1. Module One Precipitation and Agglutination Methods
CLS 404 Immunology
Immunologic Methods
Module One
Precipitation and Agglutination Methods

2. Objectives
Explain how the following physical factors can affect antigen-antibody reactions:
Ionic strength pH
Reaction time
Temperature
Number and location of antigens
Concentration ratio of antigen and antibody
Equivalence zone
Postzone
Prozone

3. Objectives
Explain the principle of each test method presented, and give a clinical use for each.
Differentiate between precipitation and agglutination reactions.
Interpret the results of Ouchterlony double diffusion.

4. Antigen/Antibody Reactions
Review – How do antigens and antibodies interact?

5. Antigen / Antibody Reactions
Antigen and antibody bind with “Lock and Key” fit.
Affinity – the total attractive force that draws antibody to antigen.
Avidity – how “tightly” antigen and antibody bind.
Antigen and antibody bonds include the following noncovalent bonds: van der Waals forces, hydrogen bonds, ionic bonds, hydrophobic bonds. AG AB

6. Antigen / Antibody Reactions
Antigen / antibody reactions are readily reversible.
Free Ag + Free Ab Ag-Ab complex

7. Antigen / Antibody Reactions
May be visualized when lattice structures form.
Sensitization: antibody attaches to antigen
Agglutination or precipitation: antibody attaches to antigens on different cells, resulting in cross-linking between cells Y Y Y Y Y Y

8. Antigen/antibody reactions are influenced by:

9. Ionic Strength
Shielding – charges that surround the Fab portion of an antibody, blocking antigen/antibody binding
Zeta potential – the difference in electrical charge between the surface of a cell and the outer layer of the ionic cloud that surrounds the cell in an electrolyte solution.
Keeps cells too far apart to allow lattice formation

10. Y Y Y Y Y Y Zeta Potential - - - - - - - - - - - - - - ++++++++++++++J Y
Zeta Potential

11. Ag/Ab binding is influenced by:
pH- optimal 6.5 to 7.5
Reaction time- depends on the immunoglobulin class and test medium
If the incubation time is too short, there will not be time for Ag/Ab interaction and lattice formation (false negative).
Prolonged incubation favors the free antigen and antibody state, also resulting in false negative test results.
Temperature- depends on the immunoglobulin class
IgM reacts best at room temperature or colder.
IgG reacts best at 36-38oC (very narrow temperature range).

12. Number of Antigens Dosage
Antibodies that react stronger with cells that have homozygous expression of an antigen than in cells that have heterozygous expression are said to show dosage
Cells with homozygous antigen expression have twice as much antigen available to participate in the reaction than do cells with heterozygous antigen expression
In a “homozygous” cell, there was only one gene for one antigen inherited. In a “heterozygous” cell, a gene was inherited along with its allele. The tow antigens must “share” the antigen locations available in the cell.

13. If there is an anti- antibody, which of these cells will yield the stronger reaction with it?
Twice as much antigen is available to react with the antibody on the cell with homozygous antigen expression than the cell with heterozygous antigen expression.
Homozygous Heterozygous

14. Y Y Y Location of Antigens
Surface antigens are more available to react than those buried within the membrane or within the cell.
Interference in ag/ab binding due to the position of other antigens Y Y Y

15. Concentration Ratio of Ag/Ab
Lattice formation is best achieved when there is a balance of antigen and antibody quantity known as the “zone of equivalence”.
Prozone – excess antibody. No cross-linkage; false negative test.
Postzone – excess antigen. No cross-linkage; false negative test.

16. Zone of Equivalence Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Postzone-antigen excess
Prozone – antibody excess

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

18. 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

19. Nephelometry
When light is passed through the solution, the precipitates cause the light to refract at various angles.
The light that is scattered at a particular angle is measured. This corresponds to the level of antigen in the sample.
Nephelometry can be used to detect serum protein levels.
Light source
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.
Used to determine serum protein levels such as immunoglobulins, complement components, C reactive protein, clotting factors and others.
Detector

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

21. Double Immunodiffusion Ouchterlony Method
The density of the precipitin line correlates with the amount of Ag/Ab complexes formed.
The pattern of precipitin lines can be interpreted.
Identity
Partial Identity
Non-identity
This test may be used to identify:
fungal antigens
antibodies to nuclear antigens

22. 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 curved line indicates the unknown patient antigen is the same as the known antigen.

23. Double Immunodiffusion Nonidentity
Here, the precipitin lines intersect.
The patient’s antigen is not the same as the known antigen.

24. Double Immunodiffusion Partial Identity
The antigens share one epitope that reacts with the antibody
One of the antigens is more complex, having a second unique epitope that also reacts with the antibody.
The precipitin lines merge, with a spur created that points to the simpler antigen.

25. Double Immunodiffusion Partial Identity
D antigen Da Db
“Shared”, simpler antigen
Da AG
Complex antigen
D AG Dc Dd
Anti-D reagent is actually a blend of antibodies with specificities to Da, Db, Dc, and Dd.
Anti-D

26. Immunoelectrophoresis
Electrophoresis is used to separate serum proteins on a gel according to differences in electrical charge.
Following electrophoresis, known antibody is applied to the gel.
Antibody diffuses through the gel.
Precipitates form where Ag/Ab complexes have been trapped in the gel.
The gel is washed to remove any unprecipitated proteins, and then stained to aid in visualization of the precipitin bands.

27. Immunoelectrophoresis
Figure 6-7
Kuby Immunology , 6th ed
©2007, WH Freeman & Co.
Used with permission

28. Quick Question
What application of immunoelectrophoresis have we previously discussed in this course?
Semi-quantitative test for determining specific heavy chain and light chain components in a monoclonal gammopathy
This test is qualitative –detects only large increases or decreases in immunoglobulin concentration
What related test also relies on the precipitation method, but uses antibodies that are directly applied to the gel rather than diffused through the gel?
Immunofixation Electrophoresis

29. Western Blot 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.
The patient’s antibody pattern is compared to a known antibody pattern.

30. 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

31. Western Blot
If a patient has antibodies to more than one antigen that an organism possesses, it is highly likely that patient has been infected by the organism.
Western Blot is used as a confirmatory test for HIV infection.

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

33. 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.

34. Direct Agglutination
Can be used to detect antigen (using known antibody as a reagent) or antibody (using known antigen as the reagent)
Bacterial antigens/antibodies
RBC antigens/antibodies
Known as hemagglutination

35. Y Y Y Y Y Quick Question Why is IgM the most effective agglutinin?
Because of its structure (pentamer) and larger size, it can easily bridge between cells Y Y Y Y Y J

36. Passive Agglutination
Antigens on a carrier molecule, such as latex, combine with patient’s sample for antibody detection.

37. Reverse Passive Agglutination
Antibody is bound to the carrier molecule, which is then mixed with patient’s sample to detect antigen.
Uses for passive agglutination include ID of bacteria, measuring hormone and drug levels, and measuring levels of some proteins.

38. Inhibition of Agglutination
Antibody reagent is combined with patient’s specimen.
If patient’s specimen contains that antigen, the antigens will react with the antibodies.
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
Uses include detecting viral antibodies and detecting hormones.

39. The End
After completing the assignments for this module, please continue to Module 2, “Labeled Methods”.