9293
8 Ayuso E et al., 2014, Human Gene Therapy 25:977–987
9 Lock M et al., 2010, Human Gene Therapy 21:1273–1285

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