1. Agglutination 1

2. Agglutination
The interaction between an antibody and a particulate antigen results in visible clumping called agglutination
Particulate antigen include:
Bacteria,
White blood cells,
Red blood cells,
Latex particles
Antibodies that produce such reactions are called agglutinins
If an agglutination reaction involves red blood cells, then it is called hemagglutination
Antigens (Bacteria)
RBCs Antigens

3. Agglutinin and Agglutinogen
It is an antibody that interacts with antigen on the surface of particles such as erythrocytes, bacteria, or latex particles to cause their agglutination in an aqueous environment
Agglutinogen
It is an antigen on the surface of particles such as red blood cells that react with the antibody known as agglutinin to produce agglutination
The most widely known agglutinogens are those of the ABO and related blood group systems

4. Applications
Agglutination reactions now have a wide variety of applications in the detection of both antigens and antibodies including:
Blood grouping,
Diagnosis of infectious & non-infectious diseases
Measure levels of certain therapeutic drugs, hormones, and plasma proteins

5. Detection of Abs or Ags
The agglutination reaction may be used to identify the antibody or antigen in a patient sample
When testing for antibody, the antigen concentration is constant for each dilution being tested
When testing for antigen, the antibody concentration is constant for each dilution being tested

6. Agglutination & Precipitation
Agglutination reactions are similar in principle to precipitation reactions; they depend on the cross linking of polyvalent antigens with the exception that:
Precipitation reactions involve soluble antigens, while agglutination involves particulate antigens
Pecipitation reactions represent a phase change, while the agglutination reactions manifest as clumping of antigen/ antibody complexes
Agglutination is more sensitive than precipitation

7. Advantages of Agglutination Techniques
The agglutination reaction has wide spread use in the clinical laboratory due to the following reasons:
They are simple
Inexpensive
Reliable
The visible manifestation of the agglutination reaction eliminates the need for complex procedures and expensive instrumentation
Numerous techniques have been described for agglutination tests, these techniques may be performed using:
Slides,
Test tubes,
or micotiter plates, depending on the purpose of the test
However the principle of the agglutination remain the same

8. Slides, Test tubes

9. Micotiter plates

10. Qualitative and Quantitative Techniques
Qualitative agglutination test
Semi-quantitative agglutination test

11. Qualitative Agglutination Test
Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody
The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen
For example, a patient’s red blood cells can be mixed with antibody to a blood group antigen to determine a person’s blood type
In a second example, a patient’s serum is mixed with red blood cells with virus Ags to assay for the presence of antibodies to that virus in the patient’s serum

12. Semi-Quantitative Agglutination Test
Agglutination tests can also be used to quantitate the level of antibodies to particulate antigens
In this test:
One makes serial dilutions of a sample to be tested for antibody
Then add a fixed number of red blood cells or bacteria or other such particulate antigen
Then determines the maximum dilution, which gives agglutination
The maximum dilution that gives visible agglutination is called the titer
The results are reported as the reciprocal of the maximal dilution that gives visible agglutination
This can be done using a microtiter plate

13. Semi-Quantitative Agglutination Test
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
Pos.
Neg.
Titer 64 8
512
<2 32
128 4
Patient 1 2 3 5 6 7
Prozone phenomenon: Too much patient antibody for amount of test. If this is suspected, dilute antibody and repeat the test.

15. Precipitation Curve
Prozone – antibody excess, many antibodies coat all antigen sites- results in false negative
Postzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative
Zone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction

16. Precipitation Curve Conc. of Ag-Ab complexes
Increasing antigen concentration (antibody concentration is constant)
Conc. of Ag-Ab complexes

17. Steps in Agglutination
Agglutination is a two-step process that results in the formation of a stable lattice network
Sensitization
The first reaction involves antigen-antibody combination through single antigenic determinants on the particle surface and is often called sensitization
Lattice formation
The second step is the formation of cross-links that form the visible aggregates
This represents the stabilization of antigen–antibody complexes with the binding together of multiple antigenic determinants
Each stage of the process is affected by different factors, and it is important to understand these in order to manipulate and enhance end points for such reactions

18. 1- Sensitization
Antibody molecules attach to their corresponding Antigenic site (epitope) on the particle
There is no visible clumping

19. 2- Lattice Formation
Crosslinking Abs
Antibody molecules crosslink the particles forming a lattice that results in visible clumping or agglutination

20. Factors that Affect Agglutination
Buffer pH
The relative concentration of Antibody and Antigen
Location and concentration of Antigenic Determinants on the Particle
Electrostatic Interactions between Particles
Electrolyte Concentration
Antibody Isotype
Temperature

21. Hemagglutination The agglutination of red blood cells by either
Direct agglutination
or indirect agglutination
Direct agg.: Ag is an intrinsic component of RBC
Indirect agg. soluble Ags are adsorbed to the RBC
There are 2 ways in which Ags can be bound to RBCs:
Spontaneous adsorption of Ags by RBCs
Covalent binding using chemical links

22. The Latex particles
Latex particles are usually prepared by emulsion polymerization
Styrene (unsaturated liquid hydrocarbon) is mixed with a surfactant (sodium dodecyl sulfate) solution, resulting emulsified in billions of micelles extremely uniform in diameter
When the polymerization is finished, polystyrene chains are arranged into the micelles with the hydrocarbon part in the center and the terminal sulfate ions on the surface of the sphere, exposed to the water phase
Surfactants are usually organic compounds that are amphiphilic, meaning they contain both hydrophobic groups (their tails) and hydrophilic groups (their heads)

23. The Latex Particles
Black ball chains represents polystyrene with sulfate free radicals (shaded balls)
Blue balls denote the sulfonic acid group of the SDS
Tail represent the hydrocarbon tail
The simplest method of attaching proteins to the particles is by passive adsorption

