The DOI matches its Crossref record; 42 of the 42 references it cites were found in a registry or an index.
Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2
Abstract
We demonstrate the rapid and reliable quantification of physical AAV-2 (adeno-associated virus type 2) particles via a novel ELISA based on a monoclonal antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at least 80+percent of all particles were empty, compared with a maximum of 50percent in wild-type AAV-2 stocks, indicating that the recombinant genomes were less efficiently encapsidated. This finding was confirmed upon titration of CsCl gradient fractions from recombinant and wild-type AAV-2 stocks. ELISA-based measurement of capsid numbers revealed a large number of physical particles with low densities corresponding to empty capsids in the recombinant, but not in the wild-type AAV-2 preparations. Moreover, additional expression of VP proteins during rAAV production was found to result in an excessive capsid formation, whilst yielding only minor increases in DNA-containing or transducing rAAV particles. We conclude that encapsidation of viral genomes rather than capsid assembly can be limiting for rAAV production, provided that a critical level of VP expression is maintained. The feasibility of quantifying AAV-2 capsid numbers via the ELISA allows determination of physical to DNA-containing or infectious particle ratios. These are important parameters which should help to optimize and standardize the production and application of recombinant AAV-2.
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In the citing paper
“ELISA involves multiple lengthy incubation and washing steps prior to analysis, for a total turnaround time of 4–5 h, or up to a full working day.26,61, 62, 63”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [61]
In the citing paper
“Specificity is high, with little cross-reactivity between serotypes, and monoclonal antibodies specific to the conformational epitope of serotypes rAAV1, -2, -3, -5, -6, -8, -9, and rh.10 are commercially available.61,62”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [61]
In the citing paper
“The assay is typically performed after a purification step but is sufficiently robust and specific to be used on samples with moderate matrix effects and free capsid proteins, like cell lysates.26,37,61,85”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [61]
In the citing paper
“Specificity and robustness to matrix effects are major advantages of ELISA, but in considering the long turnaround time, moderate throughput of only 10 samples per 96-well plate, and labor-intensive sample preparation, it is challenging to implement for routine quantification of capsid titer in a process development setting without automated liquid handling.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [61]
In the citing paper
“The quantification limit is around 108 capsid particles (cps) per milliliter, depending on serotype, and the linear range is limited to 1–2 orders of magnitude, so that pre-titration is recommended for samples of unknown titer.61,62”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [61]
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Metadata
- Authors
- Grimm, D., Kern, A., Pawlita, M., Ferrari, F. K., Samulski, R. J., & Kleinschmidt, J. A.
- Affiliations
- German Cancer Research Center (DE)
- Princeton University (US)
- University of North Carolina at Chapel Hill (US)
- Venue
- Gene Therapy
- Published
- 1999-07
- Volume
- 6
- Issue
- 7
- Pages
- 1322-1330
- ISSN
- 0969-7128 (ISSN-L)
- 1476-5462
- OpenAlex
- W2079090019
- Cited by
- 195
- References
- 42
- Citations by year
- 2026: 5 · 2025: 6 · 2024: 9 · 2023: 8 · 2022: 3 · 2021: 10 · 2020: 9 · 2019: 5 · 2018: 5 · 2017: 4 · 2016: 3 · 2015: 2 · 2014: 5 · 2013: 6 · 2012: 13
- FWCI
- 3.53
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Gene Therapy (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Bundesministerium für Bildung und Forschung
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- adeno-associated virus type 2, empty capsids, capsid assembly, viral vector production, monoclonal antibodies
- MeSH terms
- Dependovirus, Antibodies, Monoclonal, Capsid, Enzyme-Linked Immunosorbent Assay, Genetic Engineering, Genetic Vectors, Humans, Genetic Therapy
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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