RADIOIMMUNOASSAY OF DIGITALIS AND INSULIN.pptx

An immunoassay is a biochemical test that detects and quantifies specific substances (analytes) in a sample by using the specific reaction between an antibody and an antigen.An antibody is a protein that is produced by the body in response to an “invading” (foreign) substance.​

Antibodies are produced as a part of body’s immune response to protect itself.An antigen is the substance that the body is trying to “fight off” by mounting an immune response.​

For example, the drug is the antigen that binds to the antibody.​Radioimmunoassay (RIA) is an immunoassay that uses radiolabelled molecules in a stepwise formation of immune complexes.​RIA works on the principle of competitive binding between a radio-labeled antigen and an unlabeled antigen for a limited number of antibody sites, with the radioactivity measured being inversely proportional to the concentration of the antigen in the sample.​Digoxin is the primary cardiac glycoside extracted from the foxglove plant, Digitalis lanate, belonging to family Plantaginaceae.​

It is also used for drug preparations that contain cardiac glycosides, like Digoxin & Digitoxin extracted from various parts of the plant.​
It is used to increase cardiac contractility and as an antiarrhythmic agent to control the heart rate, particularly in the irregular atrial. fibrilla.Insulin is a hormone produced by the pancreas that helps regulate blood sugar by allowing glucose to enter the body's cells for energy​

​

Essential for life: You cannot live without insulin because it’s vital for cells to get energy.​

Diabetes treatment: It is primarily used to treat diabetes, a condition where the body has too much sugar in the blood. ​
Prevents complications: Managing high blood sugar with insulin can help prevent serious diabetes-related complications like heart disease, kidney problems, and nerve damage.

1. RADIOIMMUNOASSAY OF DIGITALIS AND INSULIN HONEY HETTY 2 nd SEM MPHARM 2024 DEPARTMENT OF PHARMACEUTICAL CHEMISTRY ST. JAMES’ COLLEGE OF PHARMACEUTICAL SCIENCES, CHALAKUDY 1

2. CONTENTS 2

3. IMMUNOASSAY An immunoassay is a biochemical test that detects and quantifies specific substances ( analytes ) in a sample by using the specific reaction between an antibody and an antigen. 3

4. ANTIBODY ANTIGEN An antibody is a protein that is produced by the body in response to an “invading” (foreign) substance. Antibodies are produced as a part of body’s immune response to protect itself. An antigen is the substance that the body is trying to “ fight off” by mounting an immune response. For example, the drug is the antigen that binds to the antibody. 4

5. RADIOIMMUNOASSAY PINCIPLE AND THEORY Radioimmunoassay (RIA) is an immunoassay that uses radiolabelled molecules in a stepwise formation of immune complexes.     Use of radioactive material Antigen antibody binding theory Detection of compounds RIA works on the principle of competitive binding between a radio-labeled antigen and an unlabeled antigen for a limited number of antibody sites, with the radioactivity measured being inversely proportional to the concentration of the antigen in the sample. RIA works on the principle of competitive binding between a radio-labeled antigen and an unlabeled antigen for a limited number of antibody sites, with the radioactivity measured being inversely proportional to the concentration of the antigen in the sample. Radio Immuno Assay 5

6. RIA OF DIGITALIS 6 Digoxin is the primary cardiac glycoside extracted from the foxglove plant, Digitalis lanate , belonging to family Plantaginaceae . It is also used for drug preparations that contain cardiac glycosides, like Digoxin & Digitoxin extracted from various parts of the plant.
It is used to increase cardiac contractility and as an antiarrhythmic agent to control the heart rate, particularly in the irregular atrial. fibrilla .

