Unlabeled Insulin Research Articles

An Investigation into the Acidity-Induced Insulin Agglomeration: Implications for Drug Delivery and Translation.

Insulin undergoes agglomeration with (subtle) changes in its biochemical environment, including acidity, application of heat, ionic imbalance, and exposure to hydrophobic surfaces. The therapeutic impact of such unwarranted insulin agglomeration is unclear and needs further evaluation. A systematic investigation was conducted on recombinant human insulin-with or without labeling with fluorescein isothiocyanate-while preparing insulin suspensions (0.125, 0.25, and 0.5 mg/mL) at pH 3. The suspensions were incubated (37 °C) and analyzed at different time points (t = 2, 4, 24, 48, and 72 h). Transmission electron microscopy and nanoparticle tracking analysis identified colloidally stable (zeta potential 15 ± 5 mV) spherical agglomerates of unlabeled insulin (100-500 nm). Circular dichroism established the preservation of insulin's secondary structure rich in α-helices despite exposure to an acidic environment (pH 3) for 72 h. Furthermore, fluorescence lifetime imaging microscopy illustrated an acidic core inside these spherical agglomerates, while the acidity gradually lessened toward the periphery. Some of these smaller agglomerates fused to form larger chunks with discrete zones of acidity. The data indicated a primary nucleation-driven mechanism of acid-induced insulin agglomeration under physiologically relevant conditions.

Photoacoustic imaging reveals mechanisms of rapid-acting insulin formulations dynamics at the injection site

ObjectiveUltra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog® and Lyumjev® formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. MethodsWe employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. ResultsThrough experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. ConclusionsThis study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and the formulation excipients, which could be useful to make faster and better controlled insulin formulations in the future.

FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer

Fluorescein isothiocyanate-labeled insulin (FITC-insulin) has been widely used for bioanalytical applications. Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature, there is still a need to develop a cost effective, reliable and quick labeling method for insulin. The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer. FITC labeling of insulin gives mono-, di- or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin. Mono-conjugate with unlabeled insulin, mixture of di- and tri-conjugate, and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h. Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer. Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient. However, tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.

The insulin receptor is expressed and functional in cultured blood-brain barrier endothelial cells but does not mediate insulin entry from blood to brain.

Insulin and its receptor are known to be present and functional in the brain. Insulin cerebrospinal fluid concentrations have been shown to correlate with plasma levels of insulin in a nonlinear fashion, indicative of a saturable transport pathway from the blood to the brain interstitial fluid. The aim of the present study was to investigate whether insulin was transported across brain endothelial cells in vitro via an insulin receptor-dependent pathway. The study showed that the insulin receptor was expressed at both the mRNA and protein levels in bovine brain endothelial cells. Luminally applied radiolabeled insulin showed insulin receptor-mediated binding to the endothelial cells. This caused a dose-dependent increase in Akt-phosphorylation, which was inhibited by coapplication of an insulin receptor inhibitor, s961, demonstrating activation of insulin receptor signaling pathways. Transport of insulin across the blood-brain barrier in vitro was low and comparable to that of a similarly sized paracellular marker. Furthermore, insulin transport was not inhibited by coapplication of an excess of unlabeled insulin or an insulin receptor inhibitor. The insulin transport and uptake studies were repeated in mouse brain endothelial cells demonstrating similar results. Although it cannot be ruled out that culture-induced changes in the cell model could have impaired a potential insulin transport mechanism, these in vitro data indicate that peripheral insulin must reach the brain parenchyma through alternative pathways rather than crossing the blood-brain barrier via receptor mediated transcytosis.

Visualization and Quantitative Assessment of the Brain Distribution of Insulin through Nose-to-Brain Delivery Based on the Cell-Penetrating Peptide Noncovalent Strategy.

