Development and Characterization of Guinea Pig Anti-Insulin Polyclonal Antibody.

Development and characterization of guinea pig anti-insulin polyclonal antibody against a target-specific insulin antigen. In India, an insulin immunogenicity kit for detecting insulin antibodies (neutralizing Nab) is an unmet medical need for diabetic patient's routine diagnosis. Type 1 diabetics rely on insulin injections daily basis for survival; if the body develops anti-insulin antibodies and neutralizes the exogenous recombinant insulin, glucose control is lost, and the patient eventually dies. Antibodies are excellent diagnostic reagents due to the specificity and sensitivity they provide in recognizing specific and unique target antigens. The paper describes the use of insulin as a target antigen and the development of target (insulin) specific antibodies in guinea pigs for use as a positive control for immunogenicity kit validation. Anti-insulin polyclonal antibody was raised against insulin in the Dunkin Hartley guinea pigs host. Anti-insulin antibody titer of all bleeds from four animals was tested using an indirect ELISA assay format. All four animals responded to the target-specific antigen but only one animal (#4) responded with a high-affinity antibody titer. The hyperimmune sera were purified using a protein A column. The purified anti-insulin antibody was characterized through SDS Page and western blot. The specificity, reactivity, and antibody binding efficiency were confirmed through immunoassays. Guinea pig anti-insulin polyclonal antibody developed in this study showed good specificity, reactivity, and efficiency in the immunoassays. This paper describes the development and characterization of anti-insulin antibodies for use as a control in developing a user-friendly insulin immunogenicity kit.

Serious diabetic ketoacidosis induced by insulin allergy and anti-insulin antibody in an individual with type 2 diabetes mellitus.

Diabetic ketoacidosis (DKA) is one of the most serious acute metabolic complications of diabetes mellitus, and is characterized by hyperglycemia, metabolic acidosis and increased total ketone body concentrations. The main mechanism of DKA is a lack of insulin in the body. It has been reported that some immunological response is associated with insulin therapy. Herein, we report a case of serious DKA, which was induced by insulin allergy and anti-insulin antibody. This case clearly shows that DKA can be induced by insulin allergy and anti-insulin antibodies in individuals with type 2 diabetes treated with insulin. Furthermore, we should know that as the required insulin dose might be very high under severe insulin resistance and serious DKA in such cases, we should increase the insulin dose appropriately while monitoring pH, base excess and other factors.

Production of insulin antibody associated with relapsed hodgkin's lymphoma.

A 72-year-old man with type 2 diabetes mellitus with good glucose control for 20 years and maintained on oral hypoglycaemic agents was diagnosed with Hodgkin's Lymphoma (HL) and started on insulin glargine for glycaemic control. Despite increased doses of insulin, his blood glucose levels went up dramatically. The anti-insulin antibody test proved to be positive, and Scatchard plot analysis showed 2 binding sites with relatively low-affinity constants: K 1 = 0.0032, K 2 = 0.0002 (108/M); and high binding capacities: R 1 = 98.4, R 2 = 372 (10-8 M), which were compatible with the features of antibody of insulin autoimmune syndrome (IAS). However, hypoglycaemia was not noted throughout the course of treatment. Since the insulin binding ratio of the antibody decreased from 87.3% to 62% after the termination of insulin treatment, it was suggested that the antibody reacted mainly to exogenously injected insulin. Switching insulin preparations or introducing insulin secretagogues did not improve elevated blood glucose levels. The initiation of brentuximab vedotin (BV), a therapeutic agent for relapsed HL, resulted in a remarkable improvement in glycaemic control despite the absence of insulin therapy and partial remission of HL. This case suggested that HL triggered anti-insulin antibody production, which resulted in poor glycaemic control, and that BV could be a new treatment option for autoimmune diseases associated with HL. Supplementary Information: The online version contains supplementary material available at 10.1007/s13340-021-00550-1.

Comparative study on the therapeutic effects of bone marrow mesenchymal stem cells versus platelet rich plasma on the pancreas of adult male albino rats with streptozotocin-induced type 1 diabetes mellitus.

BACKGROUND: The optimal treatment for autoimmune type 1 diabetes mellitus (T1DM) is endogenous regeneration of the pancreatic beta-cell. This can be achieved either by transplanting bone marrow mesenchymal stem cells (BMSCs) or injecting platelet-rich plasma (PRP). Current research reviewed a T1DM model and compared the effect of BMSCs on exocrine and endocrine pancreas portions versus PRP. MATERIALS AND METHODS: Rats were divided into four groups: Control group, Diabetic group (single streptozotocin dose 60 mg/kg IP), Diabetic + PRP group (PRP, 0.5 mL/kg SC twice weekly/4 weeks given to diabetic rats) and Diabetic + BMSCs group (1 mL of PKH26 labelled MSCs suspension in buffer phosphate solution, 3 × 106 cells/mL IV to diabetic rats). Glucose, amylase and lipase levels were calculated and pancreases were designed for light, electron microscopic, immunohistochemistry, morphometry and statistical analysis. RESULTS: Diabetic rats exhibited elevated glucose, decreased amylase and lipase compared to control rats. In addition, variable histological degenerative changes in the form of congested blood vessels have been identified with a significant increase in the mean area percentage of collagen, a significant decrease in the diameter of the islets, the number of cells in the islets of Langerhans and the number of zymogen granules. Ultrastructural findings exhibited distorted Golgi apparatus morphology, degenerated mitochondria, pyknotic nuclei, and few secretory beta-cell granules. CONCLUSIONS: Administration of BMSCs to diabetic group significantly increased the number of cells and diameter of Langerhans islets and the number of zymogen granules compared to Diabetic group as well as Diabetic + PRP group. BMSCs could be considered more efficient than PRP in the treatment of type 1 diabetes.

