Anti-insulin IgG Research Articles

Autoreactive antibodies control blood glucose by regulating insulin homeostasis

Homeostasis of metabolism by hormone production is crucial for maintaining physiological integrity, as disbalance can cause severe metabolic disorders such as diabetes mellitus. Here, we show that antibody-deficient mice and immunodeficiency patients have subphysiological blood glucose concentrations. Restoring blood glucose physiology required total IgG injections and insulin-specific IgG antibodies detected in total IgG preparations and in the serum of healthy individuals. In addition to the insulin-neutralizing anti-insulin IgG, we identified two fractions of anti-insulin IgM in the serum of healthy individuals. These autoreactive IgM fractions differ in their affinity to insulin. Interestingly, the low-affinity IgM fraction (anti-insulin IgMlow) neutralizes insulin and leads to increased blood glucose, whereas the high-affinity IgM fraction (anti-insulin IgMhigh) protects insulin from neutralization by anti-insulin IgG, thereby preventing blood glucose dysregulation. To demonstrate that anti-insulin IgMhigh acts as a protector of insulin and counteracts insulin neutralization by anti-insulin IgG, we expressed the variable regions of a high-affinity anti-insulin antibody as IgG and IgM. Remarkably, the recombinant anti-insulin IgMhigh normalized insulin function and prevented IgG-mediated insulin neutralization. These results suggest that autoreactive antibodies recognizing insulin are key regulators of blood glucose and metabolism, as they control the concentration of insulin in the blood. Moreover, our data suggest that preventing autoimmune damage and maintaining physiological homeostasis requires adaptive tolerance mechanisms generating high-affinity autoreactive IgM antibodies during memory responses.

IAA, GAD65 AND IA2 Antibodies In Type 1 Children and Adolescents

Objective: Diabetes mellitus (DM) is a complicated, prolonged metabolic disorder that leads to hyperglycemia and it occurrs due to complete deficiency of insulin with or with insulin resistance. The most common type of DM in children and adolescents is Type-1 DM (T1DM). The aim of this study was to determine the associations of IAA, GAD65 and IA-2A antibodies in type 1 diabetes mellitus with age, gender and duration of disease.
 Methodology: This was a retrospective study conducted in the NICH hospital at the department of endocrinology and diabetes by using non probability consecutive sampling technique, after taking ethical approval. The duration of the study was one year. A total of 143 patients with clinically diagnosed type 1 diabetes ( ?1 year to ?20 years of age, regardless of the duration of the disease) were selected for the study. All study subjects were investigated for pancreatic autoimmune markers (GAD65, IA-2A, IAAs). Pearson’s Chi square test was applied to evaluate the association.
 Results: A total of 143 children diagnosed with type 1 diabetes were enrolled for this study wherein 71(49.7%) were males and 72(50.3%) were females. Regarding the distribution of positivity of autoantibodies, 27(18.9%) had AntiInsulin IgG, 63(44.1%) had GAD65, 47(32.9%) had IA-2A and only 5(3.5%) had grey zone. There was a significant association was reported (p<0.001) in duration of diabetes and with IAA. Furthermore, there was a statistically significant association with (p=0.003) duration of diabetes and frequency of IA-2A.
 Conclusion: This study concluded that presence of autoantibodies such as insulinoma-associated antigen-2 autoantibodies, Insulin autoantibodies and glutamic acid decarboxylase 65 were found to be predominant in males than females although it was not significantly associated with age and gender. Additionally, presence of insulinoma-associated antigen-2 autoantibodies, Insulin autoantibodies significantly associated with the duration of disease.

Mature anti-insulin B cells survive and present antigen in the absence of Bruton’s tyrosine kinase.

Abstract Bruton’s tyrosine kinase (Btk) is a tec-family kinase present in B lymphocytes and innate immune cells. Btk is an important regulator of autoreactive B cells. In the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D), Btk-deficiency is protective against disease development and results in significant loss of anti-insulin IgG, even as total IgG is preserved. Anti-insulin B cells drive T1D by presenting antigen to autoreactive T cells. In a transgenic model, conventional Btk-deficiency reduces anti-insulin B cells by 95%, effectively blocking their development. However, the ability of mature anti-insulin B cells to survive or present antigen without Btk was unknown. We induced deletion of Btk using a loxP-flanked Btk mouse model paired with tamoxifen-inducible Cre. Surprisingly, these anergic anti-insulin B cells survive without Btk, as normal numbers of mature B cells were maintained in transgenic Btkflox/Cre-ERT2 animals after tamoxifen treatment. Btk-negative anti-insulin B cells also remained able to internalize and present antigen to cognate T cells and to phosphorylate phospholipase C γ2 in response to anti-IgM. These findings show that though anti-insulin B cells require Btk for their development, it is not required for mature anti-insulin B cell survival. In addition, our finding that Btk-negative anti-insulin B cells can present antigen may have implications for the use of Btk-inhibition in autoimmunity driven by this mechanism.

