A Distinctive Surface for Phospholipid Assays

Abstract

Solid-phase immunoassay is a commonly used immunological technique in many clinical and research laboratories. It is known that proteins will adsorb to a solid surface such as polystyrene. Many studies have also indicated that surface modifications such as the introduction of different functional groups onto the polystyrene surface will enhance the overall protein-binding properties of the plastic polymer (1). Nevertheless, the increased binding also means increased nonspecific binding, producing higher background signals. The currently available polystyrene surfaces work well for most protein antigens. However, these polystyrene surfaces are not adequate for the measurement of all antigenic substances. Certain antigenic lipids are not easily detected and measured using these surfaces (2). A commonly used diagnostic test for anti-phospholipid antibodies (aPLs) is an ELISA using cardiolipin, an anionic phospholipid, as the antigen in the solid-phase immunoassay (3). aPLs are antibodies associated with the clinical syndrome of thrombosis and recurrent midtrimester fetal death and other, less common manifestations, termed antiphospholipid syndrome (4). aPLs are also detected in patients with systemic lupus erythematosus and related autoimmune disorders (5)(6)(7). There are numerous critical variables for anticardiolipin assays. Critical factors that can influence ELISA results include the amount of phospholipids coated on the surface, the solvent for the phospholipids, choice of blocking agents and serum diluent, incubation temperature, and the type of microwell plates (8)(9)(10). To develop a solid-phase immunoassay optimized for phospholipids, we examined various modified polystyrene surfaces for the ability to bind protein and phospholipids, including cardiolipin. The following reagents were obtained from Sigma: cardiolipin (diphosphatidylglycerol), sodium salt from beef heart, 4.8 g/L in ethanol; o -phenylenediamine dihydrochloride (OPD), 30-mg tablets; p -nitrophenyl phosphate, disodium; and diethanolamine, ACS reagent grade. Nitrobenzoxadizole (NBD)-labeled 1-palmitoyl-2-[6-(7-nitro-2-1,3-benzoxadiazol-4-y)]- sn -glycerol 3-phosphoserine (sodium salt), M r 796.81, 1 g/L in …