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United States Department of Agriculture

Agricultural Research Service

Title: Development of An Enzyme Linked Immunosorbent Assay (Elisa) for Quantification of Skeletal Muscle Calpastatin

Authors
item Doumit, Matthew
item Lonergan, Steven - UNIV. OF NE, LINCOLN
item Arbona, Juan
item Killefer, John
item Koohmaraie, Mohammad

Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 26, 1996
Publication Date: N/A

Interpretive Summary: Inconsistency in meat tenderness is a major problem facing the meat industry. Breakdown of structural proteins results in meat tenderization during postmortem storage. This protein breakdown is caused by enzymes known as calpains. Calpastatin is a protein in muscle that inhibits calpain action, and thereby, inhibits meat tenderization. Calpastatin activity accounts for a greater proportion of the variation in beef tenderness (about 40%) than any other single measure. Thus, calpastatin has the potential to be used as a predictor of meat tenderness. However, procedures for quantification of calpastatin activity are laborious. We demonstrate the production of specific antibodies which can be used to detect calpastatin. These antibodies were used to develop a simple, rapid and sensitive method (ELISA) for quantification of calpastatin. This procedure will enable us to determine if calpastatin can be used as a predictor of meat tenderness.

Technical Abstract: An indirect antibody ELISA was developed for rapid and sensitive quantification of skeletal muscle calpastatin. Polyclonal antibodies were raised in rabbits against recombinant calpastatin, corresponding to domains 2, 3, and 4 of bovine skeletal muscle calpastatin. Western blot analysis revealed that these antibodies specifically recognize an immunoreactive calpastatin protein of approximately 130 kDa in pre-rigor skeletal muscle extracts. The intensity of the immunoreactive bands corresponds qualitatively with assayable calpastatin activity. For ELISA development, optimum dilutions of sample, primary anti-calpastatin antibody, and peroxidase-conjugated secondary antibody were determined by titration. A dilution optimum for coating of Immulon 4 (Dynatech) plates was observed when heated muscle extracts were diluted to 2-4 ug protein/mL and incubated for 2 h at 37 deg C. Optimum primary (30 ug IgG/mL) and secondary (Sigma A-6154; 1:1000 dilution) antibody incubations were for 1 h at 37 deg C. Tetramethylbenzidine was used as substrate and A450 of the stopped reaction product was recorded in an automated plate reader. Calpastatin ELISA results were linearly related to calpastatin activity (calpain inhibitory activity) of heated longissimus muscle homogenates from pre-rigor lamb (r**2 = .89; n = 40) and beef aged for 24 or 48 h (r**2 = .90; n = 47). Intra-assay CV was < 5% (n = 8) and inter-assay CV was < 6% (n = 5). This assay offers advantages of speed, simplicity and sensitivity over conventional methodology for calpastatin quantification.

Last Modified: 7/25/2014
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