Labeled assays

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