Insulin In Human Serum Research Articles

Label-free Aptasensor for the Ultrasensitive Detection of Insulin Via a Synergistic Fluorescent Turn-on Strategy Based on G-quadruplex and AIEgens.

Insulin, the only hormone regulating blood glucose level, is strongly associated with diabetes and its complications. Specific recognition and ultrasensitive detection of insulin are of clinical significance for the early diagnosis and treatment of diabetes. Inspired by aggregation-induced emission, we presented a turn-on label-free fluorescence aptasensor for insulin detection. Quaternized tetraphenylethene salt was synthesized as the fluorescence probe. Guanine-rich aptamer IGA3 was selected as recognition element. Graphene oxide was chosen as the quencher. Under optimized conditions, the fluorescence aptasensor displayed a wide linear range (1.0pM-1.0μM) with a low limit of detection (0.42pM). Furthermore, the aptasensor was successfully applied to detect insulin in human serum. Spiked recoveries were obtained in the range of 96.06%-104.26%. All these results demonstrated that the proposed approach has potential application in the clinical diagnostics of diabetes.

Colorimetric detection of insulin in human serum using GO/AuNPs/TX-100 nanocomposite.

In this study, graphene oxide/gold nanoparticles/Triton X-100 nanocomposites (GO/AuNPs/TX-100) were synthesized using the sonochemical method and their ability in ultrasound-assisted colorimetric detection of insulin was investigated. The synthesized GO/AuNPs/TX-100 nanocomposites were characterized by UV-visible absorption spectroscopy and TEM analysis. The interaction between the nanocomposites and insulin was observed by both naked eye and optical absorption spectroscopy. The GO/AuNPs/TX-100 nanocomposites displayed apparent color changes (red to blue) and absorption spectra changes (decreasing of the band around 528nm and appearance of a new red-shifted band at 640nm) in presence of insulin. The interaction mechanism of the nanocomposites and insulin was discussed. It is based on the special structure of insulin, that insulin can be easily self-assemble into the GO/AuNP/TX-100 nanocomposites and can also play the role of a bridge between two different GO/AuNPs/TX-100 nanocomposites by peptide chains. The effective parameters for insulin detection were optimized. The colorimetric method was used for quantification of insulin in the range of 2-300ngmL-1 with a detection limit of 0.1ngmL-1. Moreover, the relative standard deviation of the method was 3.1 and 2.7% (n=10) at concentrations of 50 and 200ngmL-1, respectively on the same day and 4.8% at a concentration (200.0ngmL-1) on five consecutive days. The present method was utilized for insulin assay in human blood serums with satisfactory results.

Transplantation of human dental pulp stem cells in streptozotocin-induced diabetic rats.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease caused by the destruction of pancreatic β-cells. Human dental pulp stem cells represent a promising source for cell-based therapies, owing to their easy, minimally invasive surgical access, and high proliferative capacity. It was reported that human dental pulp stem cells can differentiate into a pancreatic cell lineage in vitro; however, few studies have investigated their effects on diabetes. Our study aimed to investigate the therapeutic potential of intravenous and intrapancreatic transplantation of human dental pulp stem cells in a rat model of streptozotocin-induced type 1 diabetes. Forty Sprague Dawley male rats were randomly categorized into four groups: control, diabetic (STZ), intravenous treatment group (IV), and intrapancreatic treatment group (IP). Human dental pulp stem cells (1 × 106cells) or vehicle were injected into the pancreas or tail vein 7days after streptozotocin injection. Fasting blood glucose levels were monitored weekly. Glucose tolerance test, rat and human serum insulin and C-peptide, pancreas histology, and caspase-3, vascular endothelial growth factor, and Ki67 expression in pancreatic tissues were assessed 28days post-transplantation. We found that both IV and IP transplantation of human dental pulp stem cells reduced blood glucose and increased levels of rat and human serum insulin and C-peptide. The cells engrafted and survived in the streptozotocin-injured pancreas. Islet-like clusters and scattered human dental pulp stem cells expressing insulin were observed in the pancreas of diabetic rats with some difference in the distribution pattern between the two injection routes. RT-PCR analyses revealed the expression of the human-specific pancreatic β-cell genes neurogenin 3 (NGN3), paired box 4 (PAX4), glucose transporter2 (GLUT2), and insulin in the pancreatic tissues of both the IP and IV groups. In addition, the transplanted cells downregulated the expression of caspase-3 and upregulated the expression of vascular endothelial growth factor and Ki67, suggesting that the injected cells exerted pro-angiogenetic and antiapoptotic effects, and promoted endogenous β-cell replication. Our study is the first to show that human dental pulp stem cells can migrate and survive within streptozotocin-injured pancreas, and induce antidiabetic effects through the differentiation and replacement of lost β-cells and paracrine-mediated pancreatic regeneration. Thus, human dental pulp stem cells may have therapeutic potential to treat patients with long term T1DM.