Diagnosis of insulin autoimmune syndrome using polyethylene glycol precipitation and gel filtration chromatography with ex vivo insulin exchange.

SummaryContextInsulin‐binding antibodies may produce severe dysglycaemia in insulin‐naïve patients (‘insulin autoimmune syndrome’ (IAS) or Hirata disease), while rendering routine insulin assays unreliable.ObjectiveTo assess the performance of clinically used insulin assays and an optimal analytical approach in the context of IAS.DesignObservational biochemical study of selected patients with hyperinsulinaemic hypoglycaemia.PatientsThree patients without diabetes with recurrent spontaneous hyperinsulinaemic hypoglycaemia and ‘positive’ insulin antibodies.MeasurementsA panel of clinically used insulin assays (Siemens ADVIA ® Centaur, Siemens Immulite® 2000, DiaSorin LIAISON ® XL, PE AutoDELFIA ® and the Beckman Coulter Access® 2) were used before and after plasma dilution or polyethylene glycol (PEG) precipitation. Anti‐insulin IgG antibodies were measured by Isletest™‐IAA ELISA. Gel filtration chromatography (GFC) was undertaken with and without preincubation of plasma with exogenous insulin.ResultsDilution of IAS plasma with assay‐specific buffer increased insulin recovery, supporting negative immunoassay interference by antibodies. PEG precipitation of IAS plasma decreased insulin recovery using all assays except the Immulite® 2000. GFC discriminated high molecular weight and monomeric insulin, while ex vivo addition of exogenous insulin to plasma increased insulin bound to antibody, thereby improving the sensitivity of detection of insulin immunocomplexes.ConclusionsImmunoprecipitation with PEG must be used with caution in screening for insulin–antibody complexes as results are assay dependent. GFC with addition of exogenous insulin can identify significant insulin immunocomplexes with enhanced sensitivity, with attendant greater clinical utility and avoidance of radiolabelled reagents.

Relapsing and Remitting Severe Hypoglycemia due to a Monoclonal Anti-insulin Antibody Heralding a Case of Multiple Myeloma

We report a novel case of insulin autoimmune syndrome (IAS) presenting with hypoglycemia due to production of a monoclonal anti-insulin antibody in a patient subsequently found to have multiple myeloma (MM). The aim of the study was to describe the 5-yr clinical course of a patient with IAS and MM and to characterize the origin and function of the pathogenic antibody. We conducted a longitudinal case history with laboratory investigations to characterize the anti-insulin antibody subtype, specificity, affinity, and origin. The patient presented with IAS, which worsened during treatment of hepatitis C. The patient was then discovered to have a monoclonal gammopathy that progressed to MM. Treatment of the MM induced remission of the neoplasia and IAS, which then followed a synchronized course of progression and response to therapy. An anti-insulin IgG(3)-λ that bound specifically but with low affinity to the insulin B chain (amino acids 9-30) and that was distinct from the primary MM IgG(3)-κ clone was recovered from the patient and cloned. The antibody bound insulin and showed mutations of normal affinity maturation. We describe a case of MM heralded by IAS, where full characterization of the pathogenic antibody revealed that the monoclonal anti-insulin antibody had originated from a self-reactive clone.

