Comparing the results of converted data, all three methods provided different insulin concentrations within the same samples. analysis of basal blood and blood samples obtained during a dynamic diagnostic stimulation test (OGT) with SOX18 elevated insulin concentrations. Results Insulin values obtained from the ELISA, RIA and CLIA, investigated for analyses of basal blood Pseudouridimycin samples differed significantly between all three assays. Analyses of samples obtained during dynamic diagnostic stimulation testing with consecutively higher insulin concentrations revealed significantly (=1 7) c RIA and CLIA ( em n /em ?=?20). * em p /em ? ?0.05, ** em p /em ? ?0.01, *** em p /em ? ?0.001 Open in a separate window Fig. 4 Insulin concentrations measured in stimulated blood samples from OGT procedure by ELISA, RIA and CLIA. Data were analyzed by Wilcoxon matched-pairs rank test. a ELISA and RIA ( em n /em ?=?11) b ELISA and CLIA ( em n /em ?=?11) c RIA and CLIA ( em n /em ?=?19). * em p /em ? ?0.05, ** em p /em ? ?0.01, *** em p /em ? ?0.001 Open in a separate window Fig. 5 Scatterplot of measurement results for basal and stimulated samples ELISA compared to a RIA and b CLIA. c Scatterplot of results supplied by CLIA compared to RIA Open in a separate window Fig. 6 Bland-Altman plot of average compared to difference a ELISA minus RIA ( em n /em ?=?28), b ELISA minus CLIA ( em n /em ?=?28) and c CLIA minus RIA ( em n /em ?=?39). em Dashed line /em ?=?bias, em pointed line /em ?=?95?% limits of agreements The average recovery rate of equine insulin in 33 samples after one cycle of refreezing and thawing was 97??17?%, but the corresponding range of recovery stretched from 71 to 158?%. In total, insulin concentrations were significantly ( em p /em ?=?0.0232) lower in the second measurement (54.99??40.95 IU/mL) compared to the first (58.5??43.29 IU/mL). When samples were subdivided into concentration range groups of low (3.51C15.21 IU/mL), medium (30.42C90.09 IU/mL) and high (92.43C125.19 IU/mL) concentrations, the range as well as the mean recovery between the three concentration ranges differed, but there was no statistically significant difference between the first and second measurement when samples were clustered in subgroups depending on concentration. The mean recovery was 98??16?% (range: 79 to 129?%) in the low, 99??24?% (range: 74 to 158?%) in the medium and 92??12?% (range: 71 to 107?%) in the high concentration group. Discussion Most immunoassays used in veterinary medicine for analyzing insulin concentrations in equine serum or plasma samples were originally designed for human diagnostics and research, and specific immunoassays for the quantification of equine insulin are not commercially available. A human-specific insulin radioimmunoassay8 was used in Pseudouridimycin a variety of equine studies [3, 11, 12] in which several IR and ID diagnostic tests and related reference ranges for plasma and serum insulin had been established. Unfortunately, this assay is no longer available, thus, there is an urgent need to reevaluate the reference ranges established on analyses from samples measured with other approaches for measuring insulin. Reproducibility, recovery upon dilution and measuring range of assays The CVs calculated for the ELISA showed that insulin concentration-dependent differences in CVs occurred and that the CVs calculated by the use of equine serum samples were higher than the CVs stated in the manufacturers protocol. Very high or low insulin concentrations could not be detected by all assays due to the various analytical ranges of the assays. Therefore, as samples vary greatly in insulin concentration when obtained in fasted conditions or in dynamic diagnostic tests with induced insulin secretion, selection of an assay with an appropriate analytical range is important for the detection of very high or low insulin concentrations. Moreover, our results show that samples with high insulin concentrations, similar to those that can occur in patients with IR or ID, needed to be diluted in some cases to obtain results. Although the ELISA offered the smallest analytical range of the three assays tested, its excellent RUD results enabled the coverage of a broad range of insulin concentrations. Using Pseudouridimycin this ELISA allowed valid measurements of earlier diluted samples of dynamic diagnostic tests. By contrast, using the RIA, all samples could be measured without dilution in the analytical range of the assay. However, RUD experiments did not provide satisfactory results. In a substantial amount of samples, too high insulin concentrations were obtained for the diluted samples Pseudouridimycin compared to the related undiluted samples. The CLIA offered the widest analytical range when compared to the additional two assays, but also could not measure all high insulin concentration samples. This assay, related.