1. ENZYMES IN MEDICINE

2. ENZYMES - USE IN LABORATORY ASSAYS Enzymes isolated from different sources - used for determination of various substances in the blood, plasma/serum and urine  enzyme methods much more specific than chemical methods, the presence of relative substances with similar chemical properties does not hinder Components of commercial kits or diagnostic strips- determination of glucose - glucose oxidase, peroxidase cholesterol - cholesterol esterase, cholesterol oxidase peroxidase, urea – urease, ……. in blood, plasma, serum - proof of glucose (glucose oxidase), …….. in blood or urine (strips) Markes in the immunochemical analysis - ELISA (=enzyme-linked immunoadsorbent assay) – peroxidase, alkaline phosphatase

3. GOD POD FOR GLUCOSE PRINCIPAL : Glucose is oxidized by glucose oxidase and produces gluconate and hydrogen peroxide. The hydrogen peroxide is oxidatively coupled with 4- aminoantipyrine and phenol. The intensity of the coloured complex ( quinonimine) is proportional to the glucose concentration in the sample and can be measured photometrically at 505 nm( 500-540nm)

4. GOD Glucose + + H 2 O Gluconate + H 2 O POD 2H 2 O 2 + Phenol + H2O + 4-Aminoantipyrine Red Quinonemine + H 2 O 2

5. TOTAL CHOLESTEROL / HDL CHOLESTEROL Cholesterol esters are hydrolysed by the cholesterol esterase to give free cholesterol and fatty acid molecules. The free cholesterol gets oxidized by cholesterol oxidase to liberate cholest-4 ene-3 one and peroxide. The indicator quinonimine is formed from hydrogen peroxide and 4- aminoantipyrine in the presence of phenol and peroxidase. This coloured complex is measured at 505 nm( 500-540nm) and is directly proportional to the cholesterol concentration present in the sample.

6. CHE Cholesterol ester + H 2 O Cholesterol + FFA CHO Cholesterol + O 2 Cholest -4ene-3-one + H 2 O 2 POD 2H 2 O 2 + Phenol + 4-Aminoantipyrine Red Quinoneimine complex + H 2 O

7. TRIGLYCERIDES Triglycerides are determined after enzymatic hydrolysis with lipases. Serum TGL are hydrolysed to glycerol and FFA by lipases. In the presence of ATP and Glycerol kinase. Glycerol is converted to Glycerol-3-Phosphate which is then oxidized by GPO to give H2O2. Peroxidase catalyses the conversion of H2O2, 4-Aminoantipyrine and ESPAS to a coloured quinoneimine complex measurable at 546nm.

8. TRIGLYCERIDES Lipases Triglycerides + H2O Glycerol + Fatty acids GK Glycerol + ATP Glycerol-3-P + ADP GPO G-3-P + O2 H2O2 + DHAP POD H2O2 + 4-Aminoantipyrine + ESPAS Purplish Brown Quinoneimine complex + H2O + HCL

9. URIC ACID Enzymatic conversion of uric acid to allantoin quantitativelyproduces H 2 O 2. H 2 O 2 thus produced is measured through an indicator reaction involving peroxidase. Uricase Uric acid + O 2 + 2H 2 O Allantoin + CO 2 + H 2 O 2 Peroxidase 2H 2 O 2 + DHBS + 4AAP Red Quinoneimine Complex + HCL + 4H 2 O

10. SGPT (ALT) SGPT catalyses the transfer of amino acids from L-Alanine to 2- Oxaloglutarate. The rate of reaction is monitored using a coupling enzyme LDH. The pyruvate formed is converted to Lactate by SGPT in the presence of NAD + LDH L-Alanine + 2 Oxaloglutarate Pyruvate + L-Glutamate. SGPT Pyruvate + NADH + H + L- Lactate + NAD +

11. UREA Urea is hydrolysed to ammonia and CO2 by urease. Ammonia produced in this reaction reacts with Ketoglutarate to form glutamate in the presence of glutamate dehydrogenase. NADH is oxidized to NAD which is measured as decrease in in absorbance at 340 nm. The rate of decrease in absorbance at 340 nm is directly proportional to urea/ BUN concentration in specimen. Urease Urea + H 2 O 2NH 2 + CO 2 GLDH NH 3 + Ketoglutarate + NADH Gluatamate + NAD

