Enzyme-Linked Immunosorbent Assay (ELISA) with Covalently Bound Protein on Glass Tubes: 1. Stable Antigenicity and Binding of IgG as a Model Antigen after Repeated Use

Abstract

A modification of the ELISA procedure is described. The system is based on the covalent binding of protein to glass tubes. Human IgG was used as model antigen. Optimal conditions were tested for the removal of alkaline phosphatase-labeled antibodies from their antigen. Under such optimal conditions, a regenerable system could be created which exhibited many advantages, as compared with conventional ELISAs with antigens absorbed unspecifically to plastic surfaces. The advantages are: 1. A higher density of IgG as model antigen on the solid phase, i.e., 1 molecule IgG per 94 nm 2with glass tubes as compared with 110 nm 2with polystyrene (PS) or 143 nm 2with polyvinylchloride (PVC) microtiter plates. 2. A much smaller unspecific absorption of less than 1 % as compared with 39 % with PS-plates or 16 with PVC-plates treated under identical conditions. 3. A higher stability of the binding of the model antigen to the solid phase, i.e., a drastically reduced protein loss of less than 6 % during the first ELISA procedure (including the regeneration) and of less than 1 % during the subsequent ELISA and regeneration cycles with glass tubes as compared with 46 % (PS plates) or 55 % (PVC plates). 4. A smaller intraday variation coefficient of the ELISAs of 4.8 % with glass tubes as opposed to 9.7 % or 7.5 % with PS or PVC plates respectively. The system with covalently bound antigens on glass tubes could be used in at least 20 consecutive measuring cycles. In five consecutive cycles, a protein loss of less than 5 % was observed and the interday variation coefficient of the ELISA reaction was smaller than 5 using the same tubes repeatedly. Our results indicate that covalent linkage of protein can improve ELISA and lead to repeatedly usable systems as long as the antigen is stable against the regeneration procedure. Such an ELISA system may be helpful with highly purified proteins.