Elisa technique
- 2. And if you would count the graces of Allâh, never could you be able to count them Truly! Allâh is Oft-Forgiving, Most Merciful. (Surah An-Nahl:18) )
- 3. ELISA ENZYME LINKED IMMUNOSORBENT ASSAY By: Dr.MUHAMAD SALMAN PASHA
- 4. Enzyme-linked immunosorbant assay (ELISA) is a non-isotopic immunoassay. An enzyme is used as a label in ELISA
- 6. 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.
- 7. 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
- 9. Analyte The sample being analyzed and in immunoasssays the analyte is either Antibody or Antigen
- 10. BASIC PRINCIPLES OF ELISA
- 11. ELISA is based on the immunochemical principles of antigen-antibody reaction. The stages of ELISA are summarized: 1. The antibody against the protein to be determined is fixed on an inert solid such as polystyrene. 2. The biological sample containing the protein to be estimated is applied on the antibody coated surface. 3. The protein antibody complex is then reacted with a second protein specific antibody to which an enzyme is covalently linked. These enzymes must be easily assayable and produce preferably coloured products. Peroxidase, amylase and alkaline phosphatase are commonly used.
- 12. 4. After washing the unbound antibody linked enzyme, the enzyme bound to the second antibody complex is assayed. 5. The enzyme activity is determined by its action on a substrate to form a product (usually coloured). This is related to the concentration of the protein being estimated. The principle for the use of the enzyme peroxidase in ELISA is illustrated next.
- 14. Enzyme Product Product No. (Total Assays ¹) Absorban ce and Color Detection Limit ² Primary (1°) and Secondary (2°) Antibody Dilutions ² AP PNPP Substrat e 1- Step Sol ution Tablet Kit Tablets (105) Powder (25g) 37621 (1000 wells) 37620 (5250 wells) 34047 (5250 wells) 34045 (250,000 wells) 405nm Yellow ~10ng/well (100ng/mL) 1° 1:500 2° 1:5K to 1:20K
- 15. HRP ABTS Substrate 1-Step Solution Tablets (50) 37615 (2000 wells) 34026 (3330 wells) 410nm (650nm) Green ~250pg/well (2.5ng/mLl) 1° 1:1K 2° 1:5K to 1:50K Gal ONPG Substrate Powder (5g) 34055 410nm Yellow ~10ng/well (100ng/mL) 1° 1:500 2° 1:5K
- 16. 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
- 17. INTRODUCTION
- 18. 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
- 19. 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).
- 21. Sandwich ELISA The ELISA plate is coated with Antibody to detect specific antigen
- 22. Sandwich ELISA
- 23. 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
- 24. Indirect ELISA
- 25. DIRECT SANDWICH ELISA An antigen coated to a multiwell plate is detected by an antibody that has been directly conjugated to an enzyme. This can also be reversed, with an antibody coated to the plate and a labeled antigen used for detection, but the second option is less common. This type of elisa has two main advantages: It is faster, since fewer steps are required. It is less prone to error, since there are fewer steps and reagents.
- 28. Applications: o ELISA is widely used for the determination of small quantities of proteins (hormones, antigens, antibodies) and other biological substances. o The most commonly used pregnancy test for the detection of human chorionic gonadotropin (hCG) in urine is based on ELISA. By this test, pregnancy can be detected within few days after conception. o ELISA is also been used for the diagnosis of AIDS.
- 29. 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
- 30. 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
- 31. 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
- 32. ELISA TEST