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
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 Procedure
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
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://www.nsbri.org/HumanPhysSpace/focus4/sf-hormonal.html
ELISA
ELISA
molecular diagnostics

50. References (Continued)