Our recent work suggested that intranasal coadministration with the cell-penetrating peptide (CPP) penetratin increased the brain distribution of the peptide drug insulin. The present study aimed to distinctly certify the ability of penetratin to facilitate the nose-to-brain delivery of insulin by quantitatively evaluating the distribution characteristics in brain using radioactive (64)Cu-NODAGA-insulin. Autoradiography and analysis using a gamma counter of brain areas demonstrated that the accumulation of radioactivity was greatest in the olfactory bulb, the anterior part of the brain closest to the administration site, at 15 min after intranasal administration of (64)Cu-NODAGA-insulin with l- or d-penetratin. The brain accumulation of (64)Cu-NODAGA-insulin with penetratin was confirmed by ELISA using unlabeled insulin in which intact insulin was delivered to the brain after intranasal coadministration with l- or d-penetratin. By contrast, quantification of cerebrospinal fluid (CSF) samples showed increased insulin concentration in only the anterior portion of the CSF at 15 min after intranasal coadministration with l-penetratin. This study gives the first concrete proof that penetratin can accelerate the direct transport of insulin from the nasal cavity to the brain parenchyma. Further optimization of intranasal administration with CPP may increase the efficacy of delivery of biopharmaceuticals to the brain while reducing the risk of systemic drug exposure.

Labeling Monomeric Insulin with Renal-Clearable Luminescent Gold Nanoparticles.

In the native physiological environment, inorganic nanoparticles (NPs) often induce nonspecific protein adsorption, which could significantly alter the function of the proteins they labeled. As a result, small fluorescent dyes are still widely used in the imaging of proteins in animals due to their minimal interference with protein function. Here, we used monomeric insulin as a model and compared its bioactivity before and after labeling with renal-clearable near-infrared-emitting gold NPs. These NPs were chosen because they have high resistance to serum protein adsorption and low nonspecific accumulation. We have found that a 1:1 insulin-NP ratio can be achieved, where the insulin-NPs show minimal serum protein binding with fully retained bioactivity comparable to that of unlabeled insulin. These results show a proof of concept that renal-clearable NPs can behave like small molecules in protein labeling without changing the individual protein's function, laying down a foundation for in vivo tracking of proteins with multimodality imaging techniques.

Designed Synthesis of Aptamer-Immobilized Magnetic Mesoporous Silica/Au Nanocomposites for Highly Selective Enrichment and Detection of Insulin

We designed and synthesized aptamer-immobilized magnetic mesoporous silica/Au nanocomposites (MMANs) for highly selective detection of unlabeled insulin in complex biological media using MALDI-TOF MS. The aptamer was easily anchored onto the gold nanoparticles in the mesochannels of MMANs with high capacity for highly efficient and specific enrichment of insulin. With the benefit from the size-exclusion effect of the mesoporous silica shell with a narrow pore size distribution (∼2.9 nm), insulin could be selectively detected despite interference from seven untargeted proteins with different size dimensions. This method exhibited an excellent response for insulin in the range 2-1000 ng mL(-1). Moreover, good recoveries in the detection of insulin in 20-fold diluted human serum were achieved. We anticipate that this novel method could be extended to other biomarkers of interest and potentially applied in disease diagnostics.

‘Insulin autoantibody affinity measurement using a single concentration of unlabelled insulin competitor discriminates risk in relatives of patients with type 1 diabetes

Development of high-risk combinations of multiple islet autoantibodies and type 1 diabetes is associated with high-affinity insulin autoantibodies (IAA), but IAA affinity measurements require large serum volumes. We therefore investigated whether a simplified method of IAA affinity measurement using a low concentration of unlabelled insulin (ULI) competitor discriminated between moderate-high- and low-affinity IAA and identified individuals at highest risk of disease. Samples were assayed by radiobinding microassay using high (4·0 × 10(-5) mol/l) and low (7 × 10(-9) mol/l) ULI concentrations for competitive displacement in three cohorts of IAA-positive individuals; (1) 68 patients with newly-diagnosed type 1 diabetes; (2) 40 healthy schoolchildren; and (3) 114 relatives of patients with type 1 diabetes followed prospectively for disease development (median follow-up 13 years). IAA results obtained with low ULI were expressed as a percentage of those obtained with high ULI and this was used to classify samples as low or moderate-high affinity (0-50% and >50%, respectively). Sixty-eight patient samples were positive with high and 67 (99%) with low ULI. Forty schoolchildren were IAA-positive with high and 22 (55%) with low ULI (P < 0·001). Of the relatives, 113 were positive with high and 83 (73%) with low ULI (P < 0·001). In relatives, moderate-high affinity IAA were associated with multiple islet antibodies (P < 0·001) and greater diabetes risk than low affinity IAA (P < 0·001). A single low concentration of ULI competitor can act as a surrogate for complex IAA affinity measurements and identifies those IAA-positive relatives at highest risk of disease progression.

