EFFICIENT IMMOBILIZATION OF PROTEINS BY MODIFICATION OF PLATE SURFACEWITH POLYSTYRENE DERIVATIVES

Immobilization of proteins on microplate wells by simple adsorption (e .g., for ELISA) is convenient, but it can be inefficient, especially i f proteins are hydrophilic or small in size. This problem was alleviat ed by the use of polyvinylbenzyl lactonoylamide (PVLA). PVLA is strong ly adsorbed to the hydrophobic well surface, and its lactonamide part can be oxidized with periodate to generate aldehydo groups. Proteins a re then immobilized covalently to the aldehydo groups by reductive ami nation under mild conditions. Using this method, henceforth termed the PVLA method, alkaline phosphatase (AP) was immobilized to microplates six- to sevenfold greater than by simple adsorption (as measured by a ctivity). Similarly, the activity of immobilized mannose-binding prote in A (MBP-A) was 4- to 8-fold higher by the PVLA method than by simple adsorption. The PVLA-coated plates needed as little as 200 ng of MBP- A per well to have a sufficient amount of MBP-A immobilized for the me asurement of binding of I-125-labeled mannosylated bovine serum albumi n (I-125-Man-BSA), but unmodified plates required as much as 20 mu g/w ell MBP-A to obtain the same response. Recommended conditions for the PVLA method are 40 mu l of 2 mg/ml of PVLA for coating, 1 mM NaIO4 for the generation of the aldehydo groups, and a 2-h reductive amination at 37 degrees C between pH 8 and 9 for the protein ligation. (C) 1997 Academic Press.