1. CLS 420 Clinical Immunology & Molecular Diagnostics
Immunologic Methods
Part 2
Basic Methods

2. Objectives
Explain the principle of each method presented, and give a clinical use for each.
Contrast precipitation, agglutination and flocculation, including:
Reaction time and conditions
Antigen state
Immunoglobulin class
Lattice formation
Describe heat inactivation of patient serum, including method and purpose.
Discuss general reasons for performing immunologic tests.

3. Precipitation Based Methods
Soluble antigen combines with antibody to form aggregates which precipitate out of solution.

4. Nephelometry Y Y Antibody reagent is combined with patient sample. Y
If antigen is present in the patient’s sample, Ag/Ab complexes will form and precipitate out of solution. Y
May use known antigen as a reagent to look for antibodies in a patient’s specimen.
Click on image at right wait for animation to begin.

5. Nephelometry
When light is passed through the solution, the precipitates cause the light to scatter at various angles.
The light that is scattered at a particular angle is measured. This corresponds to the level of antigen in the sample.
Light source
Light scattered at 90 degrees in commonly measured.
Endpoint – reaction is allowed to go to completion. Problems with precipitate settling out, reducing the amount of scatter.
Rate – measures rate that scatter increases following addition of reagent.
Nephelometry can be used to quantitate serum protein levels.
Nephelometry can be used to detect serum proteins.
Detector

6. Uses fine particles of antigen to detect antibody in patient’s serum.
Flocculation
Uses fine particles of antigen to detect antibody in patient’s serum.
Most common application is testing for syphilis, detecting antibodies to reagin.
Patient’s serum is placed within a ring on a slide or card.
A measured volume of reagent containing antigen is placed in the ring.
The slide or card is rotated to mix the sample and reagent.
The reaction is examined for fine precipitates, which indicate a positive test.
Negative test Positive test
Click on images above wait for animation to begin.

7. Double Immunodiffusion Ouchterlony Method
Testing performed in agar gel.
Antigen is placed in one well.
Antibody is place in other well.
Each diffuses through gel.
If antibody is specific to that antigen, a precipitin line will form where the 2 meet. AG AB

8. Double Immunodiffusion Identity
Known antibody with multiple specificities is placed in center well. Known antigen is placed in an outside well. Patient specimen containing antigen is placed in other outside well. The pattern of precipitin lines is interpreted.
This test may be used to identify fungal antigens and antibodies to nuclear antigens.
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9. Double Immunodiffusion Nonidentity
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10. Double Immunodiffusion Partial Identity
D antigen Da Db
Da AG
D AG Dc Dd
Anti-D

11. Immunofixation Electrophoresis
Proteins separated by electrophoresis
Antiserum (antibody) is applied to the gel.
Ag/Ab complexes form in the gel.
The gel is stained to reveal precipitin bands.
Electrophoresis used to separate proteins on a gel according to size and electrical charge.
If the antibody applied to the gel is directed against a particular protein, precipitin bands form where Ag/Ab complexes have been trapped in the gel.
The gel is washed to remove any unprecipitated proteins, then stained to reveal the bands.
Application point
Anode Patient serum Cathode
(+ electrode) (- electrode)

12. IFE stained gels = Serum application point
Example of an IgG monoclonal antibody with kappa light chains
SPE = Serum Protein Electrophoresis
SPE Anti-IgG Anti-IgA Anti-IgM Anti-Kappa Anti-Lambda

13. Western Blot Negative Patient Positive Control specimen Control p24
gp 41
gp120/160
Negative Patient Positive
Control specimen Control
No bands Patient bands compared to Pos Control
Modification of IFE
Known antigens are electrophoresed to separate them.
The separated components are transferred to nitrocellulose paper by blotting the gel.
The patient’s serum is applied to the paper.
If the patient has antibodies to any of the antigens on the paper, it will form a precipitate.
Paper is washed and stained.
If antibody to more than one antigen of an organism is detected in the patient’s serum, infection with that particular organism is highly likely.

14. Agglutination Based Methods
Antibodies cause the cross-linking of particulate antigen, usually found on a cell.

15. Direct Agglutination
The antigen is a natural part of the solid’s surface.
Often performed at room temperature.
May use centrifugation to bring antigen and antibody into closer proximity.
Can be used to detect antigen or antibody
Bacterial antigens/antibodies
RBC antigens/antibodies (hemagglutination)
Click on image at right wait for animation to begin.

16. Passive Agglutination
Antigens on a carrier molecule, such as latex, combine with patient’s sample for antibody detection.
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17. Reverse Passive Agglutination
Antibody is bound to the carrier molecule, which is then mixed with patient’s sample to detect antigen.
Uses include ID of bacteria, measuring hormone and drug levels, and measuring levels of some proteins.
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18. Inhibition of Agglutination
Antibody reagent is combined with patient’s specimen.
If patient’s specimen contains the antigen for that antibody, they will react.
Reagent antigen is added.
A positive reaction will show no agglutination, because the antibodies were bound to the patient antigen before the reagent antigen was added.
A negative reaction shows agglutination between reagent antibodies and antigen. Y Y Y Y Y Y
Click on images at right wait for animation to begin.