Evidence for Metabolic Cleavage of a PEGylated Protein in Vivo Using Multiple Analytical Methodologies

PEGylation of therapeutic proteins is commonly used to extend half-lives and to reduce immunogenicity. However, reports of antibodies toward PEGylated proteins and of poly(ethylene glycol) (PEG) accumulation suggest that efficacy and safety concerns may arise. To understand the relationship among the pharmacology, immunogenicity, and toxicology of PEGylated proteins, we require knowledge of the disposition and metabolic fate of both the drug and the polymer moieties. The analysis of PEG by standard spectrophotometric or mass spectrometric techniques is problematic. Consequently, we have examined and compared two independent analytical approaches, based on gel electrophoresis and nuclear magnetic resonance (NMR) spectroscopy, to determine the biological fate of a model PEGylated protein, (40K)PEG-insulin, within a rat model. Both immunoblotting with an antibody to PEG and NMR analyses (LOD 0.5 μg/mL for both assays) indicated that the PEG moiety remained detectable for several weeks in both serum and urine following intravenous administration of (40K)PEG-insulin (4 mg/kg). In contrast, Western blotting with anti-insulin IgG indicated that the terminal half-life of the insulin moiety was far shorter than that of the PEG, providing clear evidence of conjugate cleavage. The application of combined analytical techniques in this way thus allows simultaneous independent monitoring of both protein and polymer elements of a PEGylated molecule. These methodologies also provide direct evidence for cleavage and definition of the chemical species present in biological fluids which may have toxicological consequences due to unconjugated PEG accumulation or immunogenic recognition of the uncoupled protein.

Mis-diagnosis and mis-treatment of autoimmune pancreatitis: a clinical study of 17 cases

Objective To summarize the reasons of mis-diagnosis and mis-treatment of autoimmune pancreatitis (AIP). Methods Clinical data of 17 patients with AIP,who were admitted to the hospital from May 2005 to July 2010 and experienced mis-diagnosis and mis-treatment, were retrospectively analyzed. Results The main clinical manifestations included epigastric pain (13 cases),progressive obstructive jaundice (12 cases), fever (6 cases) and weight loss (9 cases). Fifteen patients had extrapancreatic organ involvemnet, including allergic rhinitis, swelling of lymphoglandulae submaxillares, swelling of submaxillary gland, allergic asthma, rheumatoid arthritis, Sjogren syndrome, diabetes mellitus, primary sclerosing cholangitis and autoimmune hepatitis. Of these 17 cases, 11 cases presented with high serum globulin, 14 cases with high serum IgG, 13 cases with high serum γ-globulin, 13 cases with positive anti-nuclear antibody and 2 cases with positive anti-insulin IgG antibody. The abdominal imaging demonstrated that 15 patients had diffuse enlargement of the pancreas with diffuse or segmental narrowing of main pancreatic duct, narrowing of the intrapancreatic common bile duct, dilation of the proximal biliary duct and gallbladder enlargement. Focal enlargement of the pancreas was found in 2 cases. Thirteen cases were misdiagnosed as pancreatic carcinoma. Among them, 4 cases underwent pancreaticoduodenectomy and 7 cases underwent choledochojejunostomy. Two cases were misdiagnosed as end stage of cancer that lost therapeutic chance. Another 4 cases were misdiagnosed as chronic pancreatitis. Steroid therapy was administered in all patients with satisfactory response. All patients were followed-up for 15 months (ranged from 6 months to 45 months), and recurrence was found in 4 cases. Satisfactory response was found in patients treated with steroid for the second time. No pancreatic cancer was found in these patients in the follow up period. Conclusion The main causes of mis-diagnosis and mis-treatment of AIP may be contributed by difficulty in differentiating AIP from pancreatic carcinoma based on clinical manifestations and inadequate knowledge of AIP as well as insufficient attention to AIP in China. Key words: Pancreatitis; Autoimmune diseases; Diagnostic errors; Therapeutic errors

Comparing biomimetic and biological receptors for insulin sensing

Biological information such as recognition abilities of antibodies can be transferred to man-made systems by imprinting. For instance, sensor layers with insulin-selective cavities yield similar selectivity and sensitivity as IgG. True "artificial antibodies" are achieved by synthesizing "polymer copies" retaining the original binding properties of anti-insulin IgG via two-step imprinting.

A Sensitive Sandwich Enzyme Immunoassay for Measurement of Insulin on Microtiter Plates

A sensitive enzyme immunoassay for the measurement of insulin in human sera on microtiter plates was established. The assay is based on the sandwich technique with guinea pig anti-insulin IgG adsorbed at microtiter plate wells, human insulin as standard and the same anti-insulin IgG labeled with horseradish peroxidase. Standards used cover a range from 0 to 1200 pmol/l with a detection limit of 10 pmol/l. Coefficients of variation between 3-7% for intraassay precision and 5-11% for interassay precision were obtained over the concentration range of 80-1000 pmol/l. The correlation of EIA-data with those of a commercially available double antibody radioimmunoassay (r = 0.98) could be expressed by the equation: EIA = 0.97 RIA - 57 pmol/l. Normal fasting serum insulin concentrations in healthy subjects ranged from 11-165 pmol/l. In subjects with potentially diminished basal values concentrations of 10-79 pmol/l were determined. The insulin response in oral glucose tolerance tests of children was discussed, who had a constitutional tall stature or Turner's syndrome, respectively.

