Development of insulin antibodies and changes in titers over a long-term period in patients with type 2 diabetes Naoki Hattori a, ⁎, Maharani Retna Duhita a , Akira Mukai b , Megumi Matsueda b , Akira Shimatsu c a Department of Pharmaceutical Sciences, Ritsumeikan University, Shiga, Japan b Department of Medicine, Co-op Osaka Hospital, Japan c Clinical Research Institute, National Hospital Organization, Kyoto Medical Center, Kyoto, Japan abstract article info Article history: Received 12 January 2014 Received in revised form 2 March 2014 Accepted 3 March 2014 Available online 16 March 2014 Keywords: Type 2 diabetes Insulin antibodies Glargine Detemir Lispro Aspart Background: Recently, insulin analogs have become widely used for the treatment of diabetes. The aim of this study was to determine differences in the antigenicity of insulin analogs and long-term changes in titers in patients with type 2 diabetes. Methods: Insulin antibodies were examined using polyethylene glycol and protein G methods in 381 patients with type 2 diabetes. Results: Insulin antibodies were detected in 48 of 118 patients (40.7%) who used insulin, and insulin glargine and aspart were more antigenic. Insulin antibodies were unexpectedly found in seven of 263 patients (2.7%) who had never used insulin. Serum insulin concentrations in patients with insulin antibodies were significantly higher than those without them. Two years after the initial evaluation, insulin antibodies were still positive in 92.7% of patients who used insulin; while, they disappeared in all patients who had never used insulin. A patient who stopped insulin injections 6 years ago was found to be positive for insulin antibodies at the first evaluation as well as 2 years later. Conclusions: Insulin glargine and aspart induced insulin antibodies more frequently, and insulin antibodies remained in patients for a long time. Insulin antibodies should be suspected even in patients not currently on insulin therapy. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Insulin antibodies are often produced in patients with diabetes treated with insulin preparations. Recombinant human insulin is less immunogenic than porcine insulin but still produces insulin antibodies in some patients [1]. Insulin antibodies do not usually cause clinical symptoms [1] but there are several case reports concerning insulin re- sistance [2] or hypoglycemia [3]. Insulin antibodies may reduce insulin activity by competing for the insulin receptor or the insulin-antibody complex could work as a pool of insulin, leading to hypoglycemia upon the release of free insulin [1,4]. Fast-acting insulin analogs, such as insulin lispro (Humalog®) and insulin aspart (NovoRapid®), and long-acting basal insulin analogs, such as insulin glargine (Lantus®) and insulin detemir (Levemir®), are widely used for the treatment of diabetes. To facilitate or delay the absorbance of insulin, these analogs contain several modifications to the insulin molecule [5–8]. It is possible that these modifications may increase the antigenicity, leading to the production of insulin antibodies. Several investigations examined the generation of insulin antibodies in patients with diabetes who started treatment with insulin analogs [9–16]. However, there are few reports comparing insulin antibody generation among insulin analogs and examining the changes in titers of insulin antibodies over a long-term period. In the present study, we tested for insulin antibodies in patients using different insulin analogs twice in a 2-year interval. The aim of this study was to evaluate: 1) the prevalence of insulin antibodies in patients with type 2 diabetes using insulin analogs, 2) differences in the antigenicity of the analogs, 3) effects of insulin antibodies on serum insulin levels, and 4) the changes in the insulin antibody titers over a long-term period. We hypothesized that there could be some difference in antigenicity among insulin analogs and that insulin antibodies would persist as long as patients were using insulin. 2. Materials and methods 2.1. Patients Patients with type 2 diabetes who were being followed at the diabe- tes outpatient clinic of Co-op Osaka Hospital in 2011 underwent the first Clinica Chimica Acta 433 (2014) 135–138 Abbreviations: EIA, enzyme immunoassay; GLP-1, glucagon-like peptide-1; PEG, poly- ethylene glycol; NPH insulin, neutral protamine Hagedorn insulin. ⁎ Corresponding author at: Department of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu-city, Shiga 525-8577, Japan. Tel./fax: +81 77 561 2581. E-mail address: hattorin@fc.ritsumei.ac.jp (N. Hattori). http://dx.doi.org/10.1016/j.cca.2014.03.008 0009-8981/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim