AGGLUTINATION REACTIONS

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