Insulin entry into muscle involves a saturable process in the vascular endothelium

Insulin's rate of entry into skeletal muscle appears to be the rate-limiting step for muscle insulin action and is slowed by insulin resistance. Despite its obvious importance, uncertainty remains as to whether the transport of insulin from plasma to muscle interstitium is a passive diffusional process or a saturable transport process regulated by the insulin receptor. To address this, here we directly measured the rate of (125)I-labelled insulin uptake by rat hindlimb muscle and examined how that is affected by adding unlabelled insulin at high concentrations. We used mono-iodinated [(125)I]Tyr(A14)-labelled insulin and short (5 min) exposure times, combined with trichloroacetic acid precipitation, to trace intact bioactive insulin. Compared with saline, high concentrations of unlabelled insulin delivered either continuously (insulin clamp) or as a single bolus, significantly raised plasma (125)I-labelled insulin, slowed the movement of (125)I-labelled insulin from plasma into liver, spleen and heart (p < 0.05, for each) but increased kidney (125)I-labelled insulin uptake. High concentrations of unlabelled insulin delivered either continuously (insulin clamp), or as a single bolus, significantly decreased skeletal muscle (125)I-labelled insulin clearance (p < 0.01 for each). Increasing muscle perfusion by electrical stimulation did not prevent the inhibitory effect of unlabelled insulin on muscle (125)I-labelled insulin clearance. These results indicate that insulin's trans-endothelial movement within muscle is a saturable process, which is likely to involve the insulin receptor. Current findings, together with other recent reports, suggest that trans-endothelial insulin transport may be an important site at which muscle insulin action is modulated in clinical and pathological settings.

Anti-BSA antibodies are a major cause of non-specific binding in insulin autoantibody radiobinding assays

Insulin autoantibodies (IAA) are usually the first risk-markers detected during the type 1 diabetes prodrome, but precise measurement is difficult as insulin binding is often low. Non-specific binding (NSB) of 125I-labelled insulin necessitates competitive displacement with unlabelled insulin to demonstrate specificity. NSB varies with different batches of label, suggesting that it is caused by impurities in the label. Addition of bovine serum albumin (BSA) can reduce NSB, so we investigated whether BSA antibodies cause lack of specificity in IAA assays.Samples from patients with newly-diagnosed type 1 diabetes, healthy schoolchildren previously found to have raised 125I-insulin binding (≥0.4units) and IAA-negative schoolchildren were re-assayed for IAA by radiobinding microassay using commercial 125I-insulin with and without 1g/dl BSA added to the buffer.Of 100 patients, 68 were IAA-positive on re-assay with BSA compared to 72 without BSA (p=0.125). Of 154 schoolchildren who previously had raised 125I-insulin binding, only 45 had 125I-insulin binding≥0.4units on re-assay with BSA compared to 90 without BSA (p<0.001). Following competitive displacement with unlabelled insulin, 40 were IAA-positive with BSA compared to 48 without BSA (p=0.02). No IAA-negative schoolchildren were IAA-positive on re-assay. Levels of NSB were associated with antibodies binding 125I-BSA and purification of labelled insulin reduced NSB.Addition of BSA to assay buffer improves the screening efficiency of the IAA assay without reducing disease sensitivity in patients. High titre BSA antibodies interfere with IAA measurement because of 125I-BSA present in some insulin labels. Improved purification of insulin labels should obviate the need for competitive displacement.