24. Latex Agglutination
In latex agglutination procedures, an antibody (or antigen) coats the surface of latex particles (sensitized latex)
When a sample containing the specific antigen (or antibody) is mixed with the milky-appearing sensitized latex, it causes visible agglutination
Positive
Negative

25. Viral Hemagglutination
Many viruses have nonserological hemagglutinating properties
They can agglutinate RBCs in the absence of Ab (non-immune agglutination)
Mammalian reoviruses agglutinate erythrocytes through interactions between the viral surface protein sigma 1 and carbohydrate groups attached to proteins on erythrocyte membranes
Hemagglutination (HA) can be used to
determine titers of certain viruses

26. Types of Agglutination
Direct Agglutination
Indirect or Passive Agglutination
Reverse Passive Agglutination
Agglutination Inhibition
Coagglutination

27. Direct Agglutination
In this reaction the antigen is an intrinsic component of the particle
The agglutination test is used to determine whether antibody, specific for the antigen is present in the biological fluids
serum
urine
or CSF
Direct agglutination tests are used primarily for diagnosis of infectious diseases

28. Passive or Indirect Agglutination
Employs particles that are coated with antigens not normally found on their surfaces
Antigen has been affixed or adsorbed to the particle surface
A variety of particles, including erythrocytes, latex and others are used for this purpose
The use of synthetic beads or particles provides the advantage of consistency, uniformity, and stability
Passive agglutination tests have been used to detect antibodies to viruses such as:
cytomegalovirus, rubella, varicella-zoster, and HIV-1/HIV-2

29. Reverse Passive Agglutination
In reverse passive agglutination, antibody rather than antigen is attached to a carrier particle
The antibody must still be reactive and is joined in such a manner that the active sites are facing outward
This type of testing is often used to detect microbial antigens
Latex particle coated with Ab (known) + serum looking for particular Ag
If Ag present, then visible agglutination is observed

30. Reverse Passive Agglutination
Numerous kits are available for rapid identification of antigens on infectious agents
Such tests used for organisms that
are difficult to grow
or when rapid identification is required
Testing of specimens for the presence of viral antigens has still not reached the sensitivity of enzyme immunoassays
But for infections in which a large amount of viral antigen is present, such as rotavirus and enteric adenovirus in infants, latex agglutination tests are extremely useful

31. Agglutination Inhibition
Agglutination inhibition reactions are based on competition between particulate and soluble antigens for limited antibody-combining sites
The lack of agglutination is an indicator of a positive reaction
The technique is called hemagglutination inhibition if the particle in the reaction is a RBC

32. Agglutination Inhibition - Positive
Tube containing free known Ab specific for the Ag to be detected
Patient has Ag and will combine with Ab
No visible agglutination
Latex beads coated with same Ag to be detected is added
It has nothing to attach to
No visible reaction
Therefore agglutination inhibition is positive
No Agglutination Occur

33. Agglutination inhibition - Negative
Tube containing free known Ab
Patient serum does not contain Ag
therefore no combination
Latex beads coated with same Ag to be detected is added
Visible agglutination,
Therefore agglutination inhibition is negative
Agglutination Occur

34. Hemagglutination Inhibition
Antibodies to the virus in the patient serum bind to the virus; blocks binding sites on the viral surfaces
prevents the virus from agglutinating the red cells
Example
detecting antibodies to influenza
Positive
Negative
Picture public domain
Hemagglutination inhibition for detection of influenza antibodies

35. Coagglutination
The name given to systems using bacteria as the inert particles to which antibody is attached
Staphylococcus aureus is most frequently used, because it has a protein on its outer surface, called protein A which naturally adsorbs the Fc portion of antibody molecules
The Fab region is free to interact with antigens present in the applied specimens
fragment crystallizable (FC)

36. Hepatitis B Surface Antibody Detection

37. Summary
Hepatitis B surface antigen (HBsAg) is the first serologic marker, appearing in the serum 6 to 16 weeks following HBV infection
In acute cases, HBsAg usually disappears 1 to 2 months after the onset of symptoms with the appearance of hepatitis B surface antibody (anti-HBs)
Anti-HBs also appears as the immune response following hepatitis B vaccination

38. Principle
When used by recommended technique, reagent will agglutinate in presence of Abs to HBV
No agglutination generally indicates absence of Abs
Test cells are preserved avian erythrocytes coated with Ags of HB

39. Procedure for Qualitative Screening Technique
Each specimen requires 12 wells of a microtiter plate
Add 50 µl of diluent to wells A1 – A10 for each sample
Add 50 µl of sample to well A1, mix well and transfer 50 µl to well A2, mix well and transfer 50 µl to well A3, till well A10
Transfer 25 µl of A1 to B1, 25 µl of A2 to B2, till A10 to B10
Add 25 µl of +ve control to well A11 and 25 µl of –ve control to well A12
Resuspend test cells and then add 75 µl of test cells to wells A1 – A12
Tap the plate gently to mix the contents making sure to avoid cross contamination
Incubate the plate for minutes at RT keeping the plate away from heat, direct sunlight & any source of vibration
Read and record the results, the results are stable for 24 hours if the plate is covered

40. 1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
Pos.
Neg.
Sample
Sample 1 Dil.
Sample 1
Sample 2 Dil.
Sample 2

41. Interpretation of Results
Test cells
Strong Positive
Full cell pattern covering the bottom of the well
Weak Positive
Cell pattern coves 1/3 of well bottom
Intermediate
Cell pattern shows distinctly open center
Negative
Cells settled to a compact button
Reference Values
Hepatitis B Surface Antibody
Unvaccinated: negative
Vaccinated: positive