A new application of plasma exchange in insulin dependent diabetes mellitus

An insulin dependent diabetic patient was resistant to all but central venous insulin administration. For this reason plasma exchange was tried and it restored insulin responsiveness. An anti-insulin IgG antibody was identified in the patient's plasma. Plasma exchange reduced antibody levels and these correlated with daily insulin requirements. Kinetic analysis of anti-insulin antibodies, however caused us to doubt that they were the sole cause of the problem. Although the mechanism remains unclear, plasmapheresis proved to be an effective method of treating this patient's insulin resistance.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum is described. Serum samples were subjected to successive processes of the incubation with insulin, the dextran-charcoal treatment to remove free insulin, the precipitation of insulin anti-insulin antibodies by polyethylene glycol, the acid treatment of the precipitates to inactivate anti-insulin antibodies, and the measurement of insulin by sandwich enzyme immunoassay technique. By this enzyme immunoassay, anti-insulin antibodies were demonstrated in most of serum samples from patients who had been treated with insulin for 0.6–24 months. The detection limit of anti-insulin IgG in human serum was 1,000 to 3,000-fold less than that obtained by the previously reported enzyme immunoassay, in which an insulin-coated polystyrene ball was incubated with diluted serum and subsequently with (antihuman IgG γ-chain) Fab'-horseradish peroxidase conjugate. The present enzyme immunoassay may be useful for the measurement of antibodies for not only insulin but also other antigens that are not precipitated by polyethylene glycol.     

Circulating anti-insulin receptor antibodies in a patient suffering from lupus nephritis and hypoinsulinemic hypoglycaemia

A 46-year-old female patient suffering from Lupus Nephritis came to our attention in 1981 for severe recurrent hypoglycaemia; she was obliged to eat every 5-7 hr to maintain glucose values not below 1.3-1.6 mM. All known causes of hypoglycaemia were excluded by performing selective angiography of the pancreas and skull, chest and abdominal computerized tomography, as well as stimulation and suppression tests. Oral glucose tolerance, tolbutamide and intravenous insulin (0.4 U/Kg b.w.) tests demonstrated that the patient was highly insulin resistant; furthermore, studies on the patient's red blood cells suggested that her insulin receptors were completely unable to bind insulin. Studies carried out to reveal the reason for this binding inhibition demonstrated that red blood cells from normal subjects as well as adipocytes from normal rats incubated with the patient's serum did not bind insulin (50% inhibition occurring at about 1:30 serum dilution). Insulin binding inhibitors were found in the fraction of the serum precipitated by ammonium sulphate. The serum cleared of IgG fraction was unable to affect insulin binding. These data demonstrate that the serum from the female patient investigated contained anti-insulin receptor antibodies blocking the binding of insulin to its receptors. Plasmapheresis improved the patient's metabolic status. The clinical picture would suggest that recurrent hypoglycaemia was caused by anti-insulin receptor antibodies acting as insulin on target cells.     

Hypoglycemia due to serum-complexed insulin in a patient with diabetes mellitus

A 28-year-old woman with insulin-dependent diabetes mellitus presented with a "hyperlabile" state of hyperglycemia and ketoacidosis alternating with hypoglycemia. Measurements of total and free insulin levels suggested that the clinical syndrome may have been due to antibody binding of insulin. Equilibrium analysis of insulin binding to the patient's serum demonstrated two classes of anti-insulin activities. The first class was of high affinity (dissociation constant approximately equal to 10(-9) M) and low capacity (150 microU/ml). At low total serum insulin concentrations, most of the circulating insulin was bound to the high-affinity binding activity, and the patient presented with hyperglycemia or ketosis. The second class of insulin binding activity had a lower affinity (dissociation constant approximately equal to 5 X 10(-7) M). The insulin that was bound to this low-affinity serum substance still maintained biologic activity in vivo. Isophane insulin (NPH) had a markedly prolonged serum half-life, which resulted in delayed hypoglycemia. Serum insulin complexes--that is, bound insulin--may not be "inactive" but may contribute to total insulin action. A determination of insulin activity, not only free insulin levels, may help explain hypoglycemia in selected patients with diabetes mellitus.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum is described. Serum samples were subjected to successive processes of incubation with insulin, dextran-charcoal treatment to remove free insulin, precipitation of insulin-anti-insulin antibodies by polyethylene glycol, acid-treatment of the precipitates to inactivate anti-insulin antibodies and measurement of insulin by sandwich enzyme immunoassay technique. The detection limit of anti-insulin IgG in human serum was 50 pg/assay or 450 ng/l of serum. This was 1,000- to 3,000-fold less than that obtained by a conventional enzyme immunoassay, in which an insulin-coated polystyrene ball was incubated with diluted serum and subsequently with (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-insulin antibodies were demonstrated in most (89%) of serum samples from diabetic patients who had been treated with porcine insulin and porcine insulin plus bovine insulin for 0.6-10 mth, while only a small proportion (3%) of serum samples from the same patients was positive by the conventional enzyme immunoassay. Similar results were obtained with serum samples from diabetic patients who had been treated with human insulin for 0.5-8.2 mth. The present enzyme immunoassay may be useful for the measurement of antibodies not only for insulin but also other antigens which can be removed by dextran-charcoal treatment and are not precipitated by polyethylene glycol.     