Immunoassay for Insulin Using Highly-Sensitive Hydrogen Peroxide Sensors Based on Charge Accumulation Systems

A coulometric signal accumulation system to detect a trace concentration of hydrogen peroxide (H2O2) has been applied to develop a novel electrochemical enzyme immunosensing system for insulin, an important hormone for clinical diagnosis of diabetes. Glucose oxidase (GOx)-labeled anti-insulin IgG antibody was trapped on the substrate immobilized with insulin by an indirect competitive immunoassay. H2O2 generated by the enzymatic reaction with the trapped conjugate was detected by the coulometric signal accumulation system. The H2O2 generated oxidized [Os(bpy)2Cl]+ to [Os(bpy)2Cl]2+ in a horseradish peroxidase (HRP)-containing polymer layer on an electrode under the open-circuit condition, and then the [Os(bpy)2Cl]2+ accumulated was reduced by applying a negative potential. The accumulation brought about a large current response and a low detection limit for H2O2 (0.5 nM). The enhanced sensitivity for H2O2 enabled the determination of a trace concentration of insulin with the immunoassay using the antibody modified with GOx, a H2O2-producing enzyme.

Nanosieving for rapid, solution‐phase immunoassays

We demonstrate an innovative nanosieving technique for rapid solution-phase measurement of antibodies. Polyacrylamide membranes were fabricated by in situ UV lithography in glass microchannels. Membrane pore-size was optimized to exclude large species, while small proteins (<10 kDa) electrophoretically migrate through the membrane. For detection of unlabeled antibodies, buffer containing anti-insulin IgG was electrophoretically loaded into the microchannel. Owing to the large size of IgG, the antibody was retained at the membrane interface. A known concentration of FITC-labeled insulin (Ins*) was introduced. Retention of Ins* at the membrane indicated formation of Ins*-IgG complexes (see figure), as unbound Ins* migrates through the membrane when IgG is not present. Fluorescence signal at the membrane enabled IgG quantitation. The device exploits advantages of scale-dependent transport processes in confined geometries, namely for rapid immunorecognition. Unlike protein microarrays, no surface immobilization is necessary. The reported assay could allow study of previously unusable binding pairs and binding kinetics. Funding from UC Berkeley is gratefully acknowledged.

Engineered Enteroendocrine Cells Secrete Insulin in Response to Glucose and Reverse Hyperglycemia in Diabetic Mice

Type 1 diabetes is a metabolic disorder caused by loss of insulin-producing pancreatic beta-cells. Expression of insulin in non-beta-cells to create beta-cell surrogates has been tried to treat type 1 diabetes. Enteroendocrine K cells have characteristics similar to pancreatic beta-cells, such as a glucose-sensing system and insulin-processing proteases. In this study, we genetically engineered an enteroendocrine cell line (STC-1) to express insulin under the control of the glucose-dependent insulinotropic polypeptide promoter. We screened clones and chose one, Gi-INS-7, based on its high production of insulin. Gi-INS-7 cells expressed glucose transporter 2 (GLUT2) and glucokinase (GK) and secreted insulin in response to elevated glucose levels in vitro. To determine whether Gi-INS-7 cells can control blood glucose levels in diabetic mice, we transplanted these cells under the kidney capsule of streptozotocin (STZ)-induced diabetic mice and found that blood glucose levels became normal within 2 weeks of transplantation. In addition, glucose tolerance tests in mice that became normoglycemic after transplantation with Gi-INS-7 cells showed that exogenous glucose was cleared appropriately. These results suggest that engineered K cells may be promising surrogate beta-cells for possible therapeutic use for the treatment of type 1 diabetes.