Molecular imaging analysis of intestinal insulin absorption boosted by cell-penetrating peptides by using positron emission tomography

Molecular imaging technique by use of positron emission tomography (PET) is a noninvasive tool that allows one to quantitatively analyze the function of endogenous molecules and the pharmacokinetics of therapeutic agents in vivo. This technique is expected to be useful for evaluating the effectiveness of diverse drug delivery systems. We demonstrated previously that intestinal insulin absorption is increased significantly by coadministration of cell-penetrating peptides (CPPs), which are taken up effectively by several cells. However, the distribution behavior of insulin whose absorption is increased by CPPs is not clear. We used PET imaging and quantitatively analyzed the intestinal absorption and subsequent distribution of insulin and the effect of CPPs on its absorption and distribution. An unlabeled insulin solution containing tracer insulin, 68Ga-DOTA-insulin, was administered with or without CPPs into a rat ileal closed loop. PET imaging showed that the CPPs, particularly d-R8 and l-penetratin, significantly increased the 68Ga-DOTA-insulin level in the liver, kidney, and circulation. After absorption from the intestine, the 68Ga-DOTA-insulin passed rapidly through the liver and accumulated in the kidney. The increase in the hepatic and renal distribution of 68Ga-DOTA-insulin by each CPP coadministration was similar manner as that in intestinal absorption, suggesting that the increased accumulation of insulin in the liver and kidney induced by coadministration of CPPs was associated with the increased intestinal absorption of insulin. This is the first study to show that PET imaging enables one to quantitatively analyze the distribution behavior of intestinally absorbed insulin in several organs. This imaging methodology is likely to be useful for developing effective drug delivery systems targeted to specific organs.

The Insulin Immunoassay After 50 Years: A Reassessment

In 1960 Berson and Yalow published a method for the radioimmunoassay (RIA) of plasma insulin based on the concept that the extent to which unlabeled insulin displaces labeled insulin from anti-insulin antibody is proportional to the concentration of unlabeled insulin. The RIA for insulin has greatly increased knowledge of the physiology of glucose homeostasis and of the diverse causes of diabetes mellitus. Beyond this, the insight on which the RIA-or, more broadly, the competitive protein-binding assay-is based has provided the means to measure nanomolar or picomolar concentrations of a vast array of compounds in plasma and tissues. Directly or indirectly, the RIA has profoundly affected every branch of medicine. This essay reviews the ideas that were current in the medical research community when Berson and Yalow began their work and the observations and reasoning process that led them to their seminal discovery.

Microfluidic competition assay via equilibrium binding

Competitive binding of analytes in solution, to an immobilized receptor, is a preferred method for quantifying molecules in complex mixtures. We have developed a theoretical and experimental framework for the microfluidic competition assay. A mathematical model describes the transient, convection–dispersion of solutes, undergoing equilibrium binding to immobilized receptors, while entrained in a low Reynolds number incompressible fluid flowing through a microchannel. The proposed method involves monitoring of the elution profile of a reference molecule and ligand in the presence of a competitor. The time difference between the two breakthrough curves provides a measure of the unknown concentration of the competitor. Theoretical results illustrate the general method for determining the equilibrium dissociation constant ( K d) of the ligand and competitor, as well as the competitor concentration. Experimental data is presented for the binding of fluorescein labeled insulin and unlabeled insulin to a monoclonal antibody. It is found that the unlabeled insulin binds with higher affinity ( K d = 0.17 μM) than the labeled insulin ( K d = 0.76 μM). The potential advantages of the method and further improvements in the model are discussed.

Murine High Specificity/Sensitivity Competitive Europium Insulin Autoantibody Assay

Most insulin autoantibody assays for both human and animal models are in a radioassay format utilizing (125)I-insulin, but despite the radioassay format international workshops have documented difficulty in standardization between laboratories. There is thus a need for simpler assay formats that do not utilize radioactivity, yet retain the high specificity and sensitivity of radioassays. To establish an easier enzyme-linked immunosorbent assay (ELISA) for insulin autoantibodies of non-obese diabetic (NOD) mice, we used an ELISA format, competition with unlabeled insulin, europium-avidin, and time-resolved fluorescence detection (competitive europium insulin autoantibody assay). The competitive europium assay of insulin autoantibodies when applied to sera from NOD mice had high sensitivity and specificity (92% sensitivity, 100% specificity) compared to our standard insulin autoantibody radioassay (72% sensitivity, 100% specificity) in analyzing blind workshop sera. It is noteworthy that though the assay has extremely high sensitivity for murine insulin autoantibodies and utilizes human insulin as target autoantigen, human sera with high levels of insulin autoantibodies are not detected. Our results clearly indicate that low levels of insulin autoantibodies can be detected in an ELISA-like format. Combining a europium-based ELISA with competition with fluid-phase autoantigen can be applicable to many autoantigens to achieve high specificity and sensitivity in an ELISA format.