Regulation of monoclonal immunoglobulin G synthesis by antiidiotypic antibody in a patient with hypogammaglobulinemia.

The regulation of in vitro antibody synthesis by antiidiotypic antibodies was studied in a child with hypogammaglobulinemia and a serum immunoglobulin (Ig)G1 kappa M component. A rabbit antiserum was raised against the purified M component and was rendered idiotype specific by extensive absorption with Cohn fraction II and with IgG derived from the patient's parents. Hemagglutination-inhibition studies demonstrated that less than 1 in 300,000 molecules of pooled human IgG carried M component-related idiotypic determinants. 12% of the patient's B cells, but none of her T cells, expressed idiotypic determinants on their surface. Spontaneous de novo synthesis of the M component by the patient's peripheral blood lymphocytes was demonstrated in vitro and was shown to proceed independently of the polyclonal activator pokeweed mitogen. Antiidiotypic rabbit IgG, but not its F(ab')2, fragments, profoundly inhibited the synthesis of M component by the patient's peripheral blood lymphocytes. We concluded that antiidiotypic antibodies may play a role in the regulation of antibody synthesis in man.     

Novel enzyme immunoassay of anti-insulin igG in human serum

A novel enzyme immunoassay of anti-insulin IgG in human serum is described. A serum sample containing anti-insulin IgG was treated with dextran-charcoal at pH 6.0 to remove endogenous insulin and subsequently incubated with dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate. The reaction mixture was further incubated with a rabbit (antidinitrophenyl bovine serum albumin) IgG-coated polystyrene ball to trap the complex formed between anti-insulin IgG and the conjugate. After washing to eliminate nonspecific IgG in the test serum, the polystyrene ball was incubated with dinitrophenyl-L-lysine to elute the complex. The eluate was incubated with an avidin-coated polystyrene ball. Finally, the amount of human anti-insulin IgG in the complex trapped onto the avidin-coated polystyrene ball was measured by incubation with rabbit (antihuman IgG (γ-chain)) Fab'-peroxidase conjugate. This enzyme immunoassay was 1,000-fold more sensitive than the conventional enzyme immunoassay, in which an insulin-bovine serum albumin-coated polystyrene ball was incubated with a serum sample containing anti-insulin IgG and subsequently with rabbit (antihuman IgG (γ-chain)) Fab'-peroxidase conjugate. The principle of the novel enzyme immunoassay can be used to more sensitively measure antibodies for most kinds of haptens and antigens than the conventional enzyme immunoassay.     

Use of insulin immunoassays in clinical studies involving rapid-acting insulin analogues: Bi-insulin IRMA preliminary assessment.

BACKGROUND: In clinical studies involving rapid-acting analogues (RAAs), insulin immunoreactivity is frequently measured, including endogenous, regular insulin (RI) and RAA immunoreactivities. Such a procedure implies equivalent cross-reactivities of all insulins present in serum. Commercially available human insulin immunoassays have been widely used, but their limitations (including hemolysis and anti-insulin antibodies) were not fully investigated. The aims of our study were to compare cross-reactivities of RI and RAAs in buffer and in serum and to investigate insulin immunoassay pitfalls. METHODS: Cross-reactivities were assessed using Bi-insulin IRMA (Schering Cis-Bio International) in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA) and in pools of sera spiked with RI and RAAs (lispro and aspart). To investigate the influence of hemolysis, a pool of sera spiked with RAA was mixed with a concentrated hemolysate (final hemoglobin concentration 10 g/L) and incubated for 3 h at room temperature. To determine interference by anti-insulin antibodies, insulin was removed using charcoal from 18 sera with anti-insulin antibodies and from 17 sera without detectable anti-insulin antibodies. These insulin-free samples were then spiked with RI and RAAs and the immunoreactivity was determined. RESULTS: Compared with buffer, cross-reactivity in serum for RI, lispro and aspart was lower (35%, 29% and 26% lower, respectively). Hemolysis degraded almost all RI and RAAs contained in the serum (>or=95%). Anti-insulin antibody interference was significant for RI and RAAs (p相似文献   