Rabbit nasal mucosa: nanospheres coated with polypeptides bound to specific anti-polypeptide IgG are better transported than nanospheres coated with polypeptides or IgG alone

In rabbit nasal mucosa, polypeptides and polypeptide-coated nanospheres are actively transported from lumen to blood by M-cells present in specialized transport areas of the epithelium. The largest transport is shown here to occur when some molecules of the polypeptides coating the nanospheres, after adsorption, are bound to the specific anti-polypeptide IgG, e.g. when insulin is bound to the anti-insulin IgG. The transport kinetics of nanospheres coated by insulin bound to its antibody, as a function of bead concentration or of the antibody/insulin coating ratio, have been analyzed. On this basis it was possible to assess the maximal transport capacity of the epithelium and to calculate the percentage of M-cells involved.

Different kinds of polypeptides and polypeptide-coated nanoparticles are accepted by the selective transcytosis shown in the rabbit nasal mucosa

The specific transcytosis of polypeptides, demonstrated in the nasal respiratory mucosa of the rabbit, seems to be involved in antigen sampling at the airway entry, since absorption has been shown only to occur if lymphoid aggregates are present beneath the epithelium and to be proportional to aggregate volume. Nanoparticles and many polypeptides besides the two previously tested (i.e. carbocalcitonin (CCT) and adrenocorticotropic hormone) should be transportable, in agreement with the vesicular transcytosis and antigen sampling hypothesis. Thus unidirectional mucosa-submucosa and opposite fluxes ( J ms, J sm) and the corresponding net fluxes ( J net) of uncoated or polypeptide-coated polystyrene nanospheres (diameter: about 0.5 μm) have been measured with the aid of spectrophotometry and quantitative dark-field microscopy. No net transport has been observed for uncoated beads, whereas it has always been shown for polypeptide-coated beads, although to different extents. The selectivity sequence for the polypeptides tested is as follows: BSA≅enkephalin≪anti-BSA IgG≅IgA≅CCT≅insulin≤anti-insulin IgG. With the exception of BSA and enkephalin-coated beads, whose J net is very small, in all the other cases the apparent affinities for receptors seem to be equal or similar; just over 6% polypeptide coating on the nanosphere is sufficient to elicit maximal transport; finally, transport seems to require many cooperating binding sites between the single nanosphere and receptors or one or many non-cooperating binding sites, but with a threshold number of polypeptide molecules adsorbed on the nanosphere to reach a minimal binding probability.

VH gene structure predicts a large potential anti-insulin repertoire

The majority of insulin antibodies derived from immunization are IgG antibodies that cross-react extensively with the autologous hormone. To examine the relationship between VH genes expressed by such self-reactive antibodies and their germline (non-rearranged) counterparts, we used the polymerase chain reaction (PCR) to amplify and isolate the germline progenitors of anti-insulin VH genes derived from BALB/c mice immunized with beef or human insulin. Results indicate that two anti-insulin mAbs (123 and 124) express VH genes which arise from a small subset of the J558 gene family and are highly homologous to the VH gene used by the murine CD5+ B-cell tumor, BCL1. The anti-insulin IgG mAb 127 belongs to the VH-VIII (Vgam 3.2) family and the amplification and isolation of germline VH genes from this small family precisely identified only two somatic mutation events in the CDRH2 of mAb 127. Another anti-insulin mAb, 133, aslo shows two replacement substitutions in the CDRHs when compared to the germline encoded anti-dextran antibody 19.1.2. These findings indicate that the IgG response to this small self-protein uses multiple VH genes which are largely germline encoded with only a low level of somatic mutation in their CDRHs. Additionally, analysis of N-segment additions in CDRH3s indicates anti-insulin B cells may originate from both early (fetal) and adult repertoires. These data are consistent with the concept that the mechanisms of clonal anergy or deletion do not regulate anti-insulin B cells and indicate that there is a large potential VH gene repertoire for insulin.

A human anti-insulin IgG autoantibody apparently arises through clonal selection from an insulin-specific "germ-line" natural antibody template. Analysis by V gene segment reassortment and site-directed mutagenesis.