Internalization of colloidal gold-labelled insulin complexes into cultivated human smooth muscle cells.

The binding and internalization of colloidal gold-labelled insulin complexes into cultivated human arterial smooth muscle cells was studied. We could demonstrate that the colloidal gold-labelled insulin complex was bound to specific receptors on the cell surface of smooth muscle cells and internalized after 30 min at 20 degrees C. The gold-labelled insulin complexes entered the cell via vesicles and escaped from them in the cytoplasm. The biological activity of the complexes was determined by measuring their effect on DNA synthesis in smooth muscle cells and incorporation of (14C)-glucose into isolated fat cells. The binding characteristics of the complexes were examined by competitive inhibition of 125I-insulin binding to isolated fat cells in the presence of increasing concentrations of gold-labelled insulin complexes or unlabelled insulin. The gold-labelled insulin complex showed a growth promoting and metabolic effect and was able to compete with 125I-insulin for its binding sites.

Insulin Binding Sites and Action in the Adrenal Glands from Normal and Streptozotocin Diabetic Rats

Insulin binding to rat adrenal glands was studied in vivo by i.v. injection of 125I-insulin either alone or together with an excess of unlabelled hormones (insulin, glucagon, prolactin, or growth hormone). In addition, isolated glands from normal or streptozotocin diabetic rats (STZ) were incubated in vitro with 125I-insulin and varying concentrations of unlabelled insulin. Both experiments showed specific binding sites in the adrenal glands. Furthermore the glands from diabetic rats bound more insulin than the glands from controls. The insulin stimulatory effect on the deoxyglucose (14C-DG) uptake was examined in isolated glands from normal and STZ rats. Adrenal glands from control rats exhibited higher basal values of 14C-DG uptake than glands from STZ rats. Insulin induced a stimulatory effect on the 14C-DG transport in glands from both control and diabetic rats. Adrenal glands from STZ rats responded much earlier to exogenous insulin, however the maximal stimulating concentration of the hormone did not restore the 14C-DG uptake to control values. The lowest concentration of insulin that increased the 14C-DG transport was 3 X 10(-8) M. The adrenal gland must be considered a target organ for insulin by both criteria, insulin specific binding and stimulatory action on the deoxyglucose transport.

Amyloid‐β degradation by insulin‐degrading enzyme is inhibited by nitric oxide

Amyloid beta (Aβ) accumulation is a crucial step in Alzheimer.s disease (AD) development. Insulin‐degrading enzyme (IDE) degrades Aβ. We examined the effect of nitric oxide on IDE‐mediated degradation of Aβ. Partially purified rat liver IDE was treated with NO donors S‐Nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) and sodium nitroprusside (SNP), or control compounds N‐Acetyl‐D‐penicillamine (NAP) and potassium ferricyanide (KFC). IDE activity was measured by trichloroacetic acid precipitation of 125I‐amyloid β. IDE cysteine nitrosylation was measured using the biotin switch assay. Degradation of Aβ was specifically mediated by IDE, as unlabeled insulin inhibited Aβ degradation with an approximate Km 10−7 M. SNP and SNAP inhibited IDE degradation of Aβ by 50% and 25% respectively. KFC and NAP had no effects. As a possible mechanism for the inhibitory effect on Aβ degradation, we found IDE to be nitrosylated by SNAP, indicating a new posttranslational modification of the enzyme. Aβ accumulation in the AD brain contributes to the disease progression and pathology, and IDE dysfunction has been implicated as a possible reason for this accumulation of Aβ. We have shown that increased NO levels, which have been observed in patients with AD, can decrease IDE enzymatic function, potentially resulting in increases in Aβ deposition in the brain and development of AD.Funding: Veterans Affairs Merit Review

