Independent Antibody Validation

Coat an ELISA plate with protein antigen.

The competing antibody will be added to the plate and allowed to bind prior to a biotin labeled testing.

The non-bound antibodies will be washed away, leaving only the bound antibodies on the plate.

HRP labeled streptavidin will be added to the plate in order to visualize the amount of bound testing antibody through spectrophotometry.

The designated protein antigen is coated on an ELISA plate.

The testing antibody will be pre-incubated with secondary antibody conjugated to biotin.

The competing antibody will be added to the plate and allowed to bind prior to the testing antibody (with labeled secondary).

Any unbound antibody will be washed away.

HRP labeled streptavidin will be added to the plate in order to visualize the amount of bound testing antibody through spectrophotometry via Octet RED 96.

After GenScript performs epitope binning on the customer selected purified mAbs, you will receive an epitope binning report explaining which purified mAbs belong to which epitope bin. An example report is shown in table 1. All biotin labeled testing mAbs will be shown on the top of the table, while the competing mAbs will be labeled on the left side of the table. The numerical results of the direct ELISA evaluating the competition between each testing and competing antibody is shown in the corresponding section within the table. It is important to remember, that only the bound biotin labeled testing antibody was measured, therefore a low percent binding number indicates that the test mAb was unable to bind when in the presence of the competing mAb, indicating that they both bind to the same antigen and therefore in the same bin.

Table 1. Example of Epitope Binning Report. The example epitope binning report contains the percentage of testing mAb which binds to a specific epitope when a competing mAb is present. The above table indicates that mAb I, mAb II, and mAb III belong to the same bin and are independent from mAb IV.