Microalbuminuria immunoassay based on antibodies covalently conjugated to Eupergit C-coated beads

To develop a reliable, simple, and sensitive assay for microalbuminuria, based on covalent attachment of anti-HSA to oxirane-bearing polymethylmethacrylate beads (Eupergit CB6200).

Anti-HSA antibodies were coupled to CB6200 beads by reaction of their amino groups with the oxirane groups of the matrix. The capability of the beads to bind HSA from standard solutions or urine was evaluated and compared with the state of the art ELISA test.

The new bead immunoassay is sensitive and linear in the range of 1-25 mg/L, which is considered the low microalbuminuria range. When HSA levels in urine were tested, the intra- and interassay CV values ranged between 2.7 and 3.9% and between 5.6 and 6.6%, respectively. The long-term storage stability of the antibodies covalently bound on the beads was higher than of the same antibodies adsorbed on ELISA plates. After 16 wk of storage, the CV was about 7.3% with the bead assay, compared with 14% obtained for the ELISA test under the same experimental conditions.

A new procedure for microalbuminuria assay was developed, with Eupergit CB6200 beads as a solid support for covalent binding of the first antibody. Accuracy, sensitivity, reproducibility, and precision of the bead immunoassay were similar to those of commonly used immunoassays, as exemplified by the analysis of HSA in 53 clinical urine samples. The bead assay retains a low degree of variability over long storage periods, and the beads may be reapplied after a simple acid-washing procedure.