Interfacial adsorption of insulin: Conformational changes and reversibility of adsorption

The adsorption of human insulin to Teflon particles was studied with respect to conformational changes and the reversibility of adsorption was examined by total internal reflection fluorescence (TIRF). Adsorption isotherms for the adsorption of human insulin indicated high affinity adsorption, even at electrostatic repulsive conditions. The plateau value for adsorption was in accordance with a protein layer consisting primarily of insulin monomers. Conformational changes of the insulin upon adsorption, was investigated by circular dicroism (CD) and fluorescence spectroscopy. The results suggested unfolding of adsorbed insulin, as observed by a decrease in α-helix and increase in random coil conformation. The changes in protein structure was not only related to the adsorbed species being monomeric, since CD and fluorescence results were different for adsorbed insulin compared to a monomeric analog of human insulin. Furthermore, the thermal stability in the adsorbed state was changed compared to insulin in solution. On the basis of the TIRF studies with FITC-labelled insulin it was not possible to firmly conclude whether exchange between human insulin in the adsorbed state and in solution takes place, due to the limited time range investigated. However, the desorption mechanism appeared to be different with unlabelled insulin in the bulk solution compared to phosphate buffer.

Pharmacokinetics of insulin uptake by ocular tissues and the role of cerebrospinal fluid in optic nerve insulin accumulation following topical insulin application

As part of our ongoing studies concerning the efficacy of using topically applied medications to treat retinal and optic nerve diseases, we previously showed that insulin accumulated in the retina, optic nerve, and cerebrospinal fluid (CSF) following topical application. The purpose of this study was to investigate which route insulin takes to get to the posterior segment of the eye, and to specifically assess the role of the CSF in optic nerve insulin accumulation. Lewis rats that received 125I-insulin eye drops were killed at different time points and their ocular tissues counted in a gamma counter. In order to determine whether elevated levels of CSF insulin could lead to optic nerve insulin accumulation, a separate cohort of animals was injected in their lumbar cistern with unlabeled insulin and their optic nerves later assessed for the presence of insulin by enzyme-linked immunosorbent assay. All ocular tissues (except the lens) showed at least one significant time point elevation in 125I-insulin compared to baseline. Both the combined cornea/iris/ciliary body and the sclera showed a notable, overall increase in counts over baseline, with values at 20 and 30 minutes being significantly elevated. The highest numbers of counts were seen in the aqueous humor. Animals injected intralumbar cisternally with insulin showed elevated insulin concentrations in their optic nerves and cisterna magna-derived CSF that did not appear to be due to uptake of insulin from the circulation. These results support the hypothesis that the insulin that accumulates in the retina and optic nerve following topical application arrives there after diffusing through the sclera, though an intraocular route--while unlikely--cannot be ruled out.

Development and validation of radioligand binding assays to measure total, IgA, IgE, IgG, and IgM insulin antibodies in human serum.

Radioligand binding assays for total and Ig classes of insulin antibodies (IAB) were developed and validated. For each assay, insulin-extracted serum samples were incubated with radiolabeled insulin in the presence and absence of high levels of unlabeled insulin to determine nonspecific binding and total binding, respectively. To measure total IAB, antibody-bound insulin was precipitated with a polyethylene glycol solution, washed, and counted in a gamma-counter. To measure IgG IAB, samples were treated with protein G-Sepharose beads, centrifuged, washed, and counted. For the measurement of IgA, IgE, and IgM IAB, IgG was removed from the samples and treated with anti-IgA, -IgE, or -IgM conjugated to Sepharose beads, centrifuged, washed, and counted. The acid/charcoal extraction of bound and unbound insulin from serum samples was optimized. Specificity and binding capacity of the protein G and antibody-bound beads were evaluated and optimized. The linear region of the total and IgG IAB assays was determined using serum samples containing high levels of insulin antibodies. The limit of quantitation, limit of detection, and precision for all the assays were also determined.