Microliter Sample Insulin Detection Using a Screen-Printed Electrode Modified by Nickel Hydroxide.

The monitoring of insulin, which is the only hormone that helps regulate blood glucose levels in the body, plays a key role in the diagnosis and treatment of diabetes. However, most techniques today involve complicated electrode fabrication and testing processes, which are time-consuming and costly, and require a relatively large volume of sample. To overcome these drawbacks, we present here a low-cost insulin detection method based on a screen-printed electrode (SPE) modified by nickel hydroxide (Ni(OH)2). This novel method only requires 300 μL of insulin sample, and the time it takes for electrode preparation is about 12 times shorter than traditional electrode fabrication methods such as coating and sol–gel methods. The electrochemical behaviors of the Ni(OH)2-coated SPE (NSPE) sensing area in insulin aqueous solutions are studied using cyclic voltammetry, amperometric i–t curves, and electrochemical impedance spectroscopy. The results demonstrate that the NSPE sensing surface has excellent detection properties, such as a high sensitivity of 15.3 μA·μM–1 and a low detection limit of 138 nM. It takes a short time (∼10 min) to prepare the NSPE sensing surface, and only two drops (∼300 μL) of insulin samples are required in the detection process. Moreover, the selectivity of this method for insulin detection is verified by detecting mixtures of insulin and ascorbic acid or bovine hemoglobin. Finally, we discuss the potential clinical applications of this method by detecting various concentrations of insulin in human serum.

Validation of the ADVIA Centaur® XP system for the determination of insulin and its application

In this study, a direct chemiluminescent immunoassay for the determination of human serum insulin levels using the ADVIA Centaur® XP system was validated. Dilution recovery, linearity, precision, sensitivity, between analyzer variation, reference interval and stability were analyzed. The linear range of the insulin assay was from 0.64 to 277.27 mU/L. Intra- and inter-assay coefficients of variation were 3.67–7.96% and 4.66–8.69%, respectively. The lower and upper limits of quantification were 0.61 mU/L and 8872.64 mU/L, respectively. In terms of between analyzer variation, our study showed comparable results with a good correlation of r2 = 0.9934. The human serum insulin reference interval was in the range of 3.0–25.0 mU/L. Serum insulin can be kept for 7 days between 2-8 °C and 18–26 °C, and the corresponding results for −20 °C and −70 °C were 1 month and 6 months are reported. We proved that this insulin assay was robust and the analytical performance met the requirements. We successfully applied this insulin assay to a bioequivalence study of miglitol in 48 healthy Chinese subjects. The miglitol bioequivalence study was evaluated based on pharmacokinetic and pharmacodynamic parameter endpoints. The results demonstrated that the test formulation and the reference formulation were bioequivalent.

Facile Synthesis of Cu2O@TiO2-PtCu Nanocomposites as a Signal Amplification Strategy for the Insulin Detection.

