title: Use of insulin immunoassays in clinical studies involving rapid-acting insulin analogues: Bi-insulin IRMA preliminary assessment.
abstract: BACKGROUND
  In clinical studies involving rapid-acting analogues (RAAs), insulin immunoreactivity is frequently measured, including endogenous, regular insulin (RI) and RAA immunoreactivities. Such a procedure implies equivalent cross-reactivities of all insulins present in serum. Commercially available human insulin immunoassays have been widely used, but their limitations (including hemolysis and anti-insulin antibodies) were not fully investigated. The aims of our study were to compare cross-reactivities of RI and RAAs in buffer and in serum and to investigate insulin immunoassay pitfalls.
  
  
  METHODS
  Cross-reactivities were assessed using Bi-insulin IRMA (Schering Cis-Bio International) in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA) and in pools of sera spiked with RI and RAAs (lispro and aspart). To investigate the influence of hemolysis, a pool of sera spiked with RAA was mixed with a concentrated hemolysate (final hemoglobin concentration 10 g/L) and incubated for 3 h at room temperature. To determine interference by anti-insulin antibodies, insulin was removed using charcoal from 18 sera with anti-insulin antibodies and from 17 sera without detectable anti-insulin antibodies. These insulin-free samples were then spiked with RI and RAAs and the immunoreactivity was determined.
  
  
  RESULTS
  Compared with buffer, cross-reactivity in serum for RI, lispro and aspart was lower (35%, 29% and 26% lower, respectively). Hemolysis degraded almost all RI and RAAs contained in the serum (>or=95%). Anti-insulin antibody interference was significant for RI and RAAs (p<or=0.004) and correlated with anti-insulin antibody level in the serum (p<or=0.001).
  
  
  CONCLUSIONS
  In serum, RI and RAA cross-reactivities are slightly lower than in buffer. For RAA assessment, hemolysed samples should be discarded and anti-insulin antibodies should be removed from samples before immunoreactivity measurements.
authors.affiliation: Faculté de Médecine, Université Louis Pasteur, Centre National de la Recherche Scientifique, Unité Mixte de recherche 7004, Institut de Physique Biologique, Strasbourg, France. arnaud.agin@chru-strasbourg.fr
authors.forename: Arnaud
authors.initials: A
authors.lastname: Agin
authors.forename: Nathalie
authors.initials: N
authors.lastname: Jeandidier
authors.forename: Françoise
authors.initials: F
authors.lastname: Gasser
authors.forename: Daniel
authors.initials: D
authors.lastname: Grucker
authors.forename: Rémy
authors.initials: R
authors.lastname: Sapin
mesh_terms: D000906:Antibodies / Q000097:blood / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D006801:Humans; D007004:Hypoglycemic Agents / Q000097:blood / Q000276:immunology; D007328:Insulin / Q000031:analogs & derivatives* / Q000097:blood* / Q000276:immunology; D061267:Insulin Aspart; D061268:Insulin Lispro; D061389:Insulin, Regular, Pork; D011864:Radioimmunosorbent Test / Q000295:instrumentation / Q000379:methods; D011994:Recombinant Proteins / Q000097:blood / Q000276:immunology; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D007004:Hypoglycemic Agents; D007328:Insulin; D061268:Insulin Lispro; D061389:Insulin, Regular, Pork; D011994:Recombinant Proteins; C000006:insulin, neutral; D061267:Insulin Aspart