Labeled assays

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  • 1. 2. Basic ImmunologicProcedures
  • 2. Labeled Immunoassays Some antigen/antibody reactions not detectedby 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 moleculeof a complementary configuration. Usually binds to the substance the test is trying todetect. The receptor is what binds the specific targetmolecule. “Sandwich” technique is an example.
  • 4. “Sandwich” Technique ELISA
  • 5. Terminology Reactions may be competitive or non-competitive Competitive – labeled known and patientunknown 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 forthe 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 directlyrelated to the amount of antibody in patient sample.
  • 7. Terminology Heterogeneous or homogeneous Heterogeneous assays called separationassays Require multiple steps Careful washing of surface to remove unboundreagents and samples. Homogeneous assays do NOT require aseparation 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 boundantibody molecule. Must wash off unreacted substances. Type of label on known antigen will determinemethod of detection.
  • 9. Competitive Binding
  • 10. Noncompetitive Binding Patient sample added. Will react with its homologous antigen orantibody, depending upon what is being testedfor. The reaction is measured and the concentrationis 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 builtinto 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 patientsubstance Low radioactivity high amount of patientsubstance. 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 onthe 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 wasbound to the reagent antibody. Low radioactivity indicates a high concentration of patient antigen wasbound 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 excessantibody. Labeled antibody binds to both patient antigen, ifpresent, 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 replacedradioimmunoassay.
  • 20. Enzyme Immunoassay Enzymes occur naturally and catalyzebiochemical 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 quantitativeassays. Enzymes selected according to Substrate molecules converted per molecule ofenzyme. 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 alkalinephosphatase are the most popular. Highest turnover High sensitivity Easy to detect
  • 23. Heterogenous EIA Competitive Enzyme labeled antigen competes with unlabeledpatient 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 andestrogen.
  • 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 linkedimmunosorbent assay – ELISA Enzyme labeled reagent DOES NOT participatein 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, boundantibody specific for one epitope, second labeledantibody 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 differentepitope on the antigen. Add substrate, measure intensity of color.
  • 30. Rapid Immunoassays Membrane based cassettes are rapid, easy to perform and givereproducible 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 immobilizedantigen or antibody. Color change occurs.
  • 32. Homogeneous Enzyme Assay Reaction which requires NO separation ofreactants. Less sensitive BUT rapid, easy to perform andautomate. Chief use is to detect low molecular weightanalytes such as: Hormones Therapeutic drugs Drugs of abuse Can use serum or urine.
  • 33. Homogeneous Enzyme Assay Based on principle of change in enzyme activityas specific antigen-antibody combinations occur. Reagent antigen labeled with enzyme tag. Antibody binds to specific determinant sites onantigen, active site on enzyme blocked, causesmeasurable loss of activity. Free antigen competes with enzyme-labeledantigen for limited number of antibody sites. Enzyme activity directly related to patientantigen.
  • 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 bovineimmunoglobulin which can be seen as green light in thefluorescence microscope.
  • 39. Fluorescent Immunoassay Direct immunofluorescence Tagged antibody added to unknown antigen fixed toslide 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 fluorescentmolecules. A fluorescent molecule, when excited by a polarized light, will emit fluorescence with itspolarization primarily determined by the rotational motion of the molecule. Since the molecular rotation is inversely proportional to the molecular volume, the polarization isin turn related to the molecular size. A small molecule rotates fast in solution and exhibits low value of polarization whereas a largemolecule exhibits a higher polarization because of its slower motion under the same conditions. Thus, changes in fluorescence polarization can reflect the association or dissociation betweenmolecules of interest.
  • 43. Fluorescent Polarization Another picture to illustrate the principle. Measure polarized light.
  • 44. Chemiluminescent Immunoassays The process of chemiluminescence occurs when energyin the form of light is released from matter during achemical reaction.
  • 45. Chemiluminescent Immunoassays Large number of molecules capable ofchemiluminescence Luminol Acridium esters Ruthenium derivatives Nitrophenyl oxalates Use sodium hydroxide as a catalyst Light emission ranges from quick burst or flash to lightwhich remains for a longer time. Different types of instruments are required based onemission.
  • 46. Chemiluminescent Immunoassays Can be used for heterogeneous orhomogeneous assays. Can attach label to antigen or antibody. Heterogeneous assays use competitive andsandwich assay. Competitive assays used to measure smalleranalytes. Sandwich assays are used to measure largeranalytes.
  • 47. Chemiluminescent Immunoassay Many applications. Can measure antigen or antibody. Add chemiluminescently tagged analyte. Measure light which is emitted which is directly related toconcentration 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 ELISA ELISA moleculardiagnostics
  • 50. References (Continued)