Brain-specific proteins: solid-phase immunobioluminescence assay for neuron-specific enolase in human plasma.

We raised specific antibodies in rabbits against pure alpha alpha- or gamma gamma-isoenzymes of enolase (EC 4.2.1.11) purified from human brain. After being specifically immunoabsorbed by the respective Sepharose-coupled enolase isoenzymes, the purified antibodies are desorbed by acidification and coated onto polystyrene tubes. Each sample containing the isoenzyme activity to be determined is incubated in the respective coated tube. The coating is then washed, and the reaction of the antibody-bound enzyme is initiated by adding 2-phosphoglycerate, ADP, and pyruvate kinase. The accumulation of ATP is measured by following the increase in light emission in the firefly luciferase bioluminescence system. The assay is as specific as the antibody used for coating. Its detection limit is about 5 X 10(-9) U, corresponding to about 0.1 pg or 10(-18) mol of enzyme protein per assay. Activities of enolase isoenzymes in human plasma can be evaluated separately, rapidly, and precisely. We used the assay to measure enolase isoenzyme activities in plasma of patients suffering from different types of malignant tumors.