12. LACTATE Lactate oxidase promotes the oxidation of lactic acid to pyruvate and H2O2. Then peroxidase catalyzes the reaction of H2O2 with a hydrogen donor, in the presence of 4-aminophenazone to form a coloured adduct. Colour intensity measured at 55nm is proportional to the lactate concentration in the sample. Lactate Oxidase Lactate + O2 Pyruvate + H2O2 Peroxidase H2O2 + TOPS + 4-AP Chromogen

13. ELISA Enzyme Linked Immunosorbent Assay

14. Definitions  Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.

15. Definitions- cont  Antigens A substance that when introduced into the body stimulates the production of an antibody  Immunoassay A laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample

17. Definitions- cont  Analyte The sample being analyzed and in immunoasssays the analyte is either Antibody or Antigen

18. Antigen  Is present naturally in the body like hormones  Is manufactured in special disease status for example human chorionic gonadotrophin hormone (HCG) which is normally produced by cells of the placenta in pregnancy is found in the body in some types of cancer  Is not present in the body in normal condition like drugs

19. Introduction  The Antibody: An immunoglobulin, a specialized immune protein, produced because of the introduction of an antigen into the body, and which possesses the remarkable ability to combine with the very antigen that triggered its production (specific affinity)  The antibody recognises and bind to the antigenic determinant region of the antigen

21. Antibody Production  Specific antibodies are produced by injecting an antigen into a mammal, such as a mouse, rat or rabbit for small quantities of antibody, or goat, sheep, or horse for large quantities of antibody. Blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen—in the serum, which can now be called antiserum.

22. Antibody Production-cont  To obtain antibody that is specific for a single antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies

24. ELISA technique Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.  The technique is divided into 1- Competitive ELISA 2- Sandwich ELISA (also called direct ELISA) 3- Indirect ELISA

25. Competitive ELISA  The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal).

27. Sandwich ELISA  The ELISA plate is coated with Antibody to detect specific antigen

28. Sandwich ELISA  Prepare a surface to which a known quantity of capture antibody is bound.  Block any non specific binding sites on the surface  Apply the antigen-containing sample to the plate.

29. Sandwich ELISA-Cont  Wash the plate, so that unbound antigen is removed.  Apply enzyme linked primary antibodies as detection antibodies which also bind specifically to the antigen.  Wash the plate, so that the unbound antibody- enzyme conjugates are removed.

30. Sandwich ELISA-Cont  Apply a chemical which is converted by the enzyme into a coloured product.  Measure the absorbency of the plate wells to determine the presence and quantity of antigen

31. Sandwich ELISA

32. Indirect ELISA  The protein antigen to be tested for is added to each well of ELISA plate, where it is given time to adhere to the plastic through charge interactions  A solution of non-reacting protein is added to block any plastic surface in the well that remains uncoated by the protein antigen

33. Indirect ELISA-Cont  Then the serum is added, which contains a mixture of the serum antibodies, of unknown concentration, some of which may bind specifically to the test antigen that is coating the well.  Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test antigen in the well. This secondary antibody often has an enzyme attached to it

34. Indirect ELISA-Cont  A substrate for this enzyme is then added. Often, this substrate changes colour upon reaction with the enzyme. The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen.  The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change. Often a spectrometer is used to give quantitative values for colour strength

35. Indirect ELISA

36. An example of an ELISA experiment  Before starting the work read kit instruction carefully  1- The 96 well plate is labeled carefully and the first wells are used to draw the standard curve

37. An example of an ELISA experiment-Cont  The sample is added to plate in duplicate or triplicate and then the mean result is calculated  The quality control sample which is provided with the kit is treated as the test samples

38. Results  After reading the results the standard curve is drawn were the concentration is blotted on the X-axis and the absorbance on the Y-axis Concentration ng/ml Absorption nm

39. Results-cont  The standards concentrations is specified on the x-axis and the reading of each standard is specified on the y-axis and the standard curve is drawn

40. Results-cont  This standard curve is used to determine the unknown concentration of each sample by finding the opposite concentration to the absorbance Concentration ng/ml Absorption nm

41. Results-cont  The quality control sample concentration is determined from the standard curve and if the result is in the range given by the kit manufacturer the results could be accepted

42. Useful sites  http://www.edumedia-sciences.com/en/a543-direct- enzyme-linked-immunosorbent-assay-elisa  ELISA  http://www.sumanasinc.com/webcontent/animations/cont ent/ELISA.html http://www.sumanasinc.com/webcontent/animations/cont ent/ELISA.html  http://www.biology.ualberta.ca/facilities/multimedia/upload s/procedures/elisa-sound.swf http://www.biology.ualberta.ca/facilities/multimedia/upload s/procedures/elisa-sound.swf