Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay.
In some instances, even the increased resolution that may be afforded in immunoassays by the use of monoclonal antibodies fails to effect resolution among molecules that share many epitopes. An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to overcome this difficulty in the differentiation between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (1C5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, 1C5 and 2B9 were 25% cross reactive together, 1C5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D003429:Cross Reactions; D000939:Epitopes; D006801:Humans; D009132:Muscles; D009206:Myocardium / Q000378:metabolism; D009218:Myosins / Q000276:immunology*; D011863:Radioimmunoassay / Q000379:methods*; D012685:Sepharose
D000911:Antibodies, Monoclonal; D000939:Epitopes; D012685:Sepharose; D009218:Myosins