Quantitative determination of acylphosphatase levels in horse tissues by enzyme-linked immunosorbent assay

A non competitive enzyme-linked immunosorbent assay (ELISA) specific for horse muscle acylphosphatase (E.C. 3.6.1.7.) has been developed. The purified anti-acylphosphatase antibodies were immobilized by passive absorption to a solid-phase support and incubated with known and unknown amounts of antigen. The antibody-acylphosphatase complex was quantified using the same antibody conjugated to horseradish peroxidase. The assay yields positive reactions with as little as 0.05 ng of antigen, with intra- and interassay coefficients of variation of 5% and 7%, respectively. On the basis of this assay we developed a more sensitive test than the optical one, using benzoyl-phosphate as substrate, for acylphosphatase determination. By means of this test, the presence of the enzyme in horse tissue homogenates was evaluated under conditions in which the optical test failed to distinguish the acylphosphatase activity from that of other enzymes.