The lysosomal storage disorder mucopolysaccharidosis type II (MPS II) is caused by a deficiency in the activity of the lysosomal exohydrolase iduronate-2-sulphatase (IDS). MPS II patients present within a spectrum of clinical phenotypes, which reflects the dynamic balance between the level of mutant protein, its residual enzyme activity and the resultant level of storage product. In this study, we have developed an immunoquantification assay for the accurate detection of iduronate-2-sulphatase protein and applied this methodology to the analysis of mutant iduronate-2-sulphatase protein in plasma samples from MPS II patients. The detection limit for the assay was defined as 20 ng/ml for wild type iduronate-2-sulphatase, but could be extended to a detection limit of 0.3 ng/ml by heat denaturation of the protein/plasma sample. The mutant protein detected in plasma from MPS II patients displayed similar properties to heat denatured wild type iduronate-2-sulphatase, suggesting an altered protein conformation. The ratio of heat denatured to native ELISA reactivity could be used to confirm the diagnosis of MPS II (i.e., a ratio of >1 for normal protein and <or=1 for mutant protein). Notably, four of the 20 patients tested had either normal or higher than normal levels of iduronate-2-sulphatase protein, but this protein also showed evidence of conformation change. The iduronate-2-sulphatase protein level detected in plasma from MPS II patients showed little or no direct correlation with the severity of the clinical phenotype observed in these patients.