Dear Niu

What was the reason behind attachment of two antibodies using the click chemistry (i.e., DBCO-Azido reaction)? In this case, the NHS parts of linker (in both of DBCO and Azido-ended ones) react with primary amines of lysine residues not placed necessarily in Fc regions wich can negatively affect the antibody affinity toward the antigen of interest. Besides, I am wondering why you tried to bind a secondary antibody to the first antibody covalently?

Actually; Every antibody is specific for the specific antigen, Any antibody which is for specific antigen that will no work or bind with other antigen, because light chain of antibody have specific site for antigen and when we modify antibody it will lose its specific binding site , so ,you may use alternative antibodies or may there an error with the procedure of doing and change the experimental condition for working