Basic Immunologic Procedures

1. Basic Immunologic Procedures Part 6 Labeled Immunoassays Terry Kotrla, MS, MT(ASCP)BB

2. 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.

3. Terminology • Ligand is the substance to be measured. • Defined as a molecule that binds to another molecule of a complementary configuration. • Usually binds to the substance the test is trying to detect. • The receptor is what binds the specific target molecule. • “Sandwich” technique is an example.

4. “Sandwich” Technique ELISA

5. Terminology • Reactions may be competitive or non-competitive • Competitive – labeled known and patient unknown are added to reaction and “compete” for the target. • For example, looking for an antibody. • Add labeled reagent antibody of known specificity, patient sample and known antigen. • Patient antibody competes with reagent antibody for the target antigen. • Concentration is inversely proportional to results.

6. Terminology • Non-competitive • Add patient sample, for example looking for antibody, to known reagent antigen. • Reaction occurs and the concentration is directly related to the amount of antibody in patient sample.

7. Terminology • Heterogeneous or homogeneous • Heterogeneous assays called separation assays • Require multiple steps • Careful washing of surface to remove unbound reagents and samples. • Homogeneous assays do NOT require a separation step. • Mix reagents and patient sample. • Measure the labeled product.

8. Competitive Binding • Add known labeled antigen • Add unknown antigen • Will compete with each other for sites on bound antibody molecule. • Must wash off unreacted substances. • Type of label on known antigen will determine method of detection.

9. Competitive Binding

10. Noncompetitive Binding • Patient sample added. • Will react with its homologous antigen or antibody, depending upon what is being tested for. • The reaction is measured and the concentration is directly related to the detected amount.

11. 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.

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

13. Radioimmunassay (RIA) • Competitive binding • Uses Iodine 125 (I 125) as label • Radioactive label competes with patient for sites • High radioactivity, small amount of patient substance • Low radioactivity high amount of patient substance. • Refer to your textbook for diagrams.

14. Radioimmunoassay • Sensitive technique used to measure small concentrations of antigens. • Known quantity of antigen is made radioactive, usually with Iodine 125. • Known labeled antigen and patient sample added to the reagent antibody. • Known antigen will compete with the unknown patient antigen for sites on the antibody. • The bound antigens are separated from the unbound ones. • Can measure the radioactivity of labeled free antigen in the supernatant solution. • Can measure radioactivity of fixed labeled antigen to the well. • High radioactivity indicates a low concentration of patient antigen was bound to the reagent antibody. • Low radioactivity indicates a high concentration of patient antigen was bound to the reagent antibody. • Thus, the results are inversely related to the label detected. • Standards are run and results read off of standard curve.

15. Radioimmunoassay

16. Radioimmunassay

17. Radioimmunoassay Competitive

18. Immunoradiometric Assay (IRMA) • Labeled antibody plus patient antigen • Solid phase antigen added to bind excess antibody. • Labeled antibody binds to both patient antigen, if present, and bound antigen. • Spin down to separate • Labeled antibody/antigen remain in solution. • Measure radioactivity.

19. Advantages/Disadvantages • Advantages • Extremely sensitive and precise • Detects trace amounts of analytes small in size. • Disadvantages • Health hazard • Disposal problems • Short shelf life • Expensive equipment necessary • Enzyme immunoassays have largely replaced radioimmunoassay.

20. 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.

21. Enzyme Immunoassay • Techniques pose no health hazards. • Little reagent enzyme necessary. • Can be used for qualitative or quantitative assays. • Enzymes selected according to • Substrate molecules converted per molecule of enzyme. • Ease and speed of detection. • Stability. • Availability and cost

22. 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. • Highest turnover • High sensitivity • Easy to detect

23. Heterogenous EIA • Competitive • Enzyme labeled antigen competes with unlabeled patient antigen for antibody sites. • Wash to remove unbound reactants. • Add substrate which causes color change. • Results are inversely proportional to concentration. • More patient antigen bound, less color. • If little or no patient antigen bound, dark color. • Used to measure small antigens such as insulin and estrogen.

24. Competitive ELISA • Unknown antigen competes with labeled known antigen • Enzyme produces color reaction

25. Heterogenous EIA • Noncompetitive are very popular. • Often referred to as enzyme linked immunosorbent assay – ELISA • Enzyme labeled reagent DOES NOT participate in the initial antigen-antibody reaction. • Sandwich technique • Advantages • High sensitivity and specificity. • Relatively simple to perform. • Low cost.

26. Noncompetitive EIA • Variety of solid support • Microtiter plates • Nitrocellulose membranes • Magnetic beads • Procedure • Antigen bound to solid phase • Add patient sample, antibody will bind if present • Wash • Add known enzyme labeled antibody • Wash • Add substrate • Measure enzyme label

27. Positive Reaction = Color Change

28. Sandwich or Capture Assays • 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. • Antigens detected can be • Antibodies • Hormones • Proteins • Tumor markers • Microorganisms especially viruses • Enzyme label used to detect reaction

29. Sandwich or Capture Assays • 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. • Add substrate, measure intensity of color.

