AgglutinationSerology and Immunology

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

Agglutination•Agglutinins

▫Antibodies that produce such reactions

•Involves two-step process:▫Sensitization or initial binding▫Lattice formation or formation of large

aggregates

Agglutination

•Types of particles that participate in such reactions:

▫Erythrocytes▫Bacterial cells▫Inert carriers such as latex particles

Steps in Agglutination•Primary phenomenon

(SENSITIZATION) First reaction involving Ag-Ab combination Single antigenic determinant on the surface

particle

1)Initial reaction: rapid and reversible2)Cross link formation visible aggregates

(stabilization)

SENSITIZATION

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

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

•Antibody:▫Complete

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)

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

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.

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.

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

DIRECT AGGLUTINATION

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

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

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

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

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

REVERSE PASSIVE

•PRINCIPLE: latex particles coated with antibody are reacted with a patient sample containing suspected antigen

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

Agglutination Inhibition

•Pregnancy Testing-classic example of agglutination inhibition

▫Human chorionic gonadotropin (hCG) Appears in serum and urine early in

pregnancy

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

AGGLUTINATION of carriers:Negative test for hCGNOT PREGNANT

NO AGGLUTINATION of carriers:

Positive test for hCGPREGNANT

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

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

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).

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)

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.

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.

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.

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).

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

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

2+ Medium-sized aggregates; clear supernatant

3+ Several large aggregates; clear supernatant

4+ One solid aggregate; clear supernatant