Novel ultrasensitive sandwich-type electrochemical immunosensor was proposed for the quantitative detection of insulin, a representative biomarker for diabetes. To this end, molybdenum disulfide nanosheet-loaded gold nanoparticles (MoS2/Au NPs) were used as substrates to modify bare glassy carbon electrodes. MoS2/Au NPs not only present superior biocompatible and large specific surface area to enhance the loading capacity of primary antibody (Ab1) but also present good electrical conductivity to accelerate electron transfer rate. Moreover, the amino-functionalized cuprous oxide decorated with titanium dioxide octahedral composites (Cu2O@TiO2-NH2) were prepared to load dendritic platinum-copper nanoparticles (PtCu NPs) to realize signal amplification strategy. The resultant nanocomposites (cuprous oxide decorated with titanium dioxide octahedral loaded dendritic platinum-copper nanoparticles) demonstrate uniform octahedral morphology and size, which effectively increases the catalytically active sites and specific surface area to load the secondary antibody (Ab2), even increases conductivity. Most importantly, the resultant nanocomposites possess superior electrocatalytic activity for hydrogen peroxide (H2O2) reduction, which present the signal amplification strategy. Under the optimal conditions, the proposed immunosensor exhibited a linear relationship between logarithm of insulin antigen concentration and amperometric response within a broad range from 0.1 pg/mL to 100 ng/mL and a limit detection of 0.024 pg/mL. Meanwhile, the immunosensor was employed to detect insulin in human serum with satisfactory results. Furthermore, it also presents good reproducibility, selectivity, and stability, which exhibits broad application prospects in biometric analysis.

Sensitive colorimetric assay using insulin G-quadruplex aptamer arrays on DNA nanotubes coupled with magnetic nanoparticles.

Described here is a methodology for fabrication of a sensitive colorimetric nanoassay for measurement of insulin using G-quadruplex aptamer arrays on DNA nanotubes (DNTs) coupled with magnetic nanoparticles. The spectroscopic findings (e.g. visible spectra, velocity assay and limit of detection determination) indicated a highly sensitive performance of this new nanoassay in comparison to those results obtained from the insulin assay with non-arrayed aptamers. The clinical performance statistics (i.e. paired sample t-test, Bland–Altman plot and scatter diagram) from the newly developed assay and the enzyme-linked immunosorbent assay suggested its reliable precision and its acceptable repeatability for measurement of insulin in human sera. This is, to our knowledge, the first study for the application of magnetic nanoparticle-coupled DNTs for carrying G-quadruplex aptamers for detection of biomolecules (such as insulin) in human serum.

Functionalized gold and persistent luminescence nanoparticle-based ratiometric absorption and TR-FRET nanoplatform for high-throughput sequential detection of l-cysteine and insulin.

In vitro diagnostic is a crucial component of healthcare systems for early diagnosis of diseases and follow-up therapy, which generally makes clinical diagnosis faster, easier, and painless for patients. Herein, we report a dual-signaling nanoplatform for ratiometric absorption and time-resolved fluorescence resonance energy transfer based on l-cysteine-mediated aggregated gold nanoparticles and long afterglow nature of persistent luminescence nanoparticles. With this nanoplatform, we have achieved high-throughput sequential detection of l-cysteine and insulin in human serum without matrix interference. The proposed nanoplatform shows excellent linearity and precision for the determination of l-cysteine in the range of 10 nM to 5.5 μM with the limit of detection (LOD) of 2.2 nM and for the detection of insulin in the range of 12 pM to 3.44 nM with LOD of 2.06 pM. The developed dual-signaling nanoplatform has been successfully applied for the sequential determination of l-cysteine and insulin in human serum.