IgE and IgG antibody against human (recombinant DNA) insulin in patients with systemic insulin allergy

We demonstrated IgE and IgG antibodies to human (rDNA) insulin as well as to bovine and porcine insulin in the serum of two patients with systemic insulin allergy by an enzyme-linked immunosorbent assay. We also demonstrated IgE and total antibody binding to bovine insulin with the use of radioimmunoassays. Both patients had cutaneous reactivity to all three insulins. When the serum of one patient was preincubated with human, porcine, or bovine insulin, there was inhibition of binding of the patient's IgE and IgG antibodies to human insulin. The other patient had very low levels of IgE antibodies to insulin and thus only IgG inhibition was possible. Preincubation with human insulin inhibited binding of each patient's antibodies to bovine or porcine insulin. We conclude that, for these two patients, human insulin has all the antigenic determinants that bovine and porcine insulin have. Therefore, human insulin for these two patients will not eliminate insulin allergy in all patients with systemic allergy to animal insulin, because there are patients whose antibodies recognize determinants common to commercial human, bovine, and porcine insulin.     

Insulin assays: previously known and new analytical features

Insulin assays play a central role in the investigation of glucose metabolism disorders (investigation of the causes of hypoglycemia, assessment of beta-cell function and determination of pathogenesis of type 1 and type 2 diabetes), and in studies on the pharmacology of insulin itself. Greater convenience and improved reproducibility, sensitivity and specificity have been achieved with human insulin immunometric assays. Besides human insulin, insulin analogues (lispro, aspart and glargine) have been introduced to therapeutic use. The specificity of human insulin assays to these analogues should be assessed. Anti-insulin antibodies are present in a significant proportion of sera to be analyzed for insulin. The sensitivity of insulin assays to interference from these antibodies should also be assessed. Interferences from anti-insulin antibodies and hemolysis, which degrades insulin molecules, remain the main pitfalls of insulin assays. Immunometric assays have also improved the sensitivity and reproducibility of free insulin measurements. Standardization of insulin immunoassays is still called for.     

Antigenic characterization of human hepatocellular carcinoma. Development of in vitro and in vivo immunoassays that use monoclonal antibodies.

Several libraries of monoclonal antibodies have been produced by immunization of Balb/c mice with single cell suspensions of nontrypsin-treated human hepatocellular carcinoma cell (HCC) lines in order to study the antigenic properties of transformed hepatocytes. The antibodies were characterized with regards to specificity for hepatoma-associated antigens and their capability for use as reagents in radioimmunoassays (RIAs) and tumor localization in vivo. Three such antibodies namely, P215457, PM4E9917, P232524 of the IgG2a, IgG2a, and IgG1 isotypes, respectively, not only recognized separate and distinct antigenic determinants on four human hepatoma cell lines but also reacted with epitopes present on chemically induced rat hepatoma cell lines. In contrast, only 1 of 38 other human malignant and transformed cell lines demonstrated reactivity with the three antibodies; normal human tissues were also found to be unreactive. Monoclonal antibody P215457 densely stained the plasma membrane by indirect immunofluorescence, showed rapid binding activity to HCC cells in suspension, and precipitated a 50,000-mol wt cell surface protein; antibody PM4E9917 also stained the plasma membrane and precipitated a 65,000-mol wt protein, whereas P232534 recognized cytoplasmic antigenic determinants. With these antibodies "simultaneous sandwich" RIAs were established that detect soluble hepatoma-associated antigens in culture supernatants. Finally, the Fab fragment of P215457 was found to be useful in tumor localization in vivo. This antibody fragment when labeled with 131I was shown to localize by radionuclide-imaging studies in human hepatoma grown in nude mice. Thus, these investigations demonstrate that monoclonal antibodies may be produced against epitopes that reside almost exclusively on transformed hepatocytes and such antibodies may be successfully employed in the development of in vitro and in vivo immunoassays.     

