Amperometric and colorimetric enzyme immunoassay for urinary human serum albumin using a plasma-treated membrane

Abstract

By water vapour plasma glow discharge treatment, microporous hydrophobic polypropylene film was modified to make it partially hydrophilic. The plasma-treated part was a spot 6 mm in diameter and permeable to water. After treatment with octamethylenediamine and glutaraldehyde, the plasma spot membrane served as a substrate to immobilize proteins such as antibodies or enzymes. Using this membrane, an amperometric enzyme immunoassay system was established to determine human serum albumin (HSA) in urine. The measurable range with this system was from 0.5 to 100 mg l −1 HSA in urine, which is suitable for the prognosis of diabetic nephritis. The HSA values in urine samples determined by the present system showed a linear correlation with those obtained by radioimmunoassay ( r = 0.974). After keeping the antibody-immobilized plasma spot membranes at 4°C for 6 months, the same calibration graph for HSA determination was obtained. In addition, a colour-producing reaction was also performed on the immunoreacted plasma spot membrane. Reflectance spectrometric analysis demonstrated that the colour on the plasma spot membrane itself corresponded to the HSA concentration. The difference in colour between 10 and 100 mg l −1 HSA could be visually discerned.