Abstract
Background
Insulin analogues used in the treatment of insulin-dependent diabetes may result in hypoglycaemia; however, these preparations show variable cross-reactivity in many insulin assays. In order to investigate a suspected insulin overdose, it is therefore essential to characterize the cross-reactivity of these preparations in insulin assays.
Methods
The cross-reactivity of 12 commercial insulin preparations was assessed on the Beckman DxI analyser, by comparison with the insulin calibration curve corresponding to the midpoint of the dose–response curve. This was achieved by manually plotting the relative light units generated by the analyser for each preparation.
Results
All 12 insulin preparations examined showed significant cross-reactivity with the Beckman DxI insulin assay, with 11 of the insulin preparations demonstrating cross-reactivity of 83% or greater. The lowest cross-reactivity was observed with detemir with a value of 47.6%.
Conclusion
When investigating hypoglycaemia it is important to be aware of the cross-reactivity of the recombinant insulin analogues in insulin assays. This study has established the cross-reactivity of 12 preparations in the Beckman DxI insulin assay.
Introduction
The assay of insulin is pivotal to the investigation of the hypoglycaemia. Insulin excess may result from endogenous hypersecretion of insulin or exogenous use of insulin or oral hypoglycaemic drugs. Assays for insulin, however, are optimized to measure the endogenous peptide while recombinant insulin analogues, used in the treatment of insulin-dependent diabetes, have variable cross-reactivity. Owen and Roberts 1 examined the cross-reactivity of three insulin analogues (insulin aspart, insulin glargine and insulin lispro) in five commercial insulin assays, four of which were automated. The study demonstrated that the Beckman Access and Siemens Centaur assays had cross-reactivities of greater than 80% with all three insulin preparations; the Coat-A-Count assay had intermediate values between 27% and 57% while the Roche E170 and Immulite 2000 assays had low cross-reactivity. Examining the Abbott Architect insulin assay Moriyama and co-workers found that aspart, lispro and glargine all had cross-reactivities greater than 70% while the TOSOH II (IRI) insulin assay had greater than 100% recovery with aspart and lispro but <25% with glargine. 2 More recently, criticism has been made of commercial insulin assays, which now dominate the methods currently participating in the United Kingdom National External Quality Assessment Service (UK-NEQAS) insulin scheme. 3 Actrapid was found to show low cross-reactivity in seven insulin assays, of which five were automated methods. In view of these observations, the present study was performed to examine the cross-reactivity of 12 recombinant insulin preparations with the Beckman DxI automated immunoassay. This is a paramagnetic particle, chemiluminescent one-step (‘sandwich’) immunoassay.
Methods
The cross-reactivity of 12 synthetic insulin preparations in the Beckman DxI insulin assay was examined. These were as follows: Actrapid (Novo Nordisk, Crawley, West Sussex), Humulin S (Lilly, Basingstoke, Hampshire), insulin aspart; NovoRapid (Novo Nordisk), insulin lispro; Humalog (Lilly), insulin glargine; Lantus (Aventis Pharma, Guilford, Surrey), isophane insulin; Insulatard (Novo Nordisk, Guilford, Surrey), biphasic insulin lispro; Humalog Mix 25 and Humalog Mix 50 (Lilly, Guilford, Surrey), biphasic isophane insulin; Mixtard 30 (Novo Nordisk), biphasic isophane insulin; Humulin M3 (Lilly), insulin detemir; Levemir (Novo Nordisk), biphasic insulin aspart; and NovoMix 30 (Novo Nordisk). All preparations were obtained at a nominal concentration of 600 μmol/L. The insulin analogues employed in this study were pharmaceutical preparations and as such had other constituents present. These would have had a minimal effect on the insulin assays due to the fact that they were used in dilution.
