Anti-Adeno Associated Virus Total Antibody Assay
- A variety of platform has been used for the detection of total anti-AAV antibodies in animals and humans
- Two main formats have been reported in the literature
- Direct ELISA Format
- MSD Bridging Assay Format
Direct ELISA Format
- The direct ELISA format has been used to detect the total anti-AAV antibodies, anti-AAV IgG & IgM isotype characterization.
- In these assays, the specific AAV full capsids are used. For example, AAV8 capsids are used for the detection of anti-AAV8 antibodies
- The AAV capsid may be directly absorbed into the high binding 96 well plates.
- Alternately, biotin-conjugated AAV may be used as a capture. In this case, streptavidin precoated plates should be used.
- The assay is optimized with sequential steps that include: a coating of the plate with AAV capsid, the addition of sample, and finally the addition of the detection system
Total anti-AAV antibodies:
- For the detection of total antibodies, the mixture of anti-species IgGs and IgMs labeled with the detection system are used.
- The detection system may include ready to use HRP labeled anti-IgGs and IgMs (Jackson Immunresearch)
- Other chemiluminescence detection system may be used when high sensitivity is desirable
Anti-AAV IgG antibodies:
- For the detection of total antibodies, the anti-species specific IgGs labeled with the detection system are used.
Anti-AAV IgM antibodies:
- For the detection of total antibodies, the anti-species specific IgMs labeled with the detection system are used.
Buffer and Blocking System:
- Appropriate buffer system and minimal sample dilutions should be optimized to reduce matrix interference and non-specific antibody bindings.
MSD Bridging Assay Format
- In this format, the biotinylated AAV serves as a capture reagent and ruthenylated AAV serves as a detection reagent
- The MSD bridging assay format shows superior sensitivity with minimal background signal (non-specific antibody interference)
- The critical step is the optimization of labeling of biotin and ruthenium to AAVs
- May be performed in homogenous assay format and sequential assay format.
- In the homogenous assay format, the sample, capture, and detection reagents are mixed together simultaneously and allowed to form complex
- The sequential assay steps that include: a coating of the plate with AAV capsid, the addition of sample, and finally the addition of the detection system
Critical Assessment Parameters During Assay Development and Validation
Any assays used for the GLP studies and Clinical studies should be developed and validated. These assays must meet the critical assessment parameters outlined by FDA/EMA or other regulatory agencies.
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