title: Development and validation of radioligand binding assays to measure total, IgA, IgE, IgG, and IgM insulin antibodies in human serum.
abstract: Radioligand binding assays for total and Ig classes of insulin antibodies (IAB) were developed and validated. For each assay, insulin-extracted serum samples were incubated with radiolabeled insulin in the presence and absence of high levels of unlabeled insulin to determine nonspecific binding and total binding, respectively. To measure total IAB, antibody-bound insulin was precipitated with a polyethylene glycol solution, washed, and counted in a gamma-counter. To measure IgG IAB, samples were treated with protein G-Sepharose beads, centrifuged, washed, and counted. For the measurement of IgA, IgE, and IgM IAB, IgG was removed from the samples and treated with anti-IgA, -IgE, or -IgM conjugated to Sepharose beads, centrifuged, washed, and counted. The acid/charcoal extraction of bound and unbound insulin from serum samples was optimized. Specificity and binding capacity of the protein G and antibody-bound beads were evaluated and optimized. The linear region of the total and IgG IAB assays was determined using serum samples containing high levels of insulin antibodies. The limit of quantitation, limit of detection, and precision for all the assays were also determined.
authors.affiliation: Esoterix Incorporated, Calabasas, California, USA.
authors.forename: MichaeI
authors.initials: M
authors.lastname: Moxness
authors.forename: Jim
authors.initials: J
authors.lastname: Foley
authors.forename: Mark
authors.initials: M
authors.lastname: Stene
authors.forename: Deborah
authors.initials: D
authors.lastname: Finco-Kent
authors.forename: Vahe
authors.initials: V
authors.lastname: Bedian
authors.forename: Alan
authors.initials: A
authors.lastname: Krasner
authors.forename: Thomas
authors.initials: T
authors.lastname: Kawabata
mesh_terms: D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood*; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology*; D007328:Insulin / Q000276:immunology*; D011869:Radioligand Assay
chemical_list: D001323:Autoantibodies; D007136:Immunoglobulins; D007328:Insulin