19. Other Methods

20. Neutralization Positive Test Negative Test
Applications include ASO titers and anti-DNase B titers.
The presence of an antibody prevents the antigen from functioning correctly.
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21. Complement Fixation
The patient’s serum is heated at 56oC for 30 minutes to inactivate any complement present.
Patient’s treated serum is incubated with known antigen and a known quantity of guinea pig complement.
If the patient has an antibody to the antigen, they will react and the complement will bind to the Fc pieces of the antibodies.
Sheep RBCs that are coated with hemolysin are added.
The test is incubated, centrifuged and read for hemolysis.
In a positive test, the complement will have been used up by the patient’s antibody, and no hemolysis will be present.
Hemolysin = antibodies known to activate complement and cause hemolysis.
This test is not used frequently, but has been used to detect antibodies to viruses, fungus and rickettsia.

22. Complement Fixation Negative test Positive test
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23. “Labeled” Methods
Attaches a “tag” to either the antigen or antibody. This “tag” can be detected and measured.
Tests are fast, sensitive and specific.

24. Parts of a labeled assay
Analyte (labeled and unlabeled)
Specific antibody
Separation of bound and free components
Detection of label
Standards/calibrators
Radioactivity, fluorescence, chemiluminous materials and enzymes have all been used as tags.

25. Classification
Heterogeneous: Method that requires a step that separates bound analyte from unbound analyte.
Homogeneous: Method that does not require a separation step.
Often a wash step removes the unbound analyte. Other separation methods include adsorption coupled with centrifugation or filtration, magnets, or chemically modifying the test medium so that bound analytes remain in solution while Ag/Ab complexes fall out of solution.

26. Competitive EIA
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.
Click on image at right wait for animation to begin.

27. Capture (Sandwich) EIA
Patient’s sample is incubated with bound antibody.
Following a wash, a second antibody that is labeled with a chromogen is added.
The level of color development corresponds with the amount of antigen “captured”.
Click on image at right wait for animation to begin.

28. Enzyme-multiplied Immunoassay Technique (EMIT)
Y Y Y Y
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.
Commonly used to test for drugs.
This is a homogeneous competitive binding assay.
Color development is _______proportional to the concentration of antigen.

29. Direct Fluorescence Negative test Positive test
This test has been used for detection of Chlamydia, Legionella, RSV, and other antigens.
Fluorescently labeled antibody is used to detect antigen fixed to a slide.
Click on images above wait for animation to begin.

30. Indirect Fluorescence
Positive Test Negative Test
Known antigen fixed to slide
Patient’s serum added (unknown antibody)
Incubation & wash
Fluorescently labeled anti-human globulin reagent 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.
Click on images above wait for animation to begin.

31. Microparticle Capture
Uses microbeads coated with known antigen or antibody.
The beads are incubated with a fluorescently labeled analyte and the patient’s sample.
The test mixture is centrifuged (or magnetized) to collect the beads, which are then analyzed for fluorescence.
Microbeads may be made of polysaccharides, polyacrylamide or magnetizable cellulose.

32. Fluorescent Polarization
Free labeled antigen excited by polarized light emits unpolarized light.
Labeled antigen/antibody complexes excited by polarized light emit polarized light.
FPIA is a competitive binding assay in which labeled antigen competes with unlabeled (patient) antigen for antibody binding sites.
The more labeled antigen that is bound to antibody, the more polarized light is emitted. Y
Free antigen can rotate when hit by polarized light whereas Ag/Ab complexes are too large to turn that quickly.
The degree of polarization is inversely proportional to the level of patient antigen.
Used to measure hormones and therapeutic drugs.

33. Chemiluminescence
Uses chemical labels that, when oxidized, produce a substance of a higher energy level.
When this substance decays to its original state, it emits energy in the form of light.
Common label materials include:
Luminol
Acridium esters
Peroxyoxalates
Requires sophisticated instrumentation that is specific to the chemical being used.
This labeling technique can be applied to heterogeneous or homogenous assays, and may detect antigens or antibodies.
Clinical applications include detection of drugs, hormones, and viral antigens.

34. Comparing Antibody Quantities
Antibody titers

35. Antibody Titer
An antibody titration can help determine antibody concentration levels.
Twofold serial dilutions of serum containing an antibody are made, then tested against cells possessing the target antigen.
The titer is the reciprocal of the greatest dilution in which agglutination is observed.
Twofold dilutions are the most common, however other dilutions may be used.

36. Two-fold Serial Dilutions

37. 1 2 3 4 5 6 7 8 9 10 11 12 Tube Saline 0.2 ml Serum 0.2 ml of tube 2
0.2 ml
Serum
0.2 ml of tube 2
0.2 ml of tube 3
0.2 ml of tube 4
0.2 ml of tube 5
0.2 ml of tube 6
0.2 ml of tube 7
0.2 ml of tube 8
0.2 ml of tube 9
0.2 ml of tube 10
0.2 ml of 6% BSA
RBC
Suspension
0.1 ml
Final
Dilution
1:1
1:2
1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512
1:1024
control

38. Results
Titers provide more valuable information when tested in parallel with a previous titer specimen.
A comparison of the current specimen’s results and previous specimen’s current results should be made.
A change in titer of 2 or more tubes is considered to be significant.
Change of 2 tubes = 4-fold dilution

39. Reasons to perform a titer
Acute and convalescent
Prenatal
Verify past infection
Confirm vaccination

40. Primary vs. Secondary Humoral Response
IgG
IgM
IgM
IgG
First exposure
Second
exposure

41. You have finished Immunology Student Lab Lectures!
Congratulations
You have finished Immunology Student Lab Lectures!
Good Luck on your exam!