Isotope-dilution liquid chromatography-tandem mass spectrometry for sensitive quantification of human insulin in serum using derivatization-technique

An isotope-dilution mass spectrometry (IDMS) method for measuring insulin levels in human serum was developed using C-terminal-derivatization method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The carboxyl groups of Glu-C-cleavage products were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity and IDMS quantification, resulting in increases in LC-MS/MS peak areas of derivatized Glu-C-cleavage products of human insulin by ∼23-(A5–17 peptide) to 49-fold(B14–21 peptide), respectively, as compared with results observed in the absence of derivatization. Separation was achieved on a C18 column by gradient elution at 0.3 mL/min, with a mobile phase composed of 0.1% formic acid in acetonitrile and water. Validation studies of target peptides (B1–13 peptide and B14–21 peptide) revealed a linear response in the range of 0.05 ng/mL to 10 ng/mL (regression coefficient, r2 = 0.9987 and 0.9988, respectively), a relative standard deviation within and between days of <8.6%, and spike and recovery test results indicating mean recoveries ranging from 100.2% to 106.6%. Comparison with an established commercial immunoassay showed high correlation (r2 = 0.9943 and 0.9944, B1–13 peptide and B14–21 peptide, respectively) at serum concentrations of between 0.20 ng/mL and 1.51 ng/mL. These findings suggested that this IDMS-based approach was able to quantify human serum insulin with high sensitivity and precision in the reference interval and indicated a potential for determining serum-insulin reference-measurement procedures to allow traceable measurement.

Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells into Insulin-Producing Cells: Evidence for Further Maturation In Vivo.

The aim of this study was to provide evidence for further in vivo maturation of insulin-producing cells (IPCs) derived from human bone marrow-derived mesenchymal stem cells (HBM-MSCs). HBM-MSCs were obtained from three insulin-dependent type 2 diabetic volunteers. Following expansion, cells were differentiated according to a trichostatin-A/GLP protocol. One million cells were transplanted under the renal capsule of 29 diabetic nude mice. Blood glucose, serum human insulin and c-peptide levels, and glucose tolerance curves were determined. Mice were euthanized 1, 2, 4, or 12 weeks after transplantation. IPC-bearing kidneys were immunolabeled, number of IPCs was counted, and expression of relevant genes was determined. At the end of in vitro differentiation, all pancreatic endocrine genes were expressed, albeit at very low values. The percentage of IPCs among transplanted cells was small (≤3%). Diabetic animals became euglycemic 8 ± 3 days after transplantation. Thereafter, the percentage of IPCs reached a mean of ~18% at 4 weeks. Relative gene expression of insulin, glucagon, and somatostatin showed a parallel increase. The ability of the transplanted cells to induce euglycemia was due to their further maturation in the favorable in vivo microenvironment. Elucidation of the exact mechanism(s) involved requires further investigation.

Development of an improved time-resolved fluoroimmunoassay for simultaneous quantification of C-peptide and insulin in human serum

ObjectivesDiabetes mellitus is a chronic disease affecting millions of people globally and resulting in significant death rates each year. A fast, inexpensive alternative to traditional testing and monitoring techniques is desirable, since secretion of insulin and C-peptide is impaired in diabetes mellitus. Design and methodsA highly sensitive immunoassay was developed for the simultaneous measurement of C-peptide and insulin levels in human serum, utilizing dual-label time-resolved fluoroimmunoassay (TRFIA) and magnetic particle technologies. This assay was characteristic for a single-step sandwich-type immunoassay, wherein antibody-coated magnetic particles were used as the solid phase and Eu3+ and Sm3+ chelate labels were used for detection. ResultsAntibody-coated magnetic particles in a TRFIA format performed well in addressing a number of quantitative needs. ConclusionsThe results of this assay correlated well with commercial chemiluminescence assays and provided a number a advantages, including reduced sample volume, reduced reagent and personnel costs and reduced assay time, while maintaining the required clinical sensitivity.

An electrochemical mass sensor for diagnosing diabetes in human serum

An electrochemical mass sensor for clinically relevant detection of insulin in human serum conjugated to magnetic nanoparticles and captured onto antibody immobilized gold coated quartz resonators is reported for the first time.