Clinical disorders associated with autoantibodies to the insulin receptor. Simulation by passive transfer of immunoglobulins to rats.

Patients with autoantibodies to the insulin receptor (Anti-R) may exhibit either fasting hypoglycemia or hyperglycemia and extreme insulin resistance. Occasionally, both these phenomena are observed in the same patient at different times in the clinical course. In an effort to understand what determines the patient's response to Anti-R, we developed an animal model of these clinical disorders by passive transfer of Anti-R IgG to rats. IgG fractions from the plasma of Anti-R patients and control subjects were prepared by affinity chromatography with staphylococcal protein A-Sepharose. Anti-R IgG, injected into fasting rats, induced severe and persistent hypoglycemia (plasma glucose 30-60 mg/dl). Rats injected with control IgG maintained a plasma glucose within the range of 75 (fasting) to 165 mg/dl (feeding). In comparison with the effects of insulin, the hypoglycemic response to Anti-R IgG had a slower onset (2-4 h) and lasted longer (8-24 h). Similar, dose-dependent hypoglycemic responses were observed in rats whether the Anti-R IgG was derived from an insulin-resistant or hypoglycemic patient. When Anti-R IgG was administered in sufficiently high doses for several days to fed rats, persistent hyperglycemia (plasma glucose 200-400 mg/dl) developed. Based on these in vivo and previous in vitro studies, we attribute the hypoglycemic response to an insulin-like effect of Anti-R, and the hyperglycemic response to a desensitization of host tissues to the effects of insulin, with more prolonged exposure to higher levels of Anti-R.     

Characterization of a murine monoclonal antibody, 5D-4, reacting with pancreatic cancers and islets of Langerhans.

A murine monoclonal antibody (5D-4) was prepared by immunizing mice ip with human pancreatic cancer cell line (AsPC-1). The 5D-4 MAb reacted immunohistochemically with pancreatic and gastrointestinal tract tumors as well as pancreatic islets, and to a less extent with normal tissues. The 5D-4 MAb reacted not only with ca 50 KDa and 30 KDa solubilized protein from AsPC-1 cells by Western blot analysis but also with human insulin in a competition RIA. Double immunoperoxidase staining showed that the 5D-4 MAb cross-reacted with insulin but did not react with glucagon, somatostatin or pancreatic polypeptide. Immunoelectron micrograph of Langerhans island double-stained with the 5D-4 MAb and anti-insulin Ab revealed that the 5D-4 Mab recognized human insulin and ca 50 KDa and 30 KDa antigens in B-cells of islets of Langerhans. Thus, the 5D-4 Mab may be useful for the diagnosis of islet cell tumors as well as pancreatic cancers.     

Treatment of multiple myeloma with monoclonal antibodies and the dilemma of false positive M-spikes in peripheral blood

ObjectivesTo characterize the effect of three humanized IgG κ monoclonal antibodies (daratumumab, isatuximab, and elotuzumab) on the interpretation of results generated by protein electrophoresis, immunofixation, free light chain, and heavy/light chain assays performed on human serum.MethodsHealthy volunteer serum and serum from multiple myeloma patients were supplemented with clinically relevant concentrations of each of the three monoclonal antibodies. These specimens then underwent analysis via serum protein electrophoresis, immunofixation, serum free light chain quantification, heavy/light chain quantification, total IgG, and total protein. In addition, serum specimens from patients who had undergone treatment with elotuzumab for multiple myeloma underwent similar analysis.ResultsAddition of the study drugs to serum from both the healthy donor as well as multiple myeloma patients resulted in a visible and quantifiable M-protein on SPEP and a visible IgGκ band by IFE. Increases were also noted in total IgG, IgGκ, and IgGκ/IgGλ-ratios. Analysis of serum from multiple myeloma patients receiving study drug showed similar findings with an additional IgGκ band and quantifiable M-protein with similar migration patterns in specimens drawn after administration.ConclusionThe treatment of multiple myeloma patients with monoclonal antibodies results in a visible and quantifiable M-protein that has the potential to falsely indicate poor response to therapy.     

