Products

AvaBridge ELISA Kit

Complete kit for the systematic 3-D conformational comparability analysis of Avastin biosimilar molecule to

Bevacizumab (Avastin trade name).

 

 

Bevacizumab is a humanized monoclonal antibody, produced by Genentech, that inhibits vascular

endothelial growth factor A (VEGF-A). VEGF-A is a chemical signal that stimulates angiogenesis in a variety

of diseases, especially in cancer. Bevacizumab was the first clinically available angiogenesis inhibitor in the

United States and was approved by the U.S. Food and Drug Administration (FDA) for certain metastatic

cancers. It received its first approval in 2004 for combination use with standardchemotherapy for metastatic

colon cancer. This kit will allow for conformational comparison between Avastin Biosimilars and authentic

Bevacizumab.

 

Assay Principle

The assay is in a sandwich ELISA format where the plate is coated with a panel of antibodies raised against

peptides derived from the full length protein sequence of Bevacizumab. Taken individually, each of these

antibodies is strongly antigenic to the peptide sequence that was used in its production. However, when

these peptides are incorporated into a full length correctly folded protein, the antigenicity of many of them is

masked by the three dimensional structure of the protein and onlya limited number of the antibodies respond.

The result is a histogram which can be likened to a ‘fingerprint’ for correctly folded Bevacizumab. For an

Avastin Biosimilar, if the protein is correctly folded and glycosylated, the ‘fingerprint’ will match that of

Bevacizumab. If it is not correctly folded, previously masked peptide sequences will be exposed and will be

recognized by the antibody made to that exposed sequence. In this way, changes in the ‘fingerprint’

generated by the ELISA will point out differencesbetween the Biosimilar and authentic Bevacizumab.

 

The assay is performed by making a 5 μg/ml solution of Avastin Biosimilar and Bevacizumab reference

material respectively, and adding to the 96-well plate. Following a 1 hour incubation to allow capture of the

Biosimilar and Bevacizumab reference proteins by the panel of antibodies on the plate, a reporting polyclonal

anti-human IgG antibody, conjugated with biotin, is added and incubated for 1 hour to allow it to bind to any

captured proteins. After this incubation, the plate is washed anda Streptavidin-HRP (Horse Radish

Peroxidase) conjugate is added and incubated for 45 minutes. The Streptavidin-HRP conjugate will be

captured by any biotin labeled antibody bound to the plate. Following a wash step to remove unbound

conjugate, TMB substrate is added and is converted by the captured HRP to a colored product in proportion

to the amount of HRP bound to the plate. After a short incubation to allow color development, the reaction is

stopped and the intensity of the generated color is detected in amicrotiter plate reader capable of measuring

450nm wavelength. The color development will be proportional to the captured Biosimilar or Bevacizumab

reference protein. A typical ELISA with only the Bevacizumab reference protein is shown in figures 1 and 2

below. Your results may differ from this as your source sample will not be the same one that we used to

generate this plot.

 

  

 Fig. 1. Bevacizumab conformational array ELISA from Variable Region

 

 

 Fig. 2. Bevacizumab conformational array ELISA from constant region

 

 Product instruction (PDF)