1. Module Two Labeled Test Methods
CLS 404 Immunology
Immunologic Methods
Module Two
Labeled Test Methods

2. Objectives Describe the part of a labeled assay
List materials used to label analytes
Explain the principle of each test method presented and give an example of a clinical application for each test
Differentiate the following types of assays:
Competitive vs. non-competitive
Homogeneous vs. heterogeneous
Direct vs. indirect

3. “Labeled” Methods Attaches a “tag” to either the antigen or antibody.
Antigen and antibody are allowed to react.
The degree of the reaction can be determined by detecting and measuring the “tagged” material.
Analyte is usually a small molecule or present in small quantities.
Tests are fast, sensitive, and specific.

4. Parts of a Labeled Assay
Analyte
Specific antibody
Separation of bound and free components
Detection of label
Standards/Calibrators

5. Analyte The substance being measured Unlabeled in patient specimen
As a reagent, is often labeled
Common analytes include:
Bacterial and viral antigens
Hormones
Drugs
Tumor markers
Immunoglobulins

6. Specific antibody High affinity for antigen
Important for test sensitivity
Specific to the antigen in the reaction
No cross-reactivity
Often a monoclonal antibody is used

7. Separation of bound and free components
Many tests include a means of removing any unbound antigen and/or antibody.
Heterogeneous: Method that requires a step that separates bound analyte from unbound analyte.
Homogeneous: Method that does not require a separation step.

8. Separation Methods Washing to remove unbound antigen/antibody
Adsorption of unbound reactants onto talc, charcoal or cellulose followed by centrifugation or filtration to remove adsorbed materials
Chemical modification of the test medium that results in the precipitation of antigen-antibody complexes, leaving unbound analyte in solution

9. Separation Methods
Solid-phase reaction surfaces which bind labeled analyte, including:
Microtiter plates
Magnetic beads
Cellulose membranes
Spherical surface preferred, as spheres have a larger surface area

10. Labels and Their Detection
Radioactivity
Gamma counter detects gamma radiation as radioactive isotope “tag” decays
Fluorescence
Fluorochromes are excited by light of a specific wavelength
Emit light of a different wavelength
Emitted fluorescent light is detected microscopically

11. Fluorochromes Fluorescein – absorbs blue light, emits yellow-green
Rhodamine – absorbs yellow-green light, emits deep red
Since these emit light at different wavelengths, they may be combined in one assay to detect different antigens on the same cell
Phycoerythrin – emits bright red light

12. Labels and Their Detection
Enzymes
Break down a colorless chromogenic substrate
Color development is measured using a spectrophotometer
Common enzymes include:
horseradish peroxidase
alkaline phosphatase
glucose – 6- phosphate dehydrogenase (fluorometric)

13. Labels and Their Detection
Chemiluminescent materials
The “tag” molecule is excited to a state of high energy (oxidation)
As the excited molecule returns to its original lower energy state, light is emitted
May be a short burst of light
May be a longer lasting glow
Common “tag” materials include:
Luminol
Acridium esters
Peroxyoxalates

14. Standards/calibrators
Known concentrations of analyte, used to determine relationship between labeled analyte and unlabeled analyte in specimen.
Typically used to produce a curve from which analyte concentration in patient’s specimen can be determined.

15. Standard Curve Figure 6-9b Kuby Immunology , 6th ed
©2007, WH Freeman & Co.
Used with permission

16. Specific Labeled Assays

17. Competitive ELISA
Enzyme labeled antigen competes with unlabeled patient antigen for binding sites on fixed antibodies.
A chromogen is added that reacts with the enzyme.
The level of color development is inversely proportional to the level of patient antigen.
Used to measure small molecules such as insulin or estrogen.

18. Radioimmunoassay (RIA)
Competitive binding
Uses a radioactive tag rather than an enzyme
125I common tag
This method has been used for measuring hormone levels, IgE levels and other small trace analytes
Has been replaced in most laboratories by ELISA

19. Noncompetitive (Indirect) ELISA
Antigen is bound to a solid phase.
Patient’s serum is added.
If antibodies to the antigen are present in the serum, they react with the antigen bound to the solid phase
The patient’s antibody is free to bind as much antigen as possible, since the labeled antibody is not present in the test system at this point
A wash step removes unbound patient antibody. Y Y Y Y Y Y

20. Noncompetitive (Indirect) ELISAY
An anti-human globulin (AHG) antibody that has been enzyme labeled is added.
This antibody will react with the patient’s antibody, if present.
A second wash removes unbound AHG reagent.
The enzyme substrate is added.
The degree of color development is directly proportional to the amount of patient antibody in the specimen. Y Y Y Y Y Y Y Y Y
Anti-Human Globulin (AHG) is antibody to human antibodies. The Fab portion of AHG is directed at the Fc portion of the human immunoglobulin. Y

21. Noncompetitive (Indirect) ELISA
More sensitive than competitive methods.
Used to diagnose viral infections and to detect autoantibodies.

22. Capture (Sandwich) ELISA
Patient’s sample is incubated with bound antibody.
Following a wash to remove unbound antigen, a second chromogen-labeled antibody is added.
Second antibody is also specific for the antigen (analyte)
The substrate is added.
The level of color development corresponds with the amount of antigen “captured”.
Multiple uses include detecting antibodies, hormones, viruses, proteins and tumor markers.

23. Enzyme-multiplied Immunoassay Technique (EMIT)
This is a homogeneous competitive binding assay.
When antibody binds to the labeled antigen, it blocks enzymatic activity, reducing the amount of color development.
The more patient antigen that binds to the antibody, the more enzyme-tagged antigen remains free to react with the chromogen.
Color development is _______proportional to the concentration of antigen.
Directly
Commonly used to test for drugs.

24. Chemiluminescence
This labeling technique can be applied to heterogeneous or homogeneous assays.
Used to detect antigens or antibodies.
Requires sophisticated instrumentation that is specific to the chemical being used.
Clinical applications include detection of drugs and hormones.

25. Direct Immunofluorescence
Negative test Positive test
Fluorescently labeled antibody is used to detect antigen in tissue fixed to a slide.

26. Quick Question
Which paraprotein disorder uses direct immunofluorescence to detect light chain deposits in tissue?
Amyloidosis
This test has been used for detection of Chlamydia, Legionella, RSV, and other antigens.

27. Indirect Immunofluorescence
Negative Test Positive Test
Known antigen fixed to slide
Patient’s serum added (unknown antibody)
Incubation & wash
Fluorescently labeled anti-human globulin added.
Anti-Human Globulin (AHG) is antibody to human antibodies. The Fab portion of AHG is directed at the Fc portion of the human immunoglobulin.
Clinical applications of indirect fluorescence include detection of viral, treponemal, and antinuclear antibodies.

28. Please proceed to Module Three, Antibody Titers.
The End
Please proceed to Module Three, Antibody Titers.