We analyzed the structural correlates underlying the insulin-dependent selection of the specific anti-insulin IgG1 kappa mAb13-producing cell clone, derived from a patient with insulin-dependent diabetes mellitus treated with recombinant human insulin. First, we cloned the germ-line genes that putatively gave rise to the expressed VH and V kappa segments and used them to generate the full (unmutated) "germ-line revertant" of the "wild-type" (somatically mutated) mAb13, using recombinant PCR methods and an in vitro human C gamma 1 and C kappa expression system. The full "germ-line revertant" bound insulin specifically and in a dose-saturable fashion, but with a relative avidity (AVrel) more than three-fold lower than that of its wild-type counterpart (Avrel, 1.69 x 10(-8) vs 4.91 x 10(-9) g/microliters). Second, we established, by reassorting wild-type and germ-line revertant forms of the mAb13 VH and V kappa segments, that the increased Avrel for insulin of mAb13 when compared with its full "germ-line revertant" counterpart was entirely dependent on the mutations in the VH not those in the V kappa chain. Third, we determined, by site-directed mutagenesis experiments, that of the three mutations in the mAb13 VH segment (Ser-->Gly, Ser-->Thr, and Ser-->Arg at positions 31, 56, and 58, respectively), only Arg58 was crucial in increasing the mAb13 Avrel (from 1.44 x 10(-8) to 5.14 x 10(-9) g/microliters) and affinity (Kd, from 189 to 59 nM) for insulin. The affinity enhancement mediated by the VH segment Arg58 residue reflected about a threefold decrease in dissociation rate constant (Koff, from 4.92 x 10(-3) to 1.54 x 10(-3) s-1) but not an increase in association rate constant (Kon, from 2.60 x 10(4) to 2.61 x 10(4) M-1 s-1), and it contrasted with the complete loss of insulin binding resulting from the substitution of the VH segment Asn52 by Lys. The present findings suggest that human insulin, a self Ag, has the potential to recruit a natural autoantibody-producing cell precursor expressing a specific surface receptor for Ag in unmutated configuration, and drive it through affinity maturation. They also show that binding of insulin by such a receptor can be enhanced or completely abrogated by a single amino acid change.

A direct two-site peroxidase-based enzyme-linked immunosorbent assay for proinsulin.

A non-competitive sandwich assay using an anti-C-peptide IgG and an anti-insulin IgG was developed to measure fasting levels of proinsulin in human serum. The former antibody provided the lower layer in a sandwich immunoassay, the upper layer being composed of an anti-insulin IgG-horse radish peroxidase conjugate. The assay showed negligible cross reactivity at supraphysiological levels of insulin and C-peptide. The method enabled the estimation of proinsulin in fasting non-diabetic control subjects [13.7 +/- 1.6(4) pM] and in type 2 non-insulin-dependent diabetic patients [23.2 +/- 1.1(8) pM].

Analysis of the structural correlates for antibody polyreactivity by multiple reassortments of chimeric human immunoglobulin heavy and light chain V segments.

Polyreactive antibodies (Abs) constitute a major proportion of the early Ab repertoire and are an important component of the natural defense mechanisms against infections. They are primarily immunoglobulin M (IgM) and bind a variety of structurally dissimilar self and exogenous antigens (Ags) with moderate affinity. We analyzed the contribution of Ig polyvalency and of heavy (H) and light (L) chain variable (V) regions to polyreactivity in recombinatorial experiments involving the VH-diversity(D)-JH and V kappa-J kappa gene segments of a human polyreactive IgM, monoclonal antibody 55 (mAb55), and those of a human monoreactive anti-insulin IgG, mAb13, in an in vitro C gamma l and C kappa human expression system. These mAbs are virtually identical in their VH and V kappa gene segment sequences. First, we expressed the VH-D-JH and V kappa-J kappa genes of the IgM mAb55 as V segments of an IgG molecule. The bivalent recombinant IgG Ab bound multiple Ags with an efficiency only slightly lower than that of the original decavalent IgM mAb55, suggesting that class switch to IgG does not affect the Ig polyreactivity. Second, we coexpressed the mAb55-derived H or kappa chain with the mAb13-derived kappa or H chain, respectively. The hybrid IgG Ab bearing the mAb55-derived H chain V segment paired with the mAb13-derived kappa V segment, but not that bearing the mAb13-derived H chain V segment paired with the mAb55-derived kappa V segment, bound multiple Ags, suggesting that the Ig H chain plays a major role in the Ig polyreactivity. Third, we shuffled the framework 1 (FR1)-FR3 and complementarity determining region 3 (CDR3) regions of the H and kappa chain V segments of the mAB55-derived IgG molecule with the corresponding regions of the monoreactive IgG mAb13. The mAb55-derived IgG molecule lost polyreactivity when the H chain CDR3, but not the FR1-FR3 region, was replaced by the corresponding region of mAb13, suggesting that within the H chain, the CDR3 provides the major structural correlate for multiple Ag-binding. This was formally proved by the multiple Ag-binding of the originally monoreactive mAb13-derived IgG molecule grafted with the mAb55-derived H chain CDR3. The polyreactivity of this chimeric IgG was maximized by grafting of the mAb55-derived kappa chain FR1-FR3, but not that of the kappa chain CDR3.(ABSTRACT TRUNCATED AT 400 WORDS)

VH and V kappa segment structure of anti-insulin IgG autoantibodies in patients with insulin-dependent diabetes mellitus. Evidence for somatic selection.

