1. Serology and Immunology
Agglutination
Serology and Immunology

2. Agglutination
is the aggregation of particulate matter caused by the combination with specific antibody
1896: First observed by Gruber and Durham when serum antibody was found to react with bacterial cells

3. Agglutination Agglutinins Involves two-step process:
Antibodies that produce such reactions
Involves two-step process:
Sensitization or initial binding
Lattice formation or formation of large aggregates

4. Types of particles that participate in such reactions:
Agglutination
Types of particles that participate in such reactions:
Erythrocytes
Bacterial cells
Inert carriers such as latex particles

5. Steps in Agglutination
Primary phenomenon (SENSITIZATION)
First reaction involving Ag-Ab combination
Single antigenic determinant on the surface particle
Initial reaction: rapid and reversible
Cross link formation  visible aggregates (stabilization)

6. SENSITIZATION

7. Secondary phenomenon: LATTICE FORMATION
Ab + multivalent Ag  stable network (visible reaction)
conc. of Ag and Ab
Governed by physiochemical factors:
Ionic strength of milieu pH
temperature

8. Secondary Phenomenon Lattice Formation
The Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutination
If both antigen and antibody are SOLUBLE reaction will become visible over time, ie, precipitation

9. Antibody: Complete Incomplete
primary and secondary interactions  visible agg
Incomplete
Non-agglutinating  no visible aggregation
Thought to have one active site
Now known: two active sites  no lattice formed
IgG class (small size = ↓flexibility of hinge region)

10. HEMAGGLUTINATION Detects antibody to erythrocyte antigens
sufficient concentration of antibody present-> antibody cross-link= agglutination
non-reactive/insufficient antibody present= no agglutination
Binding different antigens on the RBC surface = detect antibodies to antigen other than those present in the cells

11. HEMAGGLUTINATION
chromic chloride, tannic acid, and glutaraldehyde= cross- link antigens to the cell
IgG (does not agglutinate directly)-> need enhancement medium-> AHG
AHG binds to the second antibody present on the erythrocyte.

12. DIRECT AGGLUTINATION
-test patient serum against large, cellular antigens to screen for the presence of antibodies.
Antigen is naturally present on the surface of the cells.
In this case, the Ag-Ab reaction forms an agglutination, which is directly visible.

13. DIRECT AGGLUTINATION The particle antigen may be a bacterium.
e.g.: Serotyping of E. coli, Salmonella using a specific antiserum
The particle antigen may be a parasite.
e.g.: Serodiagnosis of Toxoplasmosis
The particle antigen may be a red blood cell.
e.g.: Determination of blood groups

15. DIRECT AGGLUTINATION
These reactions can be performed on slides (rapid tests) or on microliter plates or tubes for Antibody titration if required.

17. Passive Agglutination
An agglutination reaction that employs particles that are coated with antigens not normally found in the cell surfaces
Particle carriers include:
Red blood cells
Polystyrene latex
Bentonite
charcoal

18. Passive Agglutination
Passive agglutination has been used in the detection of :
Rheumatoid factor
Antinuclear antibody in LE
Ab to group A streptococcus antigens
Ab to Trichinella spiralis

19. The latex particles are coated with IgG and mixed with the patient's serum

20. REVERSE PASSIVE
Antibody rather than antigen is attached to a carrier particle
For the detection of microbial antigens such as:
Group A and B streptococcus
Staphylococcus aureus
Neisseria meningitidis
Haemophilus influenzae
Rotavirus
Cryptococcus neoformans
Mycoplasma pneumoniae
Candida albicans

21. REVERSE PASSIVE
PRINCIPLE: latex particles coated with antibody are reacted with a patient sample containing suspected antigen

22. Agglutination Inhibition
Based on the competition between particulate and soluble antigens for limited antibody combining site
Lack of agglutination is indicator of a positive reaction
Usually involves haptens complexed with proteins

23. Agglutination Inhibition
Pregnancy Testing
-classic example of agglutination inhibition
Human chorionic gonadotropin (hCG)
Appears in serum and urine early in pregnancy

24. Agglutination Inhibition
Urine
Antiserum
No hCG in urine:
Anti-hCG free
hCG in urine:
Anti-hCG neutralized
Carriers coated with hCG added
Carriers coated with hCG added
NO AGGLUTINATION of carriers:
Positive test for hCG
PREGNANT
AGGLUTINATION of carriers:
Negative test for hCG
NOT PREGNANT

25. Coagglutination
Name given to systems using inert bacteria as the inert particles to which the antibody is attached
S.aureus: most frequently used because it has protein A in its outer surface that naturally adsorbs the Fc portion of the antibody

26. Highly specific but not very sensitive in detecting small quantities of antigen

27. Reading/Grading Agglutination Reactions
done by gently shaking the tubes containing the serum and cells, and observing the cell button as it is dispersed
Hard shaking must be avoided because this may yield to false result
Attention should also be given to whether discoloration of the supernatant is present (Hemolysis).

28. Latex Agglutination
Antibody molecules can be bound to each latex beads
It will increase the potential number of exposed antigen- binding sites.
When an antigen is present in test specimen, it may bind to the latex bead thus forming visible cross-linked aggregates.
Latex particles can be coated with antigen (pregnancy testing, rubella antibody testing)

29. Coagulation and Liposome-enhanced testing
Are variations of latex agglutination
uses antibodies bound to a particle to enhance the visibility of agglutination
is a highly specific method but may not be sensitive.

30. Pregnancy Testing Designed to detect minute amounts of hCG.
Based on the principle of agglutination between latex particles coated with anti-hCG antibodies and hCG, if present.

31. False-Positive Result
If injected with hCG to trigger ovulation or to lengthen luteal phase of menstrual cycle.
Chorioepithelioma, hydatidiform mole or ingestion of aspirin
To detect the presence of a testicular tumor in men
False Negative
Testing before reaching detectable levels of hCG.

32. Reading/Grading Agglutination Reactions
Rupture or hemolysis of red blood cells is as important as finding agglutination
The strength of agglutination is graded from 0 (negative or no agglutination) to 4+ (agglutination or all erythrocytes clumped).

33. Reading/Grading Agglutination Reactions
Pseudoagglutination or the Rouleaux Formation also occurs
Red blood cells appear as stacks of coins.
Addition of physiologic NaCl will disperse pseudoagglutination
Saline Replacement is done after pseudoagglutination is observed so that it may not give false negative result due to the dilution effect of the saline

34. GRADING AGGLUTINATION REACTIONS
GRADE
DESCRIPTION
Appearance
Negative (-)
No aggregates
Weak (+/-)
Tiny aggregates that are barely visible macroscopically; turbid and reddish supernatant 1+
A few small aggregates just visible macroscopically; turbid and reddish supernatant

35. Medium-sized aggregates; clear supernatant2+
Medium-sized aggregates; clear supernatant 3+
Several large aggregates; clear supernatant 4+
One solid aggregate; clear supernatant