Hello Fan Janice,

Since your goal is to evaluate the binding activity of antibody with the protein, coating the surface with CD5 protein is the more appropriate method. This is because CD5-Fab antibody can bind specifically to its target antigen, allowing you to measure the binding affinity and compare different batches based on their ability to bind to the immobilized CD5 protein. Since the CD5 protein is the constant factor, any differences in binding observed across different batches of CD5-Fab can be attributed to their varying binding affinities.

On the other hand, coating the plate with the CD5-Fab antibody would generally be better for detecting or capturing CD5 protein, if your goal was to capture CD5 protein from a sample. The antibody would specifically bind to the CD5 protein, allowing you to detect and quantify it in a sandwich ELISA.

So, you should coat with CD5 protein.

Best,

Hi Fan Janice

For detecting binding activity by ELISA, since the goal is to verify that the protein and antibody can indeed bind, method 1 is more suitable. Specifically, coat the plate with CD5 protein, use gradient dilutions of different CD5-Fab batches as the primary antibody, and Anti-Fab-HRP as the secondary antibody. By fitting the curve, you can calculate the EC50 values — the lower the EC50, the higher the binding activity of that Fab batch.

In contrast, method 2 primarily reflects differences in coating efficiency and artefacts from immobilization, making it an inaccurate representation of Fab binding activity in solution.

The answer to this question is provided by MedChemExpress Technical Support.

[email protected]