CHARACTERIZATION OF MONOCLONAL-ANTIBODIES TO CALPAIN-3 AND PROTEIN EXPRESSION IN MUSCLE FROM PATIENTS WITH LIMB-GIRDLE MUSCULAR-DYSTROPHY TYPE 2A

Monoclonal antibodies were raised to two regions of calpain 3 (muscle- specific calcium-activated neutral protease), which is the product of the gene that is defective in limb-girdle muscular dystrophy type 2A. The antibodies produced characteristic patterns of bands on Western bl ots: normal calpain 3 protein was represented by bands at 94 kd, plus additional fragments at similar to 60 or 30 kd, according to the antib ody used. Specificity was confirmed by the loss of all bands in patien ts with null gene mutations. The ''normal'' profile of bands was obser ved in muscle from 53 control subjects and 70 disease-control patients . Calpain 3 protein was found to be extremely stable in fresh human mu scle, with full-size protein being detected 8 hours after the muscle h ad been removed. Blots of muscle from nine limb-girdle muscular dystro phy type 2A patients with defined mutations showed variation in protei n expression, with seven showing a clear reduction in the abundance of protein detected. No simple relationship was found between the abunda nce and clinical severity. Two patients showed normal expression of th e full-size 94 kd band accompanied by a clear reduction in the smaller fragments. This pattern was also observed in one patient with an unde fined form of limb-girdle dystrophy, These results indicate that immun odiagnosis is feasible, but caution will need to be exercised with the interpretation of near-normal protein profiles.