title: A sensitive radioimmunoassay of insulin autoantibody: reduction of non-specific binding of [125I]insulin.
abstract: There has been a lack of consensus among results of assays for insulin autoantibody (IAA) carried out by different laboratories, despite a reduction in the non-specific effect using a cold insulin competitor in radioimmunoassays (RIAs) for IAA in type I diabetes. We speculated that the discrepancies are partly a result of the non-specific binding (NSB) of [125I]insulin to unidentified molecules in serum on polyethylene glycol separation, and tried to improve IAA RIAs. The molecular weight of a candidate for the factor causing NSB was estimated to be about 700 kDa by gel filtration analysis, resembling that of alpha 2-macroglobulin (a2M). Further, the addition of purified a2M to the assay resulted in an increase in NSB. Screening revealed that heterocyclic compounds, such as isothiazolinone derivatives (ProClin300), were greatly effective at reducing NSB in control subjects from 2.904+/-0.909% to 1.347+/-0.254% (n=283, mean+/-SD, p<0.0001). Using our newly developed IAA RIA with ProClin300, the sensitivity for newly diagnosed type I diabetes patients (n=55) was 32.7% and 30.9% with or without insulin competition, respectively, whereas that of the former assay without ProClin300 was only 20.0%.
authors.affiliation: Immunology Laboratory, Diagnostics Department, YAMASA Corporation, 2-10-1 Araoi-cho, Choshi, Chiba 288-0056, Japan. murayama@yamasa.com
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Murayama
authors.forename: Nobuo
authors.initials: N
authors.lastname: Matsuura
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Kawamura
authors.forename: Taro
authors.initials: T
authors.lastname: Maruyama
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kikuchi
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Fumi
authors.initials: F
authors.lastname: Nishibe
authors.forename: Atsuo
authors.initials: A
authors.lastname: Nagata
mesh_terms: D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000097:blood*; D001667:Binding, Competitive; D002648:Child; D002675:Child, Preschool; D005260:Female; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000276:immunology; D007330:Insulin Antibodies / Q000097:blood*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000737:chemistry; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000187:drug effects / Q000276:immunology
chemical_list: D001323:Autoantibodies; D007328:Insulin; D007330:Insulin Antibodies; D013844:Thiazoles; D000511:alpha-Macroglobulins; C096800:iodo(A14-tyrosyl)insulin; C011506:2-methyl-4-isothiazolin-3-one; D011092:Polyethylene Glycols