Decay‐accelerating factor (DAF) on the blood cell membranes in patients with paroxysmal nocturnal haemoglobinuria (PNH): measurement by enzyme‐linked immunosorbent assay (ELISA)

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Abstract
Summary. We developed a quantitative enzyme-linked immunosorbent assay (ELISA) for decay-accelerating factor (DAF) on blood cell membranes using monoclonal anti-DAF antibodies. DAF is an integral membrane protein of several blood cells. It regulates the C3 and C5 convertase of the classical and alternative pathways of complement activation. It is partially or completely deficient in the membranes of blood cells of patients with paroxysmal nocturnal haemoglobinuria (PNH). The ELISA we developed for DAF measurement indicated a reliable range of measurement from 2.25 to 11.25 ng of DAF. In particular, ELISA proved to be a technically simple method and its sensitivity was enhanced by using avidin-biotin complex. In this study, DAF levels in extracts of erythrocytes from 30 healthy donors and in extracts of PMN and platelets from 15 healthy donors were measured by ELISA. The DAF content of blood cells from eight patients with PNH and erythrocytes from 13 patients with anaemia were also measured. The DAF levels of normal erythrocytes, PMN and platelets were 3110.960, 28000.13900 and 3100.1370 (mean±SD) molecules/cell, respectively. In general, the DAF content of PNH cells was below the normal range, although it was within the normal range in some cases of PNH. The DAF levels of PNH-I and -II erythrocytes were estimated from the ratio of PNH-I, -II and -III erythrocytes. And, in four cases of PNH, the DAF levels of PNH-I erythrocytes separated by acidified serum lysis were measured by ELISA. In most cases of PNH, DAF was found to be partially deficient in PNH-I and PNH-II erythrocytes.

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