Quantitation of interferon-induced Mx protein in whole blood lysates by an immunochemiluminescent assay: elimination of protease activity of cell lysates in toto.
Due to the rapidly expanding usage of interferons and its costliness of therapy, it is important to evaluate the clinical efficacy of the various interferons. Directly assaying circulating interferon is technically quite difficult. Here, we present an alternate method to evaluate interferon therapy by assaying a unique protein, called Mx protein, which is a 78 kDa cytoplasmic protein selectively induced by type-1 interferon in human leukocytes. The current assay is a two-site chemiluminescent immunoassay, designed to detect Mx protein in whole blood lysates. Since the Mx protein once solubilized, is highly susceptible to proteolysis in whole blood lysates, we have devised a new procedure both to maximize its solubility and virtually eliminate its proteolytic degradation. A mouse monoclonal antibody conjugated to the derivatized-paramagnetic particles and an acridinium ester-labeled antibody serve as the solid phase capture and detector antibodies, respectively. This assay is applicable to both manual and automated modes with a detection limit of Mx protein at 20 ng/ml whole blood. Availability of a reliable assay for Mx protein should facilitate the clinical evaluation of many of the newly constructed type-1 interferons.
Oh
S K
SK
R&D Department, Ciba-Corning Diagnostics Corp., Walpole, MA 02032.
Luhowskyj
S
S
Witt
P
P
Ritch
P
P
Reitsma
D
D
Towbin
H
H
Horisberger
M
M
von Wussow
P
P
Bluestein
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000998:Antiviral Agents / Q000097:blood*; D010450:Endopeptidases; D019204:GTP-Binding Proteins / Q000032:analysis; D006358:Hot Temperature; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D016899:Interferon-beta / Q000627:therapeutic use; D008163:Luminescent Measurements; D051379:Mice; D064547:Myxovirus Resistance Proteins; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis*
D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; C579070:Mx1 protein, mouse; D064547:Myxovirus Resistance Proteins; D011506:Proteins; D016899:Interferon-beta; D010450:Endopeptidases; D019204:GTP-Binding Proteins