Cross-validation of commercial enzyme-linked immunosorbent assay and radioimmunoassay for porcine C-peptide concentration measurements in non-human primate serum.

BACKGROUND: C-peptide concentration is widely used as a marker of insulin secretion and is especially relevant in evaluating islet graft function following transplantation, because its measurement is not confounded by the presence of exogenous insulin. To address the shortage of human islet donors, the use of porcine islets has been proposed as a possible solution and the stringent pig-to-non-human primate (NHP) model is often the most relevant for pre-clinical evaluation of the potential for diabetes reversal resulting from an islet xenograft. The Millipore radioimmunoassay (RIA) was exclusively used to measure porcine C-peptide (PCP) until 2013 when the assay was discontinued and subsequently a commercially available enzyme-linked immunosorbent assay (ELISA) from Mercodia has been widely adopted. Both assays have been used in pre-clinical trials evaluating the therapeutic potential of xenograft products in reversing diabetes in the pig-to-NHP model, to interpret data in a comparable way it may be useful to perform a harmonization of C-peptide measurements. METHODS: We performed a method comparison by determining the PCP concentration in 620 serum samples collected from 20 diabetic cynomolgus macaques transplanted with adult porcine islets. All analyses were performed according to manufacturer instructions. RESULTS: With both assays, we demonstrated an acceptable detection limit, precision, and recovery. Linearity of the ELISA met acceptance criteria at all concentrations tested while linearity of the RIA only met acceptance criteria at five of the eight concentrations tested. The RIA had a detection limit of 0.16 ng/mL, and recovery ranged from 82% to 96% and met linearity acceptance criteria at 0.35 ng/mL and from 0.78 to 2.33 ng/mL. The ELISA had a detection limit of 0.03 ng/mL, and recovery ranged from 81% to 115% and met linearity acceptance criteria from 0.08 to 0.85 ng/mL. Both assays had intra-assay precision <11% and inter-assay precision <14%. PCP concentration measured by ELISA demonstrated a significant correlation with RIA (R2 =.9721, P<.0001). This strong correlation supports use of the regression equation y=2.029x+0.0897 to transform ELISA data to RIA or inversely y=0.4930x-0.0456 to convert RIA data to ELISA for direct comparison between assays in the concentration range of 0-3.0 ng/mL. Measured C-peptide concentration was lower with the ELISA than with the RIA; individual measurements plotted against the averages of the pair demonstrated that the variability from the mean strongly depended on increasing concentration. CONCLUSIONS: Porcine C-peptide can be reliably measured in NHP serum using the Mercodia ELISA, making this assay interchangeable with the Millipore RIA. Inherent differences in antibody affinity and calibration factors may explain the lower ELISA values as compared to the RIA; however without access to a traceable reference standard, it is not possible to determine which assay is most accurate. Regression modeling resulted in a correction factor appropriate for conversion of ELISA data to RIA-equivalent data facilitating comparison of assay results longitudinally and between groups.

Evaluation of commercial ELISA and RIA for measuring porcine C-peptide: implications for research.

BACKGROUND: Pre-clinical demonstration of porcine islet graft function is necessary to support the clinical transplantation of pig islets. C-peptide concentration is an especially useful marker of insulin secretion, because its measurement is not confounded by the presence of exogenous insulin. To measure porcine C-peptide (PCP), researchers in the field exclusively used the Millipore (previously Linco Research) radioimmunoassay (RIA) until 2011, when Mercodia released an alternative enzyme-linked immunosorbent assay (ELISA). (At the end of 2013, the Millipore RIA was withdrawn from the market for commercial reasons.) In our current study, to directly compare these two assays, we performed validation studies on each. We also performed interlaboratory comparisons. Then, to determine the level of agreement between the assays, we analyzed the porcine serum C-peptide concentration measurement results obtained from each assay. METHODS: Using pre-established method validation acceptance criteria, we determined and evaluated the detection limit, sensitivity, precision, linearity, and recovery of the two commercially available PCP assays described above (ELISA and RIA). After validation requirements were met, we performed a method comparison by determining C-peptide concentration in 60 serum samples collected from 31 normal, healthy adult Landrace pigs in the fasting state; a subset underwent an intravenous glucose challenge test, to stimulate the typical physiologic range of C-peptide. All analyses were performed according to manufacturer instructions. To compare the assays, we used Deming regression analysis. RESULTS: Both assays met acceptance criteria. The RIA had a sensitivity of 0.1 ng/ml; it was linear to 2.9 ng/ml. The ELISA had a detection limit of 0.03 ng/ml; it was linear to 1.2 ng/ml. Recovery ranged from 89 to 113% with both assays. The coefficient of variability was 8% in interlaboratory comparisons. Deming regression analysis directly comparing both assays revealed significant correlation between them (before log-transformation: R2=0.9803, P<0.0001; after log-transformation: R2=0.9727, P<0.0001). Measured C-peptide concentration was lower with the ELISA than with the RIA; individual measurements plotted against the averages of the pair demonstrated that the variability from the mean strongly depended on increasing concentration. To transform ELISA data, we used the standard regression equation y=2.191x+0.1119 and the log-transformed regression equation y=0.8101x+0.7502. Both the transformed and the log-transformed (exponential) values approximated the measured RIA levels with a high degree of accuracy in the concentration range of 0 to 1.0 ng/ml. CONCLUSIONS: Porcine C-peptide concentration can be reliably measured in porcine serum samples with either assay (ELISA or RIA). However, the C-peptide results generated by these two assays are not equivalent. Therefore, assay bias must be considered before directly comparing pre-clinical studies that used either of these assays. We determined that harmonization between the assays is appropriate in a specific concentration range. Outside of that range, we do not know whether a linear correction function can be more broadly applied. The variation between the two assays may be related to calibration or reagent factors. To determine which assay is truly more accurate and to effectively compare interlaboratory results, we will need a traceable reference standard.

Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

Serum cytokine profiles in healthy nonhuman primates are blunted by sedation and demonstrate sexual dimorphism as detected by a validated multiplex immunoassay.

Cytokine profiling is a valuable tool for monitoring immune responses associated with disease and treatment. This study assessed the impact of sex and sedation on serum cytokines in healthy nonhuman primates (NHPs). Twenty-three cytokines were measured from serum using a bead-based multiplex assay. Assay validation for precision, sensitivity, recovery, linearity, and stability was performed. Samples from male and female cynomolgus and rhesus macaques either cooperating or sedated were compared. All cytokines except TNFα demonstrated acceptable sensitivity and precision, with variable recovery and linearity. IFNγ, IL-2, IL-5, IL-6, IL-8, IL-12/23 (p40), IL-13, IL-15, MCP-1, TGFα, VEGF met acceptance criteria; G-CSF, IL-4, IL-10, MIP1α, sCD40L were marginal. Higher cytokine levels were observed in females and cytokine levels were blunted in sedated NHPs when compared to awake cooperating NHPs. Significant differences observed in cytokines related to sex, species, or imposed by handling highlight the importance of model design on translational relevance for clinical settings.

Bone resorption in dogs with calcium oxalate urolithiasis and idiopathic hypercalciuria.

People with calcium oxalate (CaOx) urolithiasis and idiopathic hypercalciuria (IH) often have evidence of increased bone resorption, but bone turnover has not previously been investigated in dogs with these conditions. The aim of this study was to determine whether a marker of bone resorption, ß-crosslaps, differs between dogs with CaOx urolithiasis and IH compared to controls. This retrospective, cross-sectional study used a canine specific ELISA to measure ß-crosslaps concentrations in stored frozen serum samples from 20 dogs with CaOx urolithiasis and IH and 20 breed-, sex-, and age-matched stone-free controls (18 Miniature Schnauzers, 14 Bichons Frise, and 8 Shih Tzus). Dogs with CaOx urolithiasis and IH had lower ß-crosslaps concentrations relative to controls (P = .0043), and ß-crosslaps had a moderate negative correlation with urinary calcium-to-creatinine ratios (r = -0.44, P = .0044). Miniature Schnauzers had lower ß-crosslaps concentrations than the other two breeds (P = .0035). The ELISA had acceptable intra-assay precision, but concentrations decreased when samples were repeatedly assayed over time. Assay recovery rates were also below acceptance criteria. In conclusion, Miniature Schnauzers, Bichons Frise, and Shih Tzus with CaOx urolithiasis and IH have evidence of decreased bone resorption compared to stone-free controls. This suggests that other causes of IH, such as intestinal hyperabsorption of calcium, underlie risk for CaOx urolithiasis in these breeds. Results should be confirmed in larger populations and with other ß-crosslaps assays and additional biomarkers of bone turnover. The stability of canine serum ß-crosslaps after freeze-thaw cycles and storage at various temperatures requires investigation.

Breed-specific reference intervals for assessing thyroid function in seven dog breeds.

Thyroxine (T4), free T4 (FT4), and thyrotropin (TSH) concentrations were measured in serum from 693 healthy representatives from 7 dog breeds (Alaskan Malamute, Collie, English Setter, Golden Retriever, Keeshond, Samoyed, or Siberian Husky) to determine whether breed-specific reference intervals (RIs) are warranted. Veterinarians reviewed the health history, performed a physical examination, and approved laboratory data for the enrolled dogs. Many purebred dogs had T4 and FT4 concentrations that were at, or below, the lower limits previously determined for non-breed-specific RIs. Mean concentrations of T4, FT4, and TSH varied significantly among breeds. The range of mean concentration of T4 (19.7 nmol/L [1.53 µg/dL] in English Setters to 29.0 nmol/L [2.25 µg/dL] in Keeshonds) and FT4 (12.6 pmol/L [0.98 ng/dL] in English Setters to 20.2 pmol/L [1.57 ng/dL] in Samoyeds) was considerable. Median TSH values ranged from 6.10 mIU/L (0.07 ng/mL; Alaskan Malamute and Golden Retriever) to 17.6 mIU/L (0.26 ng/mL; Collie). Mean T4 and FT4 concentrations were higher in females. Increasing age was associated with decreasing T4 and FT4, and increasing TSH concentration. The substantial ranges across breeds of measures of central tendency (mean, median) for all hormones indicate that breed-specific RIs are warranted. RIs encompassing the central 95% of reference values for all breeds combined, and for individual breeds, were calculated using nonparametric (TSH) and robust (T4, FT4) methods. Use of breed-specific RIs in combination with careful attention to the potential for pre-analytical and analytical variability in test results will improve thyroid function assessment in these breeds.