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AAV Xpress ELISAs

A growing number of academic and industrial labs are using AAV vectors for the development of gene therapies. This leads to an increase in the demand for effective and reliable analytical AAV tools for R&D and manufacturing. To enable safe and effective AAV gene therapies, a dependable and reproducible quantification of accurate rAAV titers is needed to ensure secure and reliable gene transfer.

PROGEN's AAV ELISAs are widely used for the quantification of intact AAV capsids, thus to determine the total capsid titer including full & empty capsids. In combination with additional quantification methods, characterizing different AAV titers (e.g. genomic titer), PROGEN's AAV ELISAs are a robust and reliable tool for the comprehensive characterization of AAV preparations. In order to reduce the assay time from 4-5 hours (standard AAV ELISA) to less than 2 hours, we have adjusted the kit components and the protocols for the ELISAs for AAV2, AAV8 and AAV9.

The corresponding adjustments resulted in the shortening of the incubation steps from 60 to 20 minu- tes each, thus leading to a reduction of the assay time of more than 50%. This was achieved by adapting reagent concentrations without changing the composition of the kit's core characteristics, allowing a seamless and risk-free transition of users from the standard AAV ELISAs to the express version.

Here we demonstrate that these faster ELISAs show the same accuracy and reproducibility as the corresponding standard titration ELISA kits but save a significant amount of time for the users. With these faster AAV ELISAs, users can rely on the well-established quality of PROGEN's reagents for accurate titer determination of AAV samples.

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Comparison of Standard AAV Titration ELISAs with AAV Xpress ELISAs

Comparison of AAV Xpress titration ELISA with the standard AAV titration ELISAs curves are showing the inter-assay measurements on 5 different days. No significant drop in OD or deviation of sample values was detected compared to the standard ELISAs for AAV2, AAV8 and AAV9.

Comparison of protocols for  AAV Standard and Xpress ELISA. Xpress ELISAs enable significant time saving allowing to complete 2 assays on the same day.

Inter-assay and intra-assay values of AAV Xpress ELISAs with their corresponding standard ELISA   

* Inter-assay variance was tested on 5 different days using the same components and samples

** Intra-assay variance was tested using 3 different concentrations of AAV capsids in the range of the standard curve in 24-plex each                

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Conclusion

All three AAV Xpress ELISAs have been adjusted to shorter incubation times showing no significant changes in the level of background OD values and their performance in the quantification of AAV samples. The recovery values are in exactly the same range as for the standard AAV titration ELISA, enabling a seamless transition to the new time-saving ELISA without compromising accuracy or convenience in handling.

References:

Grimm D, Kern A, Pawlita M, Ferrari F, Samulski R and Kleinschmidt J.,Titration of AAV-2 particles via a novel capsid ELISA packaging of genomes can limit production of recombinant AAV-2. Gene Therapy 6 132-1330 (1999).

Lock M, McGorray S, et al., Characterization of a recombinant Adeno-Associated Virus Type 2 reference standard material; Hum Gene Therapy 21 1273-1285 (2010).

Wobus, C.E., Hügle-Dörr, B., Girod, A., Petersen, G., Hallek, M., Kleinschmidt, J.A., Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid epitope mapping and identi- fication of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection. Journal of virology 74, 9281-9293 (2000).