Agglutination Reactions

Agglutination Reactions

December 9, 2013

Agglutination

RBC

RBC

RBC

RBC

IgM Antibody

IgG Antibody

RBC

RBC RBC

RBC RBC

RBC RBC

Agglutination Reactions

Characteristics

• is the aggregation of particulate matter due

to combination with specific antibody.

• takes place on the surface of the particle

and thus antigen must be exposed and able

to bind with antibody

• Involve particulate antigens and antibodies

Characteristics cont’d

• types of particles participating in such reactions include:

erythrocytes, bacterial cells, and inert carrier such as

latex particles.

• antigen consists of suspensions of microorganisms, cells

(rbc) or uniform particles like latex

• Antigens may be:

– On a cell (direct agglutination)

– Attached to latex spheres (indirect or passive agglutination)

Characteristics cont’d

• Agglutination reaction is aided by elevated

temperature (37-56°C) and by:

– Movement which increases the contact between

antigen and antibody.

– Clear supernatant.

– Clumps aggregate and settle as large visible

clumps.

Agglutination Requirements

• Availability of stable cell or particle

suspension

• Presence of one or more antigens close to

the surface

• Knowledge that ‘incomplete’ or non-

agglutinating antibodies are not detectable

without modifications, e.g. antiglobulin

(Coomb’s) technic

Advantages

• Agglutination of insoluble native antigens or

antigen-coated particles simple to read with or

without the aid of a microscope.

• Increased degree of sensitivity

• Great variety of detectable substances

Steps in Agglutination

1. Sensitization

• involves antigen-antibody combination through

single antigenic determinants on the particle

surface

• may be affected by the nature of the antibody

molecules, affinity and avidity of individual

antibody, and the nature of antigen-bearing

surface

Steps in Agglutination Reaction

2. Aggregative Stage

represents the sum of interaction between antibody and

multiple antigenic determinants on a particle

dependent on environmental conditions as well as the

relative concentrations of antigen and antibody

it could be enhanced by LISS (Low ionic strength saline),

neutralizes surface charges and facilitates agglutination

(albumin 5 - 50%)

Uses of Agglutination Reactions

• Aid in the identification, by means of known antisera

(serum containing antibodies speciftic for a given

antigen), microorganisms cultured from clinical

specimens.

• Help estimate the titer of antibacterial agglutinins in

the serum of patients with unknown diseases.

Types of Agglutination Reactions

• Direct Agglutination

• Passive Agglutination

• Reverse Passive Agglutination

• Agglutination inhibition

• Hemagglutination-inhibition

• Coagglutination/Conglutination

Types of Agglutination Rxns.

• Direct agglutination - occurs when antigens are found

naturally on a particle (Serotyping of Salmonella)

– e.g. identification of bacterial types

• O antigen (somatic) - compact, fine and granular

agglutination

• H antigen (flagellar) - form a loosely woven network of

clumped cells (coarse and floccular), called snowflake

agglutination

• hemagglutination (agglutination reaction which involves

rbc)

Direct Agglutination Reaction

Agglutination Reactions

• Passive agglutination - employs particles that are

coated with antigens not normally found on their

surfaces.

– Inert materials commonly used:

1. Bentonite 3. Colloidion

2. Latex particles 4. Colloidal charcoal

Agglutination Reactions

Reverse Passive agglutination - antibody rather than

antigen is attached to a carrier particle.

Agglutination inhibition - based on competition between

particulate and soluble antigens for limited antibody

combining sites, and a lack of agglutination is an indicator

of a positive reaction.

Hemagglutination inhibition reactions -tests the presence of

antibodies to certain viruses, such as rubella, mumps,

measles, influenza, and adenovirus

Hemagglutination Hemagglutination involves agglutination of RBCs.

Viral hemagglutination inhibition tests for antibodies by the

antibodies' ability to prevent viruses from agglutinating RBCs.

Figure 18.7

Neutralization Reactions Eliminate the harmful effect of a virus or exotoxin

Figure 18.8b

Agglutination Reactions

• Coagglutination/Conglutination - name given to

systems using bacteria as inert particles to which

antibody is attached (S. aureus).

– the Fc region of antibody attaches to protein A of

staphylococcal cell leaving the Fab region to combine with

the antigen

– killed staphylococcal cells coated with antibody can be used

to identify bacteria and detect soluble extracellular bacterial

antigens in specimens and body fluids.

Reading Agglutination Reactions Macroscopic +4 One solid aggregate or clump of cells.

+3 Several large aggregates, clear background.

+2 Small to medium sized aggregates, clear background.

+1 Small aggregates, turbid reddish background.

± Weak granularity in cell suspension. A few macroscopic agglutinates but

numerous agglutinates microscopically

MF Any degree of agglutination in a sea of unagglutinated cells

Hem Hemolysis is interpreted as a positive reaction and may be graded as

complete or partial; hemolysis and agglutination may be recorded on the

same tube.

Ø No agglutination, smooth reddish background

Microscopic: + Positive - aggregates of at least 3-5 cells. Ø Negative - no agglutination.

Agglutination Reactions

Microscopic Agglutination

Hemagglutination

Hemagglutination Inhibition

Agglutination Reactions

Latex

Agglutination