β-Cell Regeneration Mediated by Human Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) have been shown to ameliorate diabetes in animal models. The mechanism, however, remains largely unknown. An unanswered question is whether BMSCs are able to differentiate into β-cells in vivo, or whether BMSCs are able to mediate recovery and/or regeneration of endogenous β-cells. Here we examined these questions by testing the ability of hBMSCs genetically modified to transiently express vascular endothelial growth factor (VEGF) or pancreatic-duodenal homeobox 1 (PDX1) to reverse diabetes and whether these cells were differentiated into β-cells or mediated recovery through alternative mechanisms. Human BMSCs expressing VEGF and PDX1 reversed hyperglycemia in more than half of the diabetic mice and induced overall improved survival and weight maintenance in all mice. Recovery was sustained only in the mice treated with hBMSCs-VEGF. However, de novo β-cell differentiation from human cells was observed in mice in both cases, treated with either hBMSCs-VEGF or hBMSCs- PDX1, confirmed by detectable level of serum human insulin. Sustained reversion of diabetes mediated by hBMSCs-VEGF was secondary to endogenous β-cell regeneration and correlated with activation of the insulin/IGF receptor signaling pathway involved in maintaining β-cell mass and function. Our study demonstrated the possible benefit of hBMSCs for the treatment of insulin-dependent diabetes and gives new insight into the mechanism of β-cell recovery after injury mediated by hBMSC therapy.

Quantification of human serum insulin concentrations in clinical pharmacokinetic or bioequivalence studies: what defines the “best method”?

Historically, quantitative clinical diagnostic assays (QCDAs) have not been accepted for use in pharmacokinetic or bioequivalence studies because they do not fully comply with the US Food and Drug Administration (FDA) Guidance for Industry: Bioanalytical Method Validation (e.g., full calibration curve not generated with each analytical run). Samples from a bioequivalence study were analysed for insulin and C-peptide concentrations with QCDAs and guidance-conforming radioimmunoassays (RIAs) and the results compared across and within assays. Serum samples (n=1913) from study MKC-TI-142 were analysed first using the Roche E170 electrochemiluminescence immunoassay (ECLIA) for insulin concentration and the Immulite 2000 chemiluminescence immunoassay (CLIA) for C-peptide, and then using the corresponding Millipore RIAs. The insulin assays were highly correlated: r2=0.92 excluding samples requiring dilution and R2=0.88 including all samples. There was increasing negative bias of the ECLIA compared with the RIA with increasing insulin, especially with samples that required dilution for the RIA. The ECLIA had significantly fewer below-quantifiable-limit samples, a larger dynamic analysis range without dilution, and tighter agreement within incurred sample reanalysis (ISR) as compared with the RIA. The C-peptide assays showed good agreement but the CLIA method produced ISR results that were in closer agreement with the original values. The science indicates that the QCDAs are appropriate for the quantification of serum insulin (ECLIA) and C-peptide (CLIA) concentrations in human pharmacokinetic and bioequivalence studies even though the calibration curve is not generated in each analytical run.

In vivo non-viral gene delivery of human vascular endothelial growth factor improves revascularisation and restoration of euglycaemia after human islet transplantation into mouse liver.

Delivery of the gene for human vascular endothelial growth factor (VEGF, also known as VEGFA) to both the transplanted islets and the surrounding tissue may promote islet revascularisation and survival. We previously showed the effective delivery of VEGF gene to rat myocardium by an ultrasound-mediated gene-transfer method named ultrasound-targeted microbubble destruction (UTMD). Here we examined the effect of non-viral VEGF delivery using UTMD on transplanted islets in vivo. A marginal number of human islets were transplanted into livers of mice which were a model for diabetes. Then, non-viral plasmid vectors encoding VEGF (VEGF group, n = 11) or the gene for green fluorescent protein (GFP) (GFP group, n = 7) were introduced into the host liver by UTMD. Transplantation without gene delivery was performed as a control (no-UTMD group, n = 8). Blood glucose, serum human insulin, C-peptide levels and the revascularisation in graft islets were evaluated. Restoration of euglycaemia occurred in 13% in the no-UTMD group and 14% in the GFP group, whereas 73% mice in the VEGF group became euglycaemic at day 30 (p < 0.05 in no-UTMD vs VEGF). Serum human insulin and C-peptide were significantly higher in the VEGF group at day 32 (insulin: no-UTMD, 17 +/- 8; GFP, 37 +/- 17; VEGF, 109 +/- 26 pmol/l, respectively, p < 0.05; C-peptide: no-UTMD, 68 +/- 38; GFP, 115 +/- 58; VEGF, 791 +/- 230 pmol/l, respectively, p < 0.05). Vessel density in graft islets was significantly higher in the VEGF group (no-UTMD, 169 +/- 36; GFP, 227 +/- 39; VEGF, 649 +/- 51 counts/mm(2), respectively, p < 0.05). Delivery of VEGF gene to host liver using UTMD promoted islet revascularisation after islet transplantation and improved the restoration of euglycaemia.

