Measurement of serum myoglobin concentrations in horses by immunodiffusion

Quantitative immunodiffusion in one dimension was performed in 6-mm Duran tubes containing a 1% Nobel agar solution and various dilutions of antisera. A series of dilutions of pure myoglobin in equine sera as well as plasma from horses with rhabdomyolysis were tested. Standard curves were prepared of the migration distance of the formed precipitate from the meniscus of the gel after 3, 6, 12, and 24 hours. The clearest line of precipitate was formed with a 1:20 dilution of antisera in agar. Standard curves were nonlinear and plasma myoglobin could be detected at 2 micrograms of myoglobin/ml or greater. The test was optimal, with an error of 5.6%, when read at 24 hours at approximately 25 C. Tubes with agar could be stored for 6 months at 4 C without affecting the accuracy of the test. The specificity of myoglobin for skeletal or cardiac muscle, and its rapid clearance from serum after muscle necrosis, make it ideally suited for evaluating acute muscle damage and for testing the susceptibility of horses for rhabdomyolysis following an exercise test.