The cloning and characterization of a human insulin autoantibody in Type I diabetes mellitus.

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Title: The cloning and characterization of a human insulin autoantibody in Type I diabetes mellitus.
Authors: Yurack, Mimi L.
Date: 1993
Abstract: Insulin autoantibodies (IAA) are, by definition, present in an individual who has not received exogenous insulin. The objectives of this project were: (1) to obtain monoclonal IAA-secreting cell lines, (2) to clone and characterize by sequence analysis the variable regions of the light and heavy chains (V$\sb{\rm L}$ and V$\sb{\rm H})$ of a human monoclonal IAA, and (3) to determine the degree of restriction of the V$\sb{\rm L}$ and the V$\sb{\rm H}$ gene usage at the onset of Type I DM. Type I DM patients seropositive for IAA were identified using a competitive insulin autoantibody assay. Their lymphocytes were isolated, immortalized with Epstein-Barr Virus and cloned. Two monoclonal insulin antibody-secreting cell lines were obtained: (1) 1-0.5G1 was obtained from a Type I DM patient treated with exogenous human insulin, and (2) 3-0.5F10 was obtained from a non-insulin treated Type I DM patient; the monoclonal antibody secreted by 3-0.5F10 is therefore an IAA. The V$\sb{\rm L}$ and V$\sb{\rm H}$ cDNAs of the monoclonal IAA secreted by the 3-0.5F10 cell line were cloned and sequenced. The genes were assigned to well defined germ-line elements, the influence of somatic hypermutation in the clonal evolution of IAA was evaluated and the sequences were compared with other antibody sequences. PCR amplification using IAA V$\sb{\rm H}$ and V$\sb{\rm L}$ specific primers followed by Southern analysis using IAA specific probes was performed. All of the Type I DM patients studied at the time of diagnosis had B lymphocytes which were producing antibodies with IAA-homologous V$\sb{\rm L}$ and V$\sb{\rm H}.$ Thus, the usage of IAA-homologous V$\sb{\rm L}$ and V$\sb{\rm H}$ elements in antibodies was found to be restricted in Type I DM patients at the time of diagnosis.
URL: http://hdl.handle.net/10393/6891
http://dx.doi.org/10.20381/ruor-11508
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010
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