Measurement of Insulin Immunoreactivity in Human Plasma and Serum

Featured article: Andersen L, Dinesen B, Jorgensen PN, Poulsen F, Roder ME. Enzyme immunoassay for intact human insulin in serum or plasma. Clin Chem 1993;39:578–82.1 Our 1993 report described a monoclonal antibody–based ELISA for insulin that had greater specificity than existing RIAs. This specificity allowed determination of intact insulin only, without comeasurement of proinsulin and the conversion intermediate des(31,32)-proinsulin. In patients with type 2 diabetes or with high concentrations of insulin precursors in the blood, the assay measured intact insulin immunoreactivity from plasma and serum samples. The assay was also suitable for large-scale studies. Immunochemical methods for estimating insulin concentrations have been used for 50 years. In 1959 Berson and Yalow introduced the RIA method for the determination of insulin (1), which led to a Nobel Prize in 1977. Competitive insulin RIAs based on polyclonal antibodies had many limitations, however, including limited stability of the radioactive isotope, limited amounts of the antibodies, and unspecific determination of insulin precursors and insulin degradation products together with insulin. The assays were laborious for large-scale use. The discovery of monoclonal antibodies made it possible to produce potentially unlimited amounts of …

The investigation on the reference range and measurement stability of human serum insulin

Objective To establish the reference range of serum human serum insulin levels in our laboratory and investigate measurement stability, Methods The automated Architect i2000 chemiluminescence immunoassay, calibrator and quality control materials were used to measure concentration of serum insulin. Totally 413 cases of healthy adults were enrolled. The physiologic characteristics and the influence of laboratory markers on insulin levels were analyzed. Then the reference range in our laboratory was established. The thermal stability of insulin at different temperatures was investigated. Results Within-run imprecision and between-day imprecision of the automated Architect i2000 insulin assay were 1.67% and 2. 6%, which indicated that this system had good analytical performance. The subjects were classified into 4 groups according to age range ( 10 years). There was no significant difference among four age groups (the median is 5. 6, 5.2, 5.3 and 5. 7 mU/L) (X~2 = 1. 929,P >0.05). However, the insulin levels were higher in female ( median = 5.7 mU/L) than male ( median = 5.0 mU/L) ( Z = 3.696, P < 0.01 ). The reference range was established based on the 2. 5 and 97.5 percentile values. The reference range of insulin for female was 2. 6-11.8 mU/L and 2. 3-11.6 mU/L for men, which differed from that of other insulin assay. Insulin had a positive correlation with BMI (r =0. 115 ,P =0. 019) and triglyeeride (r =0.143 ,P = 0. 004), and a negative correlation with high density lipoprotein (r = -0.179, P = 0.000). Insulin was stable at 25 ℃ for at least 4 hours or 4 ℃ for 24 hours or could be extended to at least 7 days when stored at -20 ℃. Conclusion Gender is an important physiologic characteristic which has an impact on human serum insuhn level. Insulin reference range should be established according to gender. Insulin immunoreactivity was not very stable at different temperatures. Key words: Insulin; Reference range; Immunoassay

Treatment of type 1 diabetes by transplantation of bone-derived mesenchymal stem cells expressing human insulin gene: experiment with mice

