A whole blood immunoassay for the interferon-inducible human Mx protein.
Mx protein, an intracellular protein induced by type I interferons (IFNs), is useful as a marker for the IFN-induced state. It is detectable, for example, in leukocytes of patients undergoing IFN-alpha treatment as well as in patients suffering from viral or autoimmune diseases. For immunizations and standardizations, recombinant human MxA protein was expressed in Escherichia coli and purified from inclusion bodies by several steps of chromatography. Two monoclonal antibodies against nonoverlapping epitopes and specific for human Mx protein were selected to establish a simple two-site immunometric enzyme assay. In addition, a monoclonal antibody also reacting with Mx proteins of other species was identified. Prior to assay, whole blood samples were lysed with a nonionic detergent. The sample was incubated on wells coated with a first monoclonal antibody (1304.5.32) together with a second biotinylated monoclonal (1302.34.16), which, after washing, was revealed by an avidin-alkaline phosphatase system. Limit of detection was 5 ng/ml. In two-thirds of normal blood samples (n = 87), Mx protein levels were below 5 ng/ml; 25 samples (29%) had Mx levels between 5 and 50 ng/ml; and 4 samples (5%) were above 50 ng/ml. No Mx was found in plasma, and the mononuclear cell fraction accounted for the bulk of Mx in blood. In vitro, as determined by flow cytometry, monocytes and lymphocytes accumulated Mx protein for 24 h with similar kinetics and remained at plateau levels for more than 70 h. Monocytes contained around eight times more Mx than lymphocytes. The immunoassay was also suitable for detecting Mx after IFN induction in heparinized blood.
Towbin
H
H
Ciba-Geigy Ltd., Pharmaceuticals Research Laboratories, Basel, Switzerland.
Schmitz
A
A
Jakschies
D
D
Von Wussow
P
P
Horisberger
M A
MA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D019204:GTP-Binding Proteins; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007370:Interferon Type I / Q000494:pharmacology; D008214:Lymphocytes / Q000737:chemistry*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000737:chemistry*; D064547:Myxovirus Resistance Proteins; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D007370:Interferon Type I; C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D011506:Proteins; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins