Monoclonal Antibodies - Essay Example

Monoclonal Antibodies Monoclonal Antibodies (MAC), which have revolutionized the medical and biotechnological field from 1976, are produced throughthe in vitro technologies. Monoclonal antibodies are used to block the autoimmune processes that results in the positive outcome. Monoclonal antibodies are produced by fusing the myeloma cells with the spleen cells of the specific antigen immunized mouse. The fused cells are grown in a selective medium called the HAT medium which contains Hypoxanthine, aminopterin and thymidine. (Saha 2010). The cells are then separated and each cell is grown in the HAT medium and the antibodies secreted by each cell are assayed for their ability to bind with the specific antigen. The tests include ELISA, western blotting and immune dot- blot methods. After the assay, the cells are grown in the selective medium indefinitely. The antibodies produced by the hybridoma cells are separated from the cellular components and other growth factors using ultra filtration, dialysis, ion exchange chromatography and other separation techniques. (Saha 2010). Fig 1: Production of hybrid cells for the manufacture of Monoclonal antibodies (Saha 2010). Type II Diabetes Mellitus: Diabetes mellitus is a chronic disease which is characterized by the high sugar levels in the blood. After a meal, the glucose level in the blood increases triggering the insulin secretion by the B-cells of the pancreas. If the insulin secretion is very less or nil then the diabetes is caused. There are two types of Diabetes. Type 1 Diabetes mellitus and Type II diabetes mellitus. Type 1 Diabetes mellitus is caused by the destruction of the beta cells in the pancreas leading to the less production of insulin. Type II diabetes mellitus is a heterogeneous multicomplex disease. Type II Diabetes mellitus (T2DM) is associated with obesity and hypertension. Insulin resistance is a notable factor for the T2DM patients. Hence the understanding of the metabolic pathways using the antibodies emerged to diagnose and treat T2DM. (Ahrens 2011). Many techniques are used for the diagnosis and treatment of T2DM. Some of them are western blotting, Immunoblotting, Immunohistochemistry, Flow cytometry, Immunofluorescence and High content screening. Type I Diabetes mellitus Type II Diabetes mellitus Diagnosed in the childhood. Diagnosed in around 30 years of age. T1DM can be treated with insulin Usually treated with medication and tablets. Higher ketone levels are observed in T1DM High blood pressure or high cholesterol levels are diagnosed Generally it is not related to obesity It is related to obesity and hypoglycemia. Approximately 5 – 10 % of the diabetes mellitus patients are type I and the rest of the diabetic patients are type II. In both the types’ long term complications such as Cardiovascular disease, diabetic neuropathy, diabetic nephropathy and diabetic retinopathy are very common. (Joslin and Kahn 2005). Antibodies are immunoglobulin protein molecules that are produced by the B cells in the body. Antibodies are used to identify and neutralize the foreign bodies present in the body. Antibodies bind with the antigen by the lock and key mechanism. Each antibody has a specific target antigen. Nearly 10- 15 % of the type 2 diabetes mellitus patients are antibody positive and the treatment guidelines and identification methods vary from type 1 diabetes mellitus. (Joslin and Kahn 2005). The monoclonal antibodies were produced against the glucagon receptor by Iwanji et al. They were tested in the partially purified rat glucagon receptor. They were found to reduce the fasting glucose level when compared with the control. The first human monoclonal antibodies were produced by Bayer and Novo Nordisk in the names CIV395.7A and hGR-2F6 respectively. (Jones et al. 2012).The complete monoclonal antibodies to the human glucagon receptors were produced by Amgen Company. This antibody was found to reduce the fasting plasma glucose levels and the decrease was dose dependent. Glucose tolerance increased in the patients after a week. Fig 2: Sequence Composition of Murine, Chimeric, Partial human and complete human antibodies. (Saha 2010). Therapeutic tools: For the diagnosis of Type II Diabetes mellitus the antibodies are used as immunological markers. Some of them are Islet Cell Antibodies ( ICA), Insulin autoantibodies ( IAA), GAD autoantibodies, Glutamic acid decarboxylase, Protein tyrosine phosphatase antibodies ( IA2) and genetic markers such as HLA etc., (Jones et al, 2012). The insulin binding immunoglobulins are present in the patients who were exposed to the insulin therapy in type 2 Diabetes mellitus. These antibodies were found to be Immunoglobulin G. Antibodies were also produced against these immunoglobulins in some patients. Many factors were found to contribute to the production of insulin antibodies. (Jones et al, 2012). Some of the factors such as mode of administration, origin of the species, genetic purity and physical state of insulin induce the insulin antibodies production. Both the insulin dependent and non dependent patients were found to have the antibody production. The patients with the HLA-DR3 gene were found to produce larger amount of antibodies than the other type 2 Diabetes mellitus patients. (Jones et al, 2012). Similarly if the insulin purity is less and contaminated with other components then the chance of cross reaction and stimulation of the immune system is high. The species is the next important factor for the production of antibodies in the humans. The insulin from the other sources may induce the immune system of the humans to produce the antibodies. The foreign body may create immunogenic. To measure the concentration of the antibodies produced in the patients the techniques such as quantitative radio immunoelectrophoresis, fluid phase methods, Scatchard analysis were used. (Van Haeften 1989). The alarming rise of the Type 2 Diabetes mellitus patients in the world, with more than 20 million people in the United States alone, has brought it to the top rank. (Jones et al, 2012). The infections, brings greater risk factor for the diabetes patients and the complications associated with it are heavy. Many immune defects are also found to be linked with the diabetes, particularly with the poorly controlled diabetes. Macrophage and neutrophil functions are impaired due to the poor glucose level. The defects in the proliferation of the T-cell, B-cell and dendritic cell functions are described in the patients with diabetes. (Van Haeften 1989). Streptococcus pneumoniae causes pneumonia and meningitis and mild infections in our body. To protect the body against the carriage of the pneumococcal carriage and infections is complex and multifactorial and the mechanisms are dependent on antibody and others. S. pneumoniae surface protein A and capsular polysaccharide are used for the determination of the antibodies to the type 2 diabetes mellitus. (Van Haeften 1989). For the analysis, ELISA was used. Enzyme linked immuno sorbant assay (ELISA or enzyme assay) is the most commonly used immunological method for the identification of many diseases. Elisa is used to detect the antigens present in the given sample by using the specific antibodies. (Van Haeften 1989). The determination of the presence or absence of the antibody concentration in the blood enables us to identify the disease and understand the level of the disease.  The measurement of the antigen and antibody by using sensitive assays are the useful indicators of the immune status. (Crowther 2001). The results indicate that the patient with T2DM had the protein effect compromisation. Diabetes creates a new environment for the differentiation and functions of T-cell subsets. Interleukin – 1 (IL-1) is found to affect the insulin secretion and beta cells. The inability of the beta cells to produce insulin in the required amount causes diabetes. Since insulin resistance creates in the patients, steps were taken to increase the pancreatic beta cell expression and the reduction of interleukin- 1 receptor antagonist. Interleukin has the ability to reduce the apoptosis of beta cells. (Larsen et al. 2009). The reduction in the destruction of the beta cells increases the insulin secretion and maintains the glucose and serum levels normal in the patients. The patients were treated with the Anakinra (interleukin- 1 receptor antagonist) and placebo for 39 weeks and the C-peptide, proinsulin and insulin levels in the patients were assayed using ELISA technique. (Larsen et al. 2009). Time- resolved fluoroimmunoassay were used to determine the C-peptide levels. Anakinra treatment was found to increase the Beta –cell function and reduce the inflammatory markers in the diabetes patients. (Larsen et al. 2009). Many studies have found that insulin antibodies are developed in the type 2 diabetes patients who are exposed to insulin therapy. Hypoglycemic attacks were more common in the patients with the insulin therapy.to overcome the insulin antibodies, and to reduce the insulin binding capacity to the antibodies, the recombinant IGF-1 was used instead of insulin. The treatment with recombinant IGF-1 reduced the insulin antibodies and reduced the insulin binding capacity of antibodies. (Hirano, Arima and Oiso 2008). Insulin resistance (IR) is one of the major risk factor associated with rheumatoid arthritis. Insulin resistance has a close association with the tumor necrosis factor alpha (TNF- α). (Larsen et al. 2009). TNF- α is one of the mediators of insulin resistance. As a result, obesity occurs in the healthy and arthritis patients. Hence the anti TNF- α therapy for insulin resistance was analyzed. The insulin resistance was analyzed using the Homeostasis model assessment. The therapy concluded that insulin resistance improved in the healthy individuals than the arthritis patients. Obesity related problems were found to be reduced in the arthritis patients. The anti TNF- α therapy failed to improve the IR in the type 2 Diabetes mellitus patients. Adipose tissue which is the source of inflammation contains inflammatory cytokine. The TNF- α and interleukin – 6( IL- 6) stimulates the c-Jun amino- terminal kinase (JNK) and the I- kB kinase – β (IKK- β)/ nuclear factor – kB ( NF-kB) pathways. (Strvropoulos- Kalinoglou et al. 2012). This stimulation of TNF- α and IL-6 up regulates the mediators of inflammation and leads to the insulin resistance. Anti TNF- α therapy reduces these adiposekines and increases the serum resisting levels. (Strvropoulos- Kalinoglou et al. 2012). Anti TNF- α therapy also reduces the lipolysis in the muscle by inhibiting the TNF- α actions. The study thus concludes that anti TNF- α therapy alone cannot reduce the obesity and insulin resistance. Additional measures must be taken to combat Insulin resistance in the obese Rheumatoid arthritis patients. Chromium is found to increase the glucose metabolism in the type II diabetes mellitus patients. The deficiency of chromium has lead to impaired glucose tolerance in the patients. The administration of chromium chloride in the diet has resulted in the increase of glucose metabolism. The mechanism of action of chromium on glucose metabolism is not yet known. Similarly vanadium was found to increase the glucose uptake, amino acid and lipid metabolism, thyroid functions, enhance insulin sensitivity and so on. (Okochi and Okpuzor 2005). The active form of Nicotinamide is Nicotinamide adenine dinucleotide ( NAD) and the phosphate derivative( NADP). (Strvropoulos- Kalinoglou et al. 2012). These two co enzymes are essential for the carbohydrate and lipid metabolism. Administrating a regular dose of nicotinamide as supplement have found to be effective in the newly diagnosed diabetes patients and in the patients who have positive islet cell antibodies. (Okochi and Okpuzor 2005).   Insulin like growth factor I (IGF-I) is produced by hepatocytes, pancreas and tissues. IGF-I stimulates the islet cell replication and prevents the autoimmune beta cell destruction and delay of onset of diabetes. The pancreatic islet – specific IGF-1 over expression promoted the islet cell regeneration and a speedy recovery from diabetes. (Robertson et al. 2008). The laboratory results showed that the deficiency of the IGF-I factor resulted in the increase of the islet beta cell mass and reduced early diabetes.over expressing the chronic IGF-I resulted in the insulin like effects on glucose disposal and glucose production in the rats. (Robertson et al. 2008). They were also found to have resistant to streptozotocin- induced diabetes and prevented weight loss and death due to diabetes. Thus it was concluded that IGF-I is useful for beta cell mass production and not for insulin secretion or hepatic glucose production. Glutamic Acid Decarboxylase antibody test (GADA test) is used for the detection of the type of diabetes present in the human. This test is the most common test to diagnose the type 2 diabetes mellitus in the patients. This test is performed to check the presence of antibodies for GAD. GADA test is performed before insulin therapy. (Lundgren et al. 2010). Islet Cell Antibody (ICA) antibodies are present in the human body before the onset of the disease. These antibodies form a heterogeneous group comprising of Insulin Auto- antibodies (IAA), GADA and IA2. (Lundgren et al. 2010). These antibodies appear in the body at different times and at different periods of the diabetes mellitus. Immunofluorescence technique using the frozen sections can be used for the detection along with western blotting and immunohistochemistry. Insulin Autoantibody (IAA): They are the first antibodies appearing at the asymptomatic period. They are found in the majority of the children who are prone to this disease. The latent autoimmune diabetes of Adults (LADA) is found to be present in 20% of the Type 2 Diabetes mellitus patients. (Hirano, Arima and Oiso 2008). The serological marker is used for the identification and diagnosis of Type 2 DM. Insulin auto antibodies are present in nearly half of the individuals with the type 2 Diabetes mellitus. (Hirano, Arima and Oiso 2008). These antibodies do not affect the glucose levels in the blood, but causes insulin resistance and frequent hypoglycemia attack in the patients. Insulin- associated Tyrosine Phosphatase Antibody test (IA2): The main domain of this antibody is the phosphatase domain and it is mainly detected in the type 1 Diabetes mellitus patients. Similarly Glutamic acid decarboxylase 65 (GAD 65) is a type of major islet cell antigen, whose antibodies in the serum act as the immunological markers for the autoimmune disease. (Thomas et al. 2012). These islet cell antibodies are detected by the ELISA technique. GAD 65: Glutamic acid decarboxylase (GAD) is the rate-limiting enzymes used in the production of gamma aminobutyric acid (GABA). (Thomas et al. 2012). There are two forms of GAD: they are GAD65 and GAD67. They are found at the neurons. The auto antibodies of GAD 65 and GAD67 are found to be associated with the type 1 diabetes and latent autoimmune diabetes in adults. (Yarlagaclcla et al. 2011). The healthy individuals and chronic psychotic disorder patients were analyzed for the presence of auto antibodies in the blood sample. It was concluded that there were no difference in the results and there was no significant result for the presence of GAD65/67 antibodies in the sample. (Yarlagaclcla et al. 2011). Recombinant insulin like growth factor has the similar properties of insulin growth factor – I (IGF-I) and the structures are similar. The recombinant IGF-1 (rhIGF-I) is found to improve the glucose control and insulin sensitivity at liver and muscle in the patients with Type 2 Diabetes mellitus. Mouse impaired with the IGF-I and insulin signaling was chosen for an experiment. This mouse had the problems of insulin resistance and had the risk of Type 2 Diabetes mellitus. (Yarlagaclcla et al. 2011). The treatment with rhIGF- I enabled the mouse to decrease the fat mass and increase the lean body mass. Insulin levels, fatty acid and triglyceride levels were not affected in this treatment. This treatment had no changes on overall insulin sensitivity and activity. These were confirmed using the pyruvate and glutamine tolerance tests. This experiment concluded that improvement in hyperglycemia by gluconeogenesis inhibition occurred in the mouse and the study of functional IGF-I receptor in skeletal muscle for better analysis was suggested to improve the insulin sensitivity of Type 2 Diabetes mellitus. (Pennisi et al. 2006). References: Ahrens, B, 2011. Antibodies in Metabolic Diseases, New Biotechnology, Vol.28, No. 5, pp: 530 – 537. Crowther. J., 2001. The ELISA Guidebook, Humana Press. Joslin, EP and Kahn, CR, 2005. Joslin’s Diabetes Mellitus. Ed. by C. Ronald Kahn et al. Lippincott Wilkins. Jones, RM, Mark Jones, R, Thurston, DE, Rotella, D, Guccione, S, Martinez, A, Fox, D and Ganellin, R., 2012. New Therapeutic Strategies for Type 2 Diabetes: Small Molecule Approaches, Royal Society of Chemistry. Hirano, M, Arima, H and Oiso, Y, 2008. Immunological Insulin Resistance due to Insulin Antibodies developed after Cessation of Insulin Therapy in a Patient with Type 2 Diabetes, Diabetes care, Vol.31. No.11. pp. e84. Larsen, CM, Faulenbach, M, Vaag, A, Ehses ,JA, Donath, MY and Mandrup-Poulsen, T, 2009. Sustained effects of Interleukin-1 Receptor Antagonist Treatment in Type 2 Diabetes, Diabetes care, Vol.32, No.9, pp. 1663-8. Lundgren, VM, Isomaa, BO, Lyssenko, V, Laurila, E, Korhonen, P, Groop, LC and Tuomi, T, 2010. GAD Antibody Positivity Predicts Type 2 Diabetes in an Adult Population, Diabetes, Vol.59, No.2, pp: 416 – 422. Okochi, VI and Okpuzor, J. 2005. Micronutrients as Therapeutic Tools in the Management of Sickle Cell Disease, Malaria and Diabetes, African Journal of Biotechnology, Vol.4, No.13, pp: 1568 – 1579. Pennisi, P,  Gavrilova ,O, Setser-Portas, J, Jou ,W, Santopietro ,S, Clemmons, D, Yakar, S and, LeRoith, D., 2006. Recombinant Human Insulin-like Growth Factor-I Treatment inhibits Gluconeogenesis in a Transgenic Mouse Model of Type 2 Diabetes Mellitus, Endocrinology, Vol.147, No.6, pp: 2619- 2630. Robertson, K, Lu, Y, Jesus, KD, Li, B, Su, Q, Lund, PK and Liu, JL, 2008. A General and Islet cell-enriched Overexpression of IGF-I results in Normalized Islet Cell Growth, Hypoglycemia, and Significant Resistance to Experimental Diabetes, American Journal of Physiology Endocrinology and Metabolism, Vol.294, pp: E928-E938. Stavropoulos-Kalinoglou, A, Metsios, GS, Panoulas, VF, Nightingale, P, Koutedakis, Y and Kitas, GD, 2012. Anti-tumour Necrosis factor Alpha Therapy improves Insulin Sensitivity in Normal-weight but not in Obese Patients with Rheumatoid Arthritis, Arthritis Research & Therapy, Vol.14, No.4, R160. Saha, GB, 2010. Fundamentals of Nuclear Pharmacy, Springer. Van Haeften, TW, 1989. Clinical Significance of Insulin Antibodies in Insulin- treated Diabetic patients, Diabetic care, Vol.12, No.9, pp: 641 – 648. Thomas, N, Jeyaraman, K, Asha, HS and Velavan, J, 2012. A Practical Guide to Diabetes Mellitus, JP Medical Ltd. Yarlagaclcla, A, Taylor, JH Jr, Hampe, CS, Hampe, CS, Alfson, E and Clayton, AH, 2011. GAD65 antibodies, Chronic psychosis, and Type 2 Diabetes Mellitus, Innovations in Clinical Neuroscience, Vol.8, No.8, pp: 34 - 36. Read More