Influence of some factors on immunoassays of human myoglobin

Six ELISA variants exploiting two monoclonal antibodies, one rabbit antibody and their peroxidase conjugates were applied in assays of purified human myoglobin, apomyoglobin and the protein in human muscle extracts. The myoglobin was accurately determined with monoclonal antibody no. 82 used for coating of ELISA plates while assays performed with monoclonal antibody no. 49 or rabbit antibody used for coating were weak or none. Determinations of human apomyoglobin with ELISA variants were somewhat more sensitive than those of myoglobin. Obtained in this work results were compared with those done using commercial Seratec kit for immunoassay of human myoglobin. Addition to the muscle extracts not only concentrated salts but also acetone, ethanol, sodium dodecyl sulfate or some other denaturing agents markedly increased assays of myoglobin by ELISA with monoclonal antibody no. 49 and antibody no. 82 conjugated with peroxidase. Removal of acetone or ammonium sulfate from extracts resulted in dramatic decrease of the estimated myoglobin. Filtration of the extract through Bio-Gel A5m column did not affect low assays of myoglobin in fractions without pretreatment with acetone. Myoglobin was isolated from human heart extract by immunoaffinity chromatography on Sepharose-antibody no. 82 column and the isolated protein was identified by gel electrophoresis and Western blot.