1. 2. Basic Immunologic
Procedures

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.ht
m
 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