7. PRINCIPLE OF RIA OF DIGITALIS The assay is based on the use of 125-I labelled digoxin and of a gel equilibration technique for the separation of antibody- bound and free digoxin. Digoxin in serum samples competes with radio-labelled (125-I) digoxin derivative for binding sites on the antibody to digoxin. The unbound digoxin is then separated from bound form. It is then quantified by counting radioactivity & concentration of unlabelled digoxin in serum. Sample is calculated by comparison to digoxin standards. 7

8. MATERIALS REQUIRED FOR ASSAY 8

9. PROCEDURE FOR ASSAY STANDARD CURVE     9

10. UNKNOWN  10

11. RIA OF INSULIN Insulin is  a hormone produced by the pancreas that helps regulate blood sugar by allowing glucose to enter the body's cells for energy Essential for life : You cannot live without insulin because it’s vital for cells to get energy. Diabetes treatment: It is primarily used to treat diabetes, a condition where the body has too much sugar in the blood. Prevents complications: Managing high blood sugar with insulin can help prevent serious diabetes-related complications like heart disease, kidney problems, and nerve damage. 11

12. A sensitive radioimmunoassay has been developed for the detection of Insulin chains in the Plasma of normal and diabetic human subjects. The S-Sulphonated derivatives of bovine insulin A and B chains were Purified, iodinated and used for immunoassay by double antibody technique. 12

13. MATERIALS 13

14. STANDARD A set of six insulin standards in the range of 0-200 (μ 10/mL (0, 7.5, 12.5, 25, 50, 100 and 200 μ 10/mL) were prepared by dissolving Procine insulin in bovine serum depleted of insulin by activated charcoal. I nsulin s tandards were calibrated against WHO International Inference Preparation. 14

15. Preparation  of Radiolabelled Insulin:   Procaine Insulin was  radiiodinated with 125- I using      chloramine –T-oxidation Labelled insulin was Purified by gel  filteration  over a  Siphadex  G-75 column 15

16. PROCEDURE 16

17. ASSAY PROCEDURE Incubation of 200 μL of standard /sample along with 100 μ L of anti- proline insulin antibody applied coupled to magnetizable cellulose particles overnight at room temperature with gentle stirring. This was followed by further addition af 100 μ L tracer with second incubation for 4hr at room temperature under gentle stirring. At the end of incubation 1mL of wash buffer was added and the assay tubes containing reaction mixture were placed in a magnetic rack for 15 min. The particles were allowed to form a Firm pellet. The supernatant was discarded by gently inverting the rack. The radioactivity were measured using multiwell gamma counter calibrated for 125 I. A standard curve was constructed by plotting (% B)/ B D (y-axis) versus insulin concentration (x-axis). 17

18. The insulin concentration in test sample was estimated from a Standard curve by interpolation. Insulin concentration % B/B D 18

19. APPLICATIONS OF RIA BLOOD BANKING : Detection of Presence of Hepatitis B Surface antigen (HBsAg) in donated blood. DIAGNOSIS OF ALLERGIES: Detect inhalant allergens (antibody) ENDOCRINOLOGY : Detect physiology of Endocrine functions. PHARMACOLOGY : Detect of Drug Abuse or Drug poisoning Study drug kinetics 19

20. ONCOLOGY :    Narcotic drug detection Tracking of leukemia virus Research with neurotransmitter ANALYSIS OF HORMONES, VITAMINS, METABOLITES, DIAGNOSTIC MAKERS : ACTH, FSH, Triiodothyronine (T3) and Thyroxine (T4), Glucagons , Insulin, Testosterone, Vitamin B12, Prostaglandins, Glucocorticoids DIAGNOSTIC PROCEDURES FOR DETECTING INFECTION : HIV, Hepatits A, B etc THERAPEUTIC DRUG MONITORING : Barbiturates, Morphine, Digoxin 20

21. REFERENCES Vogel HG, Vogel WH. Drug Discovery and Evaluation Springer Science and Business Media 2013: 1000-11 . Berson , Solomon A, and Rosalyn S. Yalow. “Quantitative aspects of the relation between Insulin and Insulin binding antibody”. The journal of clinical investigation 1959: 226-41 Parija SC. Textbook of microbiology and immunology. 4th ed. Singapore, Springer; 2024: 301-11 Goldsmith SJ. Radioimmunoassay: review of basic principles. Semin Nucl Med. 1975;5(2):125–52 21

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