30. Rapid Immunoassays • Membrane based cassettes are rapid, easy to perform and give reproducible results. • Popular in POCT and home use. • Designed to be single use and disposable. • Membrane coated with antigen or antibody produces color reaction.

31. Rapid Immunoassays • Immunochromatography • Apply sample to one end, migrates forward. • Sample dissolves labeled antigen or antibody to which it binds. • Migrates towards detection zone where it will bind to immobilized antigen or antibody. • Color change occurs.

32. Homogeneous Enzyme Assay • Reaction which requires NO separation of reactants. • Less sensitive BUT rapid, easy to perform and automate. • Chief use is to detect low molecular weight analytes such as: • Hormones • Therapeutic drugs • Drugs of abuse • Can use serum or urine.

33. Homogeneous Enzyme Assay • Based on principle of change in enzyme activity as specific antigen-antibody combinations occur. • Reagent antigen labeled with enzyme tag. • Antibody binds to specific determinant sites on antigen, active site on enzyme blocked, causes measurable loss of activity. • Free antigen competes with enzyme-labeled antigen for limited number of antibody sites. • Enzyme activity directly related to patient antigen.

34. Fluorescent Immunoassay

35. Fluorescent Immunoassay Markers • Fluorophores or fluorochromes • Ability to absorb energy and emit light • Two most commonly used: • Fluorescein – green • Tetramethylrhodamine – red • Tests may be qualitative or quantitative

36. Fluorescent Immunoassay • Complex must form for fluorescence to occur.

37. Fluorescence

38. Fluorescent Immunoassay • Antibodies and bacteria are fixed on a glass-plate. • The surplus i.e. non-bounded antibodies are washed out, antibody-bacteria-complexes ("sandwiches") remain. • The "sandwich" becomes visible by adding fluorescent anti bovine immunoglobulin which can be seen as green light in the fluorescence microscope.

39. Fluorescent Immunoassay • Direct immunofluorescence • Tagged antibody added to unknown antigen fixed to slide • If patient antigen present = fluorescence • Indirect immunofluorescence – sandwich assay • Patient plus known fixed antigen • Allow to react and wash off unbound reactants • Add tagged anti-antibody • Fluorescence

40. Fluorescent Immunoassay

41. Positive Immunofluorescence • Cryptosporidium parvum oocysts • Photo Credit: H.D.A Lindquist, U.S. EPA

42. Fluorescent Polarization • Fluorescence polarization is a measure of the time-averaged rotational motion of fluorescent molecules. • A fluorescent molecule, when excited by a polarized light, will emit fluorescence with its polarization primarily determined by the rotational motion of the molecule. • Since the molecular rotation is inversely proportional to the molecular volume, the polarization is in turn related to the molecular size. • A small molecule rotates fast in solution and exhibits low value of polarization whereas a large molecule exhibits a higher polarization because of its slower motion under the same conditions. • Thus, changes in fluorescence polarization can reflect the association or dissociation between molecules of interest.

43. Fluorescent Polarization • Another picture to illustrate the principle. • Measure polarized light.

44. Chemiluminescent Immunoassays • The process of chemiluminescence occurs when energy in the form of light is released from matter during a chemical reaction. 

45. Chemiluminescent Immunoassays • Large number of molecules capable of chemiluminescence • Luminol • Acridium esters • Ruthenium derivatives • Nitrophenyl oxalates • Use sodium hydroxide as a catalyst • Light emission ranges from quick burst or flash to light which remains for a longer time. • Different types of instruments are required based on emission.

46. Chemiluminescent Immunoassays • Can be used for heterogeneous or homogeneous assays. • Can attach label to antigen or antibody. • Heterogeneous assays use competitive and sandwich assay. • Competitive assays used to measure smaller analytes. • Sandwich assays are used to measure larger analytes.

47. Chemiluminescent Immunoassay • Many applications. • Can measure antigen or antibody. • Add chemiluminescently tagged analyte. • Measure light which is emitted which is directly related to concentration although competitive binding assays are available.

48. Chemiluminescent Immunoassays • Best known application of chemiluminescense is luminol • Luminol reacts with the iron in blood hemoglobin.

49. References • http://web.indstate.edu/thcme/PSP/labtests/precip.htm • http://www.gla.ac.uk/departments/immunology/education/nursing/lectures/antibody.htm • http://www.cellsalive.com/mac.htm • http://jeeves.mmg.uci.edu/immunology/Assays/Assays.htm • http://www.medschool.lsuhsc.edu/microbiology/DMIP/dmex03.htm • http://www.tulipgroup.com/Common/html/TurbidTech.pdf • http://departments.oxy.edu/biology/Franck/Bio222/Lectures/Feb1lecture.htm • http://www.mercodia.se/global/mainpage.asp?page_id=41 ELISA • http://www.clinprointl.com/technical.htm ELISA • http://www.nsbri.org/HumanPhysSpace/focus4/sf-hormonal.html • http://ccm.ucdavis.edu/cpl/Tech%20updates/TechUpdates.htm molecular diagnostics

50. References (Continued) • http://www.liv.ac.uk/~agmclen/Medpracs/practical_5/theory_5.html • http://www.fao.org/docrep/W0049E/w0049e06.htm • http://www.genwaybio.com/gw_file.php?fid=6056