Relationship between anti-insulin antibody production and severe insulin resistance in a diabetic cat.

A 5-year-old castrated male domestic shorthair cat was diagnosed with diabetic ketoacidosis and severe insulin resistance. Although the conventional treatment for diabetic ketoacidosis was provided, the cat required frequent hospitalization because of severe dehydration and repeated diabetic ketoacidosis. We detected anti-insulin antibodies for human in this cat. Serum insulin-binding IgG levels were markedly elevated compared with those in healthy cats and other diabetic cats. We initiated prednisolone to suppress the effects of anti-insulin antibodies. After initiation of prednisolone, the cat was gradually recovered with increasing activity and appetite. Furthermore, satisfactory glycemic control was achieved with combined subcutaneous injection of insulin detemir and insulin degludec.

Relationship between anti-insulin antibody production and severe insulin resistance in a diabetic cat.

A 5-year-old castrated male domestic shorthair cat was diagnosed with diabetic ketoacidosis and severe insulin resistance. Although the conventional treatment for diabetic ketoacidosis was provided, the cat required frequent hospitalization because of severe dehydration and repeated diabetic ketoacidosis. We detected anti-insulin antibodies for human in this cat. Serum insulin-binding IgG levels were markedly elevated compared with those in healthy cats and other diabetic cats. We initiated prednisolone to suppress the effects of anti-insulin antibodies. After initiation of prednisolone, the cat was gradually recovered with increasing activity and appetite. Furthermore, satisfactory glycemic control was achieved with combined subcutaneous injection of insulin detemir and insulin degludec.

Polyglycidyl methacrylate based immunoaffinity cryogels for insulin adsorption.

Immunoaffinity chromatography (IAC) is a kind of bioaffinity chromatography which used antibodies or antibody-related molecules as the stationary phase. IAC is used by many applications for analytical, clinical and diagnostic purposes, particularly preferring in analytical purposes on one-step separation and purification of target compounds. Moreover, immunoaffinity chromatography is used in antibody enrichment and separation of cells. IAC columns are usually applied in the antibody experiments due to powerful and selective binding of antibodies and/or their target antigens. Antigen or antibody molecules could be immobilized to the solid support. Therefore, target antibody or cell is purified. Specific bioligands can be immobilized directly on glycidyl based polymeric material with simple acid-base catalyst. In this study, polyglycidyl methacrylate based therefore cryogels were prepared and anti-insulin antibodies were immobilized on porous surface of cryogels. Swelling test, Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and scanning electron microscopy (SEM) were conducted to characterize cryogels developed. To optimize separation conditions, effects of pH, initial insulin concentration, flow rate, salt concentration, contact time and temperature on insulin adsorption capacity were examined. The results indicated that the immunoaffinity cryogel developed here could be classified as good alternative with prominent properties such as high reusability and cost-friendly adsorbent and would be one of the primary reports for immunoaffinity purification of insulin molecules in not only lab-scale but also for industrial purposes.

Acute onset disseminated superficial porokeratosis associated with exacerbation of diabetes mellitus due to development of anti-insulin antibodies.

The development of disseminated superficial porokeratosis is occasionally observed in association with renal transplant, autoimmune diseases and various hematological disorders, suggesting a certain immunosuppression may trigger a widespread abnormal keratinization. Here we report a case of sudden onset disseminated superficial porokeratosis associated with an exacerbation of diabetes mellitus due to an anti-insulin antibody formation. To our knowledge, this is the first report of disseminated superficial porokeratosis in a patient with severe diabetes mellitus.

Factors Associated with the Development of Anti-insulin Antibody in Diabetic Children / 대한소아내분비학회지

PURPOSE: Anti-insulin antibodies develop within several months of initiation of insulin therapy in most of diabetic patients. The purpose of this study is to observe the relationship between the clinical factors and development of anti-insulin antibody METHODS: Serum was collected from 116 diabetic patients and 47 nondiabetic children for the measurement of anti-insulin antibody titer by radioimmunoassay (RIA). Retrospective analysis of the medical records of clinical factors were evaluated. RESULTS: There was no relationship of anti-insulin antibody titer with age, duration, HbA1c, insulin dose, and BMI in diabetic children. There was no difference in anti-insulin antibody titer according to the sex, the presence of family history, the presence of DKA, the presence of complications, the presence of puberty, species of insulin, duration of disease in diabetic children. The titers of anti-insulin antibody were significantly higher in type 1 diabetic children(30.3+/-17.9% in type 1 and 16.5+/-7.0% in type 2, P7%. The positive rates of anti-insulin antibody were higher in male patients with diabetes(73.2% in male and 53.3% in female, P7%). CONCLUSION: The results suggests that anti-insulin antibody developed more likely in type 1 DM and less likely in DM patients whose control had been good and who used less insulin doses, which remains to be studied further with more patients for longer duation.