In some patients with insulin-dependent (type I) diabetes mellitus (IDDM), autoantibodies to insulin are present at diagnosis. After initiation of the treatment with not only animal but also human insulin, anti-insulin, mainly IgG, autoantibodies become a major component of the autoimmune response in virtually all IDDM patients. Their structure, however, is still relatively unknown. We analyzed the structure of the VH and V kappa segments of three human IgG mAb derived from three IDDM patients. The sequences of VH genes of two IgG, mAb13 and mAb48, were 98.3 and 96.6% identical with those of the H11 and 1.9III genes (VHIII family), respectively. The sequence of the VH gene of the third IgG, mAb49, was 98.6% identical with that of the 51p1 gene (VHI family). All three IgG mAb used V kappa III segments. The V kappa III gene sequences of mAb13 and mAb49 were 97.9 and 98.9% identical, respectively, to that of the kv3g gene; the mAb48 V kappa gene sequence was 96.5% identical to that of the kv328 gene. The VH and/or V kappa segments of these anti-insulin IgG mAb are similar to Ig V genes expressed in the fetal, and adult normal and autoimmune B cell repertoires. The nucleotide differences displayed by the three anti-insulin IgG mAb VH gene sequences, when compared with those of the closest reported germ-line genes, were concentrated in the CDR (6.2 x 10(-2) and 0.8 x 10(-2) difference/base in CDR and FR, respectively; p < 0.01, chi 2 test), and yielded a significantly higher putative replacement (R) to silent (S) mutation ratio in the CDR (12.0) than in the framework (0.2). The concentration of nucleotide differences in the CDR and their high R:S putative mutation ratios were consistent with the hypothesis that these expressed VH genes underwent a process of somatic mutation and Ag-driven clonal selection. That such differences constituted somatic point-mutations was formally proved in IgG mAb13, by differentially targeted PCR amplification and Southern blot hybridization of the mAb13-producing cell line DNA. The putative germ-line gene that gave rise to the expressed VH segment was cloned using genomic DNA from PMN of the same patient whose B cells were used for the generation of this mAb. Overall, in the anti-insulin IgG mAb VH and V kappa III genes, the (putative and verified) somatic point-mutations yielded 27 amino acid replacements, of which 14 nonconserved. Four of these resulted in positively charged residues, three Arg and one His.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin antibodies: reduction in various anti-insulin IgG subclasses with human insulin therapy.

The insulin-induced antibodies decrease on substitution of bovine-porcine with human insulin. In order to test whether there is a preferential reduction of certain anti-insulin IgG subclass, we investigated the composition of insulin antibodies. In serum samples collected from 23 bovine-porcine insulin-treated patients with clinical manifestations of insulin-induced immune problems (12 insulin resistant cases, 11 with lipodystrophy and/or local insulin allergy), the amounts of 125I-bovine insulin bound with IgG1, IgG2, IgG3 and IgG4 subclasses were 27.0 +/- 4.7, 7.8 +/- 2.3, 39.0 +/- 11.6 and 55.1 +/- 10.9 ng/ml respectively. Thus, there was an abundance of insulin antibodies of IgG4 subclass, and a relative paucity of IgG2. When the bovine-porcine insulin was changed to rDNA human insulin in 15 patients, the total insulin-binding IgG antibodies decreased by 58.6% with concomitant reductions in IgG1 (from 26.7 +/- 6.6 to 16 +/- 5.3), IgG3 (from 5.9 +/- 3.1 to 4.3 +/- 2.1), and IgG4 (from 46.4 +/- 15.6 to 18.4 +/- 7.9 ng of insulin bound/ml) subclasses. These observations suggest that in a selected subset of patients with insulin-induced immune problems, the bovine-porcine insulin induces antibodies of all four IgG subclasses but the IgG4 response predominates. With the substitution to human insulin, a reduction in the immune stimulus occurs followed with decreases in the IgG1, IgG3 and IgG4 subclasses.