DEVELOPMENT OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) FOR QUANTIFICATION OF SKELETAL-MUSCLE CALPASTATIN

An indirect antibody ELISA was developed for rapid and sensitive quant ification of skeletal muscle calpastatin. Polyclonal antibodies were r aised in rabbits against recombinant calpastatin, corresponding to dom ains 2, 3, and 4 of bovine skeletal muscle calpastatin. Western blot a nalysis revealed that these antibodies specifically recognize an immun oreactive calpastatin protein of approximately 130 kDa in prerigor ske letal muscle extracts. The intensity of the immunoreactive bands corre sponds qualitatively with assayable calpastatin activity. For ELISA de velopment, optimum dilutions of sample, primary anti-calpastatin antib ody, and peroxidase-conjugated secondary antibody were determined by t itration. A dilution optimum for coating of Immulon(R) 4 (Dynatech) pl ates was observed when heated muscle extracts were diluted to 2 to 4 m u g of protein/mL and incubated for 2 h at 37 degrees C. Optimum prima ry (30 mu g IgG/mL) and secondary (Sigma A-6154; ; 1:1000 dilution) an tibody incubations were for 1 h at 37 degrees C. Tetramethylbenzidine was used as substrate and A(450) of the stopped reaction product was r ecorded in an automated plate reader. Calpastatin ELISA results were l inearly related to calpastatin activity (calpain inhibitory activity) of heated longissimus muscle homogenates from prerigor lamb (r(2) = .8 9; n = 40) and beef aged for 24 or 48 h (r(2) = .90; n = 47). Intra-as say CV was < 5% (n = 8) and inter-assay CV was < 6% (n = 5). This assa y offers advantages of speed, simplicity, and sensitivity over convent ional methodology for calpastatin quantification.