Selection Guide

GenScript provides two different kinds of AAV antibodies. One type recognizes a conformational epitope of the assembled capsid of a certain serotype. These antibodies are suitable for Sandwich ELISA analysis to quantify AAV titer, or Dot Blot analysis to quickly evaluate virus expression level.

GenScript has developed ready-to-use antibody pairs to facilitate your research.

The other type of AAV antibodies recognizes linear epitopes of the three AAV capsid proteins VP1, VP2, and VP3. These antibodies are suitable for Western Blot analysis. They can be used to evaluate the expression level of VP1/VP2/VP3 in AAV production or identify the presence of capsid proteins along the AAV production and purification process.

Application

• Capsid proteins VP1, VP2, and VP3 characterization

  The AAV capsid consists of a total of 60 molecules of viral proteins (VPs). It is composed of three capsid proteins - VP1, VP2, and VP3 in a ratio of 1:1:10. The apparent molecular weights of VP1, VP2, and VP3 are 87 kDa, 73 kDa, and 62 kDa respectively. The capsid proteins are crucial for cell transduction efficiency. Insufficient VP2 and VP3 is believed to be the main cause of ineffective packaging. Excessive incorporation of VP3 may decrease transduction efficiency. The optimization of the three capsid proteins ratio is important in the AAV production process. Polyacrylamide gel electrophoresis (PAGE) and Western blot (WB) are effective methods to detect the composition of VP1, VP2, and VP3 proteins during initial optimization and the quality control of recombinant AAV production process. GenScript provides Anti-AAV Antibody for the characterization of capsid proteins VP1, VP2, and VP3 (Figures 1 and 2).

  

  Figure 1. VP1, VP2, VP3 protein Analysis of AAV Capsids by Western blot
  
  

  Western blot analysis of VP1, VP2, VP3 proteins of AAV type 1, 2, 5, 6, 8, 9, DJ and rh.1 was conducted using MonoRab™ AAVX VP1/VP2/VP3 Antibody (5G4), mAb, Rabbit (GenScript, A02206, 0.1 µg/mL). The signal was developed with Peroxidase Conjugated Goat Anti-Rabbit IgG.
  
  

  Predicted Size for VP1, VP2 and VP3: 87, 72 and 62 kDa
  Observed Size for VP1, VP2 and VP3: 87, 72 and 62 kDa

  

  Figure 2. VP1 protein Analysis of AAV Capsids by Western blot
  
  

  Western blot analysis of VP1 protein of AAV type 1, 2, 5, 6, 8, 9, DJ and rh.1 was conducted using MonoRab™ AAVX VP1 Antibody (24F5), mAb, Rabbit (GenScript, A02207, 0.1 µg/mL). The signal was developed with Peroxidase Conjugated Goat Anti-Rabbit IgG.
  
  

  Predicted Size: 87 kDa
  Observed Size: 87 kDa

• Physical titer quantitation

  Three plasmid system in combination with HEK293 cells is a common mothed to generate rAAV. The detection of virus particle titer (physical titer) is important in the process of rAAV production and the principle of AAV titer quantitation with AAV antibody is described below (Figure 3). AAV monoclonal antibody is used to prepare the immunoassay to measure rAAV physical titer. GenScript provides a variety of Anti-AAV antibodies for the quantitation of AAV physical titer (Figures 4 and 5).

  

  Figure 3. Principle of AAV titer quantitation with AAV antibody

  

  Figure 4. Standard Curve of AAV2 Capsids by Sandwich ELISA
  
  

  The Sandwich ELISA assay was developed by Anti-AAV2 (intact particle) Antibody (2C4), mAb, Mouse (GenScript, A02159-40; 2 μg/mL) and Anti-AAV2 (intact particle) Antibody (2C4) [Biotin], mAb, Mouse (GenScript, A02160-40; 2 μg/mL) as the capture and detection antibody, respectively.

  

  Figure 5. Standard curve of AAV8 by Sandwich ELISA
  
  

  The AAV8 Sandwich ELISA assay was developed by using MonoRab™ AAV8 (intact particle) Antibody (75F2), mAb, Rabbit (GenScript, A02204; 2.5 μg/mL) and MonoRab™ AAV8 (intact particle) Antibody (72E8), mAb, Rabbit (GenScript, A02205; 1 μg/mL) as the capture and detection antibody, respectively.

• Immunogenicity assay

  For patients in gene-engineered treatment, pre-existing AAV antibodies in the body may cause a safety risk. Pre-existing antibodies against AAV in patients can be attributed to natural infection as well as AAV vector administration. It is necessary to thoroughly characterize and reduce the immunogenicity of rAAV products. Anti-drug antibody (ADA) assays are key tools to measure pre-existing AAV antibodies, one anti-AAV antibody assay format is described in Figure 6. Anti-AAV Antibody can be used as positive control or to prepare the standard curve in ADA assay (Figures 7 and 8).

  

  Figure 6. Anti-AAV antibody assay format

  

  Figure 7. Standard Curve of AAV2 Capsids by Indirect ELISA
  
  

  ELISA binding of MonoRab™ AAV2 (intact particle) Antibody (33G5), mAb, Rabbit (GenScript, A02203) with AAV2.
  Coating antigen: AAV2, 1 µg/mL.
  MonoRab™ AAV2 (intact particle) Antibody (33G5), mAb, Rabbit (GenScript, A02203) dilution start from 1,000 ng/mL.

  

  Figure 8. Standard Curve of AAV8 Capsids by Indirect ELISA
  
  

  ELISA binding of MonoRab™ AAV8 (intact particle) Antibody (75F2), mAb, Rabbit (GenScript, A02204) with AAV type 8.
  Coating antigen: AAV type 8, 1 µg/mL.
  MonoRab™ AAV8 (intact particle) Antibody (75F2), mAb, Rabbit (GenScript, A02204) dilution start from 1,000 ng/mL.