Dilutions of all recombinant insulins were made in commercially produced stripped serum (Intergen Seracon 11, Serologicals Corporation, Charlottesville, VA, USA). Initially 10-fold dilutions of each preparation were made giving a nominal insulin concentrations of 60 μmol/L, eight further dilutions were then made to give a final nominal concentration of 6 pmol/L. These dilutions were analysed on the Beckman DxI along with the calibration curve. The relative light units (RLUs) for the Beckman standards and the synthetic insulin dilutions were plotted against the concentration for each product. To obtain the percentage cross-reactivity, the mid-curve RLU was first obtained for the Beckman insulin (NIBSC 66/304) standard curve as follows: ([mean RLU of highest Beckman standard − mean RLU of zero standard]/2) + mean RLU of zero standard). The mid-curve RLU was read off of the insulin calibration curve and represents the concentration of insulin at 50% binding. The RLU values for the other insulin curves were compared at this RLU. The % cross-reactivity of insulin analogue = (insulin standard concentration at the mid-curve RLU/concentration of synthetic insulin at mid-curve RLU) × 100.
Results
All of the insulin analogues examined showed significant cross-reactivity in the Beckman DxI insulin assay (see Table 1). The four short-acting preparations (Actrapid, Humulin S, NovoRapid and Humalog), the one long-acting insulin, Lantus, and five of the intermediate-acting compounds (Mixtard 30, Insultard, Humulog Mix 25, NovoMix 30 and Humulog Mix 50) all showed cross-reactivity greater than 100% in the DxI insulin assay. One intermediate-acting analogue, Humulin M3, showed 83.3% cross-reactivity and one long-acting preparation, Detemir 100, showed only 47.6% cross-reactivity. Measured and expected insulin concentrations were similar when insulin analogues were each added to a sample containing endogenous insulin (results not shown).
Cross-reactivities of 12 insulin preparations with the Beckman DxI insulin assay
Conclusion
When investigating a suspected insulin overdose it is vital that the cross-reactivity of the recombinant insulin analogues has been established for the insulin assay to be employed. This study has demonstrated that the Beckman DxI insulin assay has cross-reactivity of 83% or greater with 11 of 12 insulin preparations examined. This confirms and extends the observations of Owen and Roberts, 1 who demonstrated with the Beckman Access insulin assay, that aspart (NovoRapid), lispro (Humalog) and glargine (Lantus) have a high degree of cross-reactivity. In addition, nine further insulin preparations, not previously examined on the Beckman Access assay system, have been tested here. Importantly Actrapid, previously shown to have low cross-reactivity with seven other insulin assays (Seimens Centaur, Dako, Seimens Immulite and Immulite 2000, In-house DELFIA, Roche Elecys and PerkinElmer DELFIA), demonstrated greater than 100% cross-reactivity with the Beckman DxI. 3 Detemir has a fatty acid side chain linked to the amino acid at position B28. This showed lower cross-reactivity than the other commercial insulin preparations with the DxI assay, suggesting that one of the antibodies employed in the assay binds at or near the site of this modification.
Over-recovery of a number of the insulin analogues was observed in this study and, in part, this may be a standardization issue. The Beckman insulin assay is consistently negatively biased on the UK-NEQAS (Guildford) scheme and a study by Rodriquez-Calaleiro et al. 4 demonstrated a 17% negative bias in comparison with isotope dilution liquid chromatography/tandem mass spectrometry. In addition the structure of the insulin analogues may contribute to over-recovery. Lispro and aspart both contain modifications to the B chain (B28 Asp for Pro in aspart; B28: 29 Pro/Lys swapped in lispro) in an area of the insulin molecule thought to be involved in antibody recognition (see results of detemir). Insulatard and Actrapid are both described as recombinant insulin (human sequence); however, the reduced binding of Actrapid in many immumoasays 3 indicates that this molecule is not identical to the standard used for calibration of some insulin assays (e.g. pancreatic insulin 66/304).
In conclusion, the cross-reactivity of 12 commonly used insulin preparations has been established for the Beckman DxI insulin assay. This information is important in the evaluation of patients with a suspected insulin analogue overdose.
DECLARATIONS