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Comparison of three different methods for the quantification of equine insulin

View Article: PubMed Central - PubMed

ABSTRACT

Background: Exact analysis of equine insulin in blood samples is the key element for assessing insulin resistance or insulin dysregulation in horses. However, previous studies indicated marked differences in insulin concentrations obtained from sample analyses with different immunoassays. Most assays used in veterinary medicine are originally designed for use in human diagnostics and are based on antibodies directed against human insulin, although amino acid sequences between equine and human insulin differ. Species-specific assays are being used more frequently and seem to provide advantages compared to human-specific assays. The aim of this study was to compare three immunoassays, one porcine-specific insulin enzyme-linked immunosorbent assay (ELISA), advertised to be specific for equine insulin, one porcine-specific insulin radioimmunoassay (RIA) and one human-specific insulin chemiluminescence immunoassay (CLIA), all three widely used in veterinary laboratories for the analysis of equine insulin. Furthermore, we tested their clinical applicability in assessing insulin resistance and dysregulation by analysis of basal blood and blood samples obtained during a dynamic diagnostic stimulation test (OGT) with elevated insulin concentrations.

Results: Insulin values obtained from the ELISA, RIA and CLIA, investigated for analyses of basal blood samples differed significantly between all three assays. Analyses of samples obtained during dynamic diagnostic stimulation testing with consecutively higher insulin concentrations revealed significantly (p < 0.001) lower insulin concentrations supplied by the CLIA compared to the ELISA. However, values measured by ELISA were intermediate and not different to those measured by RIA. Calculated recovery upon dilution, as a marker for assay accuracy in diluted samples, was 98 ± 4 % for ELISA, 160 ± 41 % for RIA and 101 ± 11 % for CLIA.

Conclusions: Our results indicate that insulin concentrations of one sample measured by different methods vary greatly and should be interpreted carefully. Consideration of the immunoassay method and reliable assay-specific reference ranges are of particular importance especially in clinical cases where small changes in insulin levels can cause false classification in terms of insulin sensitivity of horses and ponies.

No MeSH data available.


Related in: MedlinePlus

Linearity upon dilution in porcine-specific insulin ELISA. Linear regression analysis indicates best-fit line of Y = 0.9987 x – 0.7385 (r2 = 0.9994, P = < 0.0001)
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Fig1: Linearity upon dilution in porcine-specific insulin ELISA. Linear regression analysis indicates best-fit line of Y = 0.9987 x – 0.7385 (r2 = 0.9994, P = < 0.0001)

Mentions: In re-validation experiments, intra-assay CV was 4.61 % at low insulin concentrations (2.34 μIU/mL) and 1.91 % at medium insulin concentrations (108.81 μIU/mL) using the equine-specific insulin ELISA. The inter-assay CV was 5.27 %, 3.24 % and 3.17 % for the low (reference value 5.03 μIU/mL insulin), medium (reference value 18.84 μIU/mL insulin) and high (reference value 60.84 μIU/mL insulin) commercial controls, respectively. Inter-assay CVs for equine serum samples were 7.34 % and 4.83 % for low (mean: 10.53 μIU/mL) and high concentrations (mean: 109.98 μIU/mL), respectively. The RUD in the first revalidation experiment was 95 % (range from 94 to 96 %), calculated in five dilution steps. The linearity upon dilution was excellent (r2 = 0.9994; p < 0.0001), showing a strong relationship between the calculated and measured concentrations of the diluted samples (Fig. 1).Fig. 1


Comparison of three different methods for the quantification of equine insulin
Linearity upon dilution in porcine-specific insulin ELISA. Linear regression analysis indicates best-fit line of Y = 0.9987 x – 0.7385 (r2 = 0.9994, P = < 0.0001)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016943&req=5

Fig1: Linearity upon dilution in porcine-specific insulin ELISA. Linear regression analysis indicates best-fit line of Y = 0.9987 x – 0.7385 (r2 = 0.9994, P = < 0.0001)
Mentions: In re-validation experiments, intra-assay CV was 4.61 % at low insulin concentrations (2.34 μIU/mL) and 1.91 % at medium insulin concentrations (108.81 μIU/mL) using the equine-specific insulin ELISA. The inter-assay CV was 5.27 %, 3.24 % and 3.17 % for the low (reference value 5.03 μIU/mL insulin), medium (reference value 18.84 μIU/mL insulin) and high (reference value 60.84 μIU/mL insulin) commercial controls, respectively. Inter-assay CVs for equine serum samples were 7.34 % and 4.83 % for low (mean: 10.53 μIU/mL) and high concentrations (mean: 109.98 μIU/mL), respectively. The RUD in the first revalidation experiment was 95 % (range from 94 to 96 %), calculated in five dilution steps. The linearity upon dilution was excellent (r2 = 0.9994; p < 0.0001), showing a strong relationship between the calculated and measured concentrations of the diluted samples (Fig. 1).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Exact analysis of equine insulin in blood samples is the key element for assessing insulin resistance or insulin dysregulation in horses. However, previous studies indicated marked differences in insulin concentrations obtained from sample analyses with different immunoassays. Most assays used in veterinary medicine are originally designed for use in human diagnostics and are based on antibodies directed against human insulin, although amino acid sequences between equine and human insulin differ. Species-specific assays are being used more frequently and seem to provide advantages compared to human-specific assays. The aim of this study was to compare three immunoassays, one porcine-specific insulin enzyme-linked immunosorbent assay (ELISA), advertised to be specific for equine insulin, one porcine-specific insulin radioimmunoassay (RIA) and one human-specific insulin chemiluminescence immunoassay (CLIA), all three widely used in veterinary laboratories for the analysis of equine insulin. Furthermore, we tested their clinical applicability in assessing insulin resistance and dysregulation by analysis of basal blood and blood samples obtained during a dynamic diagnostic stimulation test (OGT) with elevated insulin concentrations.

Results: Insulin values obtained from the ELISA, RIA and CLIA, investigated for analyses of basal blood samples differed significantly between all three assays. Analyses of samples obtained during dynamic diagnostic stimulation testing with consecutively higher insulin concentrations revealed significantly (p&thinsp;&lt;&thinsp;0.001) lower insulin concentrations supplied by the CLIA compared to the ELISA. However, values measured by ELISA were intermediate and not different to those measured by RIA. Calculated recovery upon dilution, as a marker for assay accuracy in diluted samples, was 98&thinsp;&plusmn;&thinsp;4&nbsp;% for ELISA, 160&thinsp;&plusmn;&thinsp;41&nbsp;% for RIA and 101&thinsp;&plusmn;&thinsp;11&nbsp;% for CLIA.

Conclusions: Our results indicate that insulin concentrations of one sample measured by different methods vary greatly and should be interpreted carefully. Consideration of the immunoassay method and reliable assay-specific reference ranges are of particular importance especially in clinical cases where small changes in insulin levels can cause false classification in terms of insulin sensitivity of horses and ponies.

No MeSH data available.


Related in: MedlinePlus