معاونت بهداشتی دانشگاه علوم پزشکی اصفهان گروه امور آزمایشگاهها مرداد 1392

1. معاونت بهداشتی دانشگاه علوم پزشکی اصفهانگروه امور آزمایشگاههامرداد 1392

2. 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.

3. 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

4. Definitions- cont • Analyte The sample being analyzed and in immunoasssays the analyte is either Antibody or Antigen • Avidity: The combined strength of multiplebondinteractions with an antigen. • Affinity : The Strength of Interaction between a molcol of antibady and an epitope.

5. 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

6. 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

8. Labeled Immunoassays • Some antigen/antibody reactions not detected by precipitation or agglutination. • Looking for very small amounts. • Measured indirectly using a labeled reactant. • Referred to as receptor-ligand assays

9. Labels • Used to detect reaction which has occurred. • Most common are: • Radioactive • Enzymes • Fluorescent • Chemiluminescent

10. Enzyme Immunoassay • Enzymes occur naturally and catalyze biochemical reactions. • Enzymes are • Cheap • Readily available • Have a long shelf life • Easily adaptable to automation. • Automation relatively inexpensive.

11. Enzyme Immunoassay • Enzymes used include: • Horseradish peroxidase • Glucose-6-phosphate dehydrogenase • Alkaline phosphatase • Β-D-galactosidase • Horseradish peroxidase and alkaline phosphatase are the most popular.

12. 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 3- Indirect ELISA

13. 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.

15. Sandwich ELISA • The ELISA plate is coated with Antibody to detect specific antigen

16. Sandwich ELISA-Cont • Antibody bound to solid phase. • If looking for antigen must have multiple epitopes, bound antibody specific for one epitope, second labeled antibody added specific for a different epitope.

17. Sandwich ELISA-Cont • Antigens detected can be • Antibodies • Hormones • Proteins • Tumor markers • Microorganisms especially viruses • Enzyme label used to detect reaction

18. Sandwich ELISA-Cont • Add patient sample with antigen. • Antigen will bind to antibody bound to solid phase. • Add enzyme labeled antibody directed against a different epitope on the antigen. • Wash the plate, so that the unbound antibody-enzyme conjugates are removed. • Add substrate, measure intensity of color.

19. Sandwich ELISA

20. 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

21. 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

22. 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.

23. Indirect ELISA-Cont • 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

24. Indirect ELISA

25. 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

26. 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

27. Standards or Calibrators • Substance of exact known concentration. • Usually run for each new lot number • Based on results create standard curve. • Standard curve used to “read” results or built into machine to provide results.

28. 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 Absorption nm Concentration ng/ml

29. 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

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

31. 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

32. Competitive and Noncompetitive Immunoassays • The measurement of analyte in an immunoassay is achieved by using either • a competitive or • a noncompetitive format.

33. Competitive format • unlabelled analyte (usually antigen) in the test sample is measured by its ability to compete with labeled antigen in the immunoassay. • The unlabeled antigen blocks the ability of the labeled antigen to bind because that binding site on the antibody is already occupied.

34. Con’ted • Thus, in a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present. The amount of antigen in the test sample is inversely related to the amount of label measured in the competitive format (Figure 1-7).

35. one step competitive format • In the one step competitive format (see Figure 1-8), both the labeled antigen reagent (Ag*) and the unlabeled specimen (or test sample analyte) compete for a limited amount of antibody.

36. two step competitive format • In the two step competitive format, the antibody concentration of the reaction solution is present in excess in comparison to the concentration of antigen. Antibody reagent is first incubated with specimen containing antigens of interest; then in the second step, labeled antigen is added. Remember that in the competitive format, less bound labeled antigen indicates more antigen present in the test sample. Two step competitive assay formats provide several fold improved assay sensitivity compared to one step assay formats.

38. Noncompetitive (Sandwich) Method • Noncompetitive assay formats generally provide the highest level of assay sensitivity and specificity and are applied to the measurement of critical analytes such as cardiac and hepatitis markers. This format is referred to as a “sandwich” assay because analyte is bound (sandwiched) between two highly specific antibody reagents (Figure 1-10).

40. Noncompetitive assay • Noncompetitive assay formats can also utilize either one step or two step methods, as with the competitive assay. • The two step assay format employs wash steps in which the sandwich binding complex is isolated and washed to remove excess unbound labeled reagent and any other interfering substances. • The two step noncompetitive format usually offers the highest specificity and sensitivity of all the assay formats discussed here.