To evaluate the effect of treatment of type 1 diabetes by transplantation of bone-derived stem cells expressing human insulin gene. Murine bone marrow-derived stem cells expressing green fluorescent protein (GFP-mMSCs) were isolated from 4/6-week-old GFP mice and transfected with a recombinant retrovirus-murine stem cell virus (MSCV) encoding human insulin gene, thus constructing the GFP-mMSCs-MCV-insulin. 16 C57BL/6J mice were injected with streptozotocin so as to establish models of type 1 diabetes and then randomly divided into 4 equal groups: Group A, undergoing injection into the liver with GFP-mMSC-MCV-insulin 1 week after the establishment of the model, Group B, undergoing intrahepatic transplantation of the GFP-mMSCs transfected with blank vector, Group C, undergoing intrahepatic transplantation of untransfected GFP-mMSCs, and Group D, undergoing intrahepatic transplantation of phosphate-buffered saline (PBS). Another 4 normal mice were used as controls and underwent intrahepatic transplantation of PBS too. After the transplantation the blood glucose, serum insulin, and body weight were detected everyday. 6 weeks later immunohistochemistry was used to detect the expression of human insulin in the mice liver tissues. The body weight of Group A increased by 6% within 6 weeks after treatment, and the average blood glucose level 7 d and 42 d after transplantation were (10.4 +/- 2.8) mmol/L and (6.5 +/- 0.9) mmol/L respectively, both significantly lower than those of Group D [(26.8 +/- 2.5) mmol/L and (25.4 +/- 4.1) mmol/L respectively, both P < 0.05]. Immunohistochemistry showed secretion of human insulin in serum and liver. The clinical manifestations of diabetes can be relieved effectively by intrahepatic transplantation of mMSCs expressing human insulin gene. This study implies a novel approach of gene therapy for type 1 diabetes.

Fully automated chemiluminescence immunoassay of insulin using antibody-protein A-bacterial magnetic particle complexes.

We report a fully automated sandwich immunoassay for the determination of human insulin using antibody-protein A-bacterial magnetic particle (BMP) complexes and an alkaline phosphatase-conjugated secondary antibody. BMPs bearing protein A-MagA inserted on the external surface of the membrane were prepared in the Magnetospirillum sp. AMB-1 transconjugant for a protein A-magA fusion gene. MagA protein was used as an anchor to attach protein A onto the membrane. Protein A-BMP complexes harvested from transconjugant AMB-1 were subsequently complexed with anti-human insulin antibodies by specific binding between the Z domain of protein A and the Fc component of IgG to form the antibody-protein A-BMP complexes. The complexes were quite monodisperse after the binding of the antibody. The BMPs' monodispersity resulted in high signal and low noise in the immunoassay. The luminescence intensity ((kilocounts/s)/microg of antibody) from antibody-protein A-BMP complexes after immunoreaction was higher than that from BMPs chemically conjugated to an antibody. This was explained by a difference in dispersion. The fully automated sandwich immunoassay system using antibody-protein A-BMP complexes made possible precise assays of human insulin in serum.

Enzyme immunoassay for intact human insulin in serum or plasma

We describe an enzyme-linked two-site immunoassay for quantitation of intact insulin in human serum and plasma. The method uses two murine monoclonal antibodies that bind to two different epitopes on the insulin molecule. The immunoassay is specific. Human proinsulin is not bound by the antibodies, and the binding of partially processed proinsulin intermediates is believed to be of minor clinical importance. The relative response of human, bovine, and porcine insulin is 1, 1, and 3, respectively. The assay is sensitive (detection limit 5 pmol/L), accurate (101% recovery with 50 pmol/L insulin added to samples, 95% with 100 pmol/L, and 89% with 300 pmol/L), and fast (results within 3 h), and has a high analytical capacity (done in microtiter plates). The working assay range selected is 5-600 pmol/L, corresponding to a clinically useful range. Because of its specificity, this two-site immunoassay gives results that are lower than those obtained by using a competitive radioimmunoassay, both in normal individuals and in non-insulin-dependent diabetics.