AAV ELISAs


Data Comparability

 
Intra-Assay Variance

The intra-assay variance is tested by applying three sample dilutions in 24 replicates to the same microtiter plate in a single run. Recovery has to be within our internal acceptance range of +/- 30% while observed CVs are usually < 5%. 

Inter-Assay Variance

The inter-assay variance is determined by measuring a comprehensively characterized internal control in six ELISA plates from the same lot on six different days. Observed CVs are usually < 7%.

Lot-to-lot Consistency

We focus on ensuring the lot-to-lot consistency of our AAV ELISA kits by using independent internal controls.

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Matrix Effects of Additives

The determination of AAV capsid titers from e.g. cell extract can be influenced by several conditions such as the composition of your lysis buffer. For example, high salt concentrations in your buffer might inhibit adequate capsid detection (Grimm, D. et al. Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes can limit production of recombinant AAV-2. Gene Ther.6, 1322–30 (1999).)

For more information, you can refer to the table showing the analysis of matrix effects of different additives on the AAV ELISAs. Data on cumulative effects has not been collected, therefore tolerated concentrations might differ from the concentrations indicated in this table.

How PROGEN developed an internal gold standard

International standard material is only available for serotype AAV2 and AAV8. To ensure we can provide AAV ELISA kits with consistent quality for each serotype, PROGEN has established internal gold standards. Based on these gold standards each kit is thoroughly calibrated to ensure your data will be consistent, accurate and reproducible.

2.1. Genome titer - qPCR/dPCR

Due to the high variabilities of PCR-based methods, the genome titer is determined by three independent laboratories in an average of 15-20 different runs.

2.2. Full vs empty - Neg. stain & cryo EM

Full vs empty AAV capsid ratios are determined by negative stain and cryo EM, which both show similar results for the ratio analysis. Although both methods would be equally suitable for determing the ratio of DNA-filled and empty AAV particles, to ensure the best possible result for the internal gold standard both methods are used.

3. Alignment to the internal gold standard

The standards included in the AAV ELISA kits have an assigned, lot-specific titer which has been carefully aligned to the corresponding internal gold standard.

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