01- Insulin ELISA AFM STUDY MATERIAL.pptx

Insulin ELISA AFM STUDY MATERIAL

1. Determination of blood Insulin concentration by enzyme linked immunosorbent assay (ELISA) Prepared and presented By Dr. Mohamed Agha

2. Cases CASE I : A 50-year-old female presents to your clinic with complaints of excessive thirst , fluid intake , and urination . She denies any urinary tract infection symptoms. She reports no medical problems, but has not seen a doctor in many years. On examination she is an obese female in no acute distress. Her physical exam is otherwise normal. The urine analysis revealed high glucose level , and a serum random blood sugar level was 320 mg/dl . What is the most likely diagnosis? A case of type II DM (might be insulin resistance state).

3. CASE II An 82-year-old white woman presented to her primary care physician with history of episodic confusion and somnolence. The episodes occurred typically in the morning, just after waking. They lasted minutes and were relieved when she ate her breakfast or had juice . As she was waiting for check-out, she developed confusion, a capillary blood glucose test was performed, and she was noted to have a plasma glucose level of 28 mg/dl. She was given juice and her symptoms resolved after a few moments. What is the most likely diagnosis? A case of insulinomas (islet cell tumors of the pancreas).

4. Insulin Human insulin is a peptide hormone composed of 51 amino acids. It is produced in the islets of Langerhans in the pancreas. It is a hormone central to regulating carbohydrate and fat metabolism in the body.

5. Diseases associated with insulin disturbances Elevated insulin levels are seen with: Insulinomas Insulin resistance, such as in obesity, Diabetes (Type 2) and metabolic syndrome. Decreased insulin levels are seen with: Diabetes (Type 1) Pancreatic diseases such as chronic pancreatitis (including cystic fibrosis) and pancreatic cancer

6. Enzyme Linked Immunosorbent Assay (ELISA) Definition: This is a sensitive and specific enzyme-based immunoassay that is used for quantification of a substance even of picograms (10 -12 g) range (ex. hormones, drugs, pathogens). The name suggests three components: Antibody. Solid phase (sorbent). Enzymatic amplification.

7. Principle: Antigen: a high molecular weight (>5000) substance recognized by the immune system of an organism as foreign. The organism reacts to antigen by producing a specific antibody. Hapten: a low molecular weight (<5000) substance can´t stimulate the production of antibodies. It must be carried by a specific carrier to stimulate the production of a specific antibody.

8. Antibody (immunoglobulin): a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. recognizes a unique part of the foreign target, termed an antigen. ELISA: is based on an antigen-antibody (immunological) reaction

9. Equipment: Microtiter plate Microplate sealing tape Automatic pipettes

10. ELISA washer ELISA reader

11. Types of ELISA: Indirect ELISA Sandwich ELISA Competitive ELISA

12. Indirect ELISA Used to measure antibody levels.   The antibody reacts with specific antigen attached to the solid phase.   Enzyme activity measured is proportional to the amount of specific antibody in the original serum.   Color produced is directly proportional to antibody amount.

13. Indirect ELISA

14. Sandwich ELISA The double antibody method. Used to measure antigen levels. The unknown antigen reacts with specific antibody attached to the solid phase. Enzyme activity measured is proportional to the amount of specific antigen in the original solution. Color produced is directly proportional to antigen amount.

15. Sandwich ELISA

16. Competitive ELISA Enzyme- labeled antigen and the unknown amount of the antigen in the sample react with a specific antibody attached to a solid phase. Enzyme activity measured is proportional to the proportion of labelled antigen in mixture of labelled and unlabelled antigen. Color produced is inversely proportional to unlabelled antigen amount.

17. Principle of the insulin ELISA Based on the sandwich principle . The microtiter wells are coated with a monoclonal antibody directed towards Insulin molecule. Samples and standards are pipetted into these wells, along with the enzyme conjugate ( monoclonal antibody against human insulin, which has horseradish peroxidase ( HRP ) enzyme covalently linked to it). The amount of bound HRP complex is proportional to the concentration of Insulin in the sample.

18. After washing to remove unbound materials, a substrate for HRP is added.  The substrate is converted to a colored compound by the action of HRP.  The intensity of colour developed is proportional to the concentration of Insulin in the patient sample.

19. Assay Procedure