Elicited antibody nature of human monoclonal protein with anti-streptolysin O activity--analysis with monoclonal anti-idiotype antibody

Sera from 7 patients with multiple myeloma having antistreptolysin O (ASO) activity in high titers were detected by a streptolysin O (SLO) inhibition assay. However, activity was in low titer when assayed by a passive agglutination assay. The discrepancy between these 2 assays raised some doubts as to whether these monoclonal proteins (M.protein) bond to SLO in the same manner as elicited antibodies. Immunochemical analysis and idiotope analysis using monoclonal antibody to one of these M.proteins strongly suggest that M.protein with ASO activity bind to SLO in a manner similar to elicited antibody. The discrepancy between the 2 assays might be due to differences in the antigenic structure of different forms of the SLO molecule.     

细胞角蛋白20单克隆抗体纯化方法的探讨

[目的]探讨细胞角蛋白20（CK20）单克隆抗体纯化的方法。[方法]采用SPA-Sepharose CL-4B为固定相的亲和层析法，将含有CK20单抗的小鼠腹水过柱后，用pH6．0、4．0和3．03个范围0．1mol／L枸橼酸分步洗脱，并以双向琼脂扩散法和Western-blotting对获得的单抗加以鉴定。[结果]以枸橼酸3个pH范围洗柱时分别收集5ml、4ml和20ml洗脱液，双向琼脂扩散法鉴定结果是：pH6．0时洗脱液中单抗亚型是IgG1、pH4．0时为IgG2a、pH3．0时为IgG2b，Western-blotting结果表明所获抗体具有较高免疫活性。[结论]该方法简便、特异，并可直接获得不同亚型单抗，适合腹水中CK20单克隆抗体纯化。     

Mouse monoclonal antibodies reacting with M blood group-related antigens

Four mouse monoclonal antibodies were produced with specificities related to human blood group M antigen. The antibodies react in direct hemagglutination systems, and their specificities were investigated by their reactions with variant and enzyme-modified red cells. The effects of temperature and pH on their hemagglutination reactions also were investigated, and all four were murine IgG antibodies. Physicochemical and serologic investigations showed them to be four distinct antibodies, and each one could be used for M blood grouping under appropriate conditions.     

Autoimmune insulin syndrome

Initially described in Japan, the autoimmune insulin syndrome is caused by the presence of anti-insulin antibodies in patients who have never received insulin. This syndrome accounts for spontaneous or reactive hypoglycaemia with very high levels of total immuno-reactive insulin. Discordance between the levels of immunoreactive insulin and C peptide indicate the possible presence of anti-insulin antibodies; this can avoid an incorrect diagnosis of insulinoma. These autoimmune hypoglycaemias often present a difficult diagnostic problem in distinguishing them from factitious hypoglycaemia. The course of the autoimmune insulin syndrome is usually favourable, with a spontaneous rapid diminution of the levels of anti-insulin antibodies. The reasons for the appearance of anti-insulin antibodies and the exact mechanisms of the hypoglycaemia remain uncertain. However, the frequent association of the autoimmune insulin syndrome with certain autoimmune diseases suggest a common immune dysfunction. Drugs containing a sulphydryl group have been implicated in the aetiology of this syndrome.     

Successful immunosuppressive therapy in a patient with autoantibodies to insulin receptors and immune complex glomerulonephritis

Altered mental status, acanthosis nigricans, immune complex glomerulonephritis with nephrotic syndrome, fasting hypoglycemia, and postprandial hyperglycemia associated with anti-insulin receptor antibodies (type B insulin resistance) developed in a 43-year-old black woman who initially was treated for diabetes mellitus. Her HLA phenotype was A2, A29, Bw45(w6), B13(w4), Cw3, DR1 (DQw1). Her serum contained immune complexes, low complement levels, and antibodies that bound to the glomerular mesangium of mouse kidney. All clinical and serologic abnormalities resolved with combination cyclophosphamide and glucocorticoid treatment, and low doses of these agents have maintained the remission for more than a year.     

MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins

BackgroundPlasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M−protein). Some patients have more than one identifiable M−protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M−proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M−protein, (2) an M−protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M−protein with light chain glycosylation, or (4) two distinct biclonal M−proteins.MethodsPatient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M−proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples.ResultsEighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated.ConclusionsWe demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M−proteins is useful for monitoring of patients with PCDs.