You Measured Your AAV Titer.
But What Did You Actually Measure?

AAV titer is not a single measurement. Capsid ELISA measures viral particles, while ddPCR measures vector genome copies. Neutralization and infectivity assays answer yet another set of questions. Understanding these differences is essential when designing an AAV characterization strategy.

AAV Titer Is Not One Number

Vector titer is one of the first measurements performed after AAV production, but the word “titer” can describe different analytical endpoints.

These measurements provide complementary rather than interchangeable information.

1 — Capsid ELISA: Measuring Viral Particles

Capsid ELISA uses antibodies recognizing assembled AAV capsids to quantify physical viral particles.

For example, an AAV2 sandwich ELISA available through antibodies-online is designed to detect intact wild-type and recombinant virions as well as assembled AAV capsids.

This makes capsid ELISA useful for monitoring vector production and purification, but it does not determine whether each detected particle contains a vector genome.

2 — ddPCR: Measuring Vector Genomes

Droplet digital PCR provides absolute quantification of vector genome copy number.

While capsid ELISA examines the physical particle, ddPCR provides information about the nucleic-acid component of the vector preparation.

3 — Why Measure Both?

An AAV preparation can contain genome-containing particles as well as empty capsids.

Comparing capsid and genome titers can therefore provide a practical indicator of the relationship between physical particles and packaged genomes.

This ratio should not be considered identical to a direct structural full/empty measurement, and additional characterization may be appropriate depending on the development stage.

4 — Neutralization: What About Pre-Existing Immunity?

Vector quality is only one part of an AAV program. Pre-existing antibodies against AAV can interfere with vector transduction.

Neutralization assays can therefore be used to evaluate immune responses that may affect AAV performance.

5 — Infectivity: Does the Vector Actually Function?

The presence of a capsid and a packaged genome does not by itself demonstrate functional vector activity in a relevant cell system.

Infectivity and cell-based assays add the biological question: can the vector enter cells and produce the expected functional outcome?

Building an AAV Characterization Workflow

Serotype-Specific Tools

The appropriate analytical reagent depends on the AAV serotype and the question being asked.

The current antibodies-online portfolio highlighted in its October newsletter includes antibodies for AAV1, AAV2, AAV4, AAV5, AAV8 and AAV9, with capsid ELISA options for AAV2, AAV5, AAV8 and AAV9.

Recombinant capture/detection antibodies and neutralizing clones are also available for selected serotypes.

Developing or Characterizing an AAV Vector?

Dana Bioscience can help identify serotype-specific AAV antibodies, capsid ELISA kits, neutralizing antibodies and gene-therapy analytical services.

Share your AAV serotype, sample type, analytical objective, sample number and development stage, and we can help evaluate suitable products and testing options.