AGGLUTINATION

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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

RBCs Antigens

Antigens (Bacteria)

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Agglutinin and Agglutinogen

• Agglutinin:• 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

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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

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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

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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

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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

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Slides, Test tubes

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Micotiter plates

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Qualitative and Quantitative Techniques

• Qualitative agglutination test• Semi-quantitative agglutination test

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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

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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

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Semi-Quantitative Agglutination Test

1/2

1/4

1/8

1/16

1/32

1/64

1/12

8

1/25

6

1/51

2

1/10

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Pos

.

Neg

.

Titer

64

8

512

<2

32

128

32

4

Patient

1

2

3

4

5

6

7

8

14

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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

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Precipitation Curve

Increasing antigen concentration (antibody concentration is constant)

Co

nc.

of

Ag

-Ab

co

mp

lexe

s

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Steps in Agglutination• Agglutination is a two-step process that results in the

formation of a stable lattice network

1. Sensitization• The first reaction involves antigen-antibody combination through

single antigenic determinants on the particle surface and is often called sensitization

2. 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

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1- Sensitization

• Antibody molecules attach to their corresponding Antigenic site (epitope) on the particle

• There is no visible clumping

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2- Lattice Formation

• Antibody molecules crosslink the particles forming a lattice that results in visible clumping or agglutination

Crosslinking Abs

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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

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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:1. Spontaneous adsorption of Ags by RBCs

2. Covalent binding using chemical links

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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

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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

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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

Negative

Positive

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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

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Types of Agglutination

• Direct Agglutination• Indirect or Passive Agglutination• Reverse Passive Agglutination• Agglutination Inhibition • Coagglutination

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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

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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

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• 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

Reverse Passive Agglutination

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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

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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

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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

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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

Positive Negative

Hemagglutination inhibition for detection of influenza antibodies

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

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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

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Hepatitis B Surface Antibody Detection

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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

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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

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Procedure for Qualitative Screening Technique

1. Each specimen requires 12 wells of a microtiter plate

2. Add 50 µl of diluent to wells A1 – A10 for each sample

3. 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

4. Transfer 25 µl of A1 to B1, 25 µl of A2 to B2, till A10 to B10

5. Add 25 µl of +ve control to well A11 and 25 µl of –ve control to well A12

6. Resuspend test cells and then add 75 µl of test cells to wells A1 – A12

7. Tap the plate gently to mix the contents making sure to avoid cross contamination

8. Incubate the plate for 30-45 minutes at RT keeping the plate away from heat, direct sunlight & any source of vibration

9. Read and record the results, the results are stable for 24 hours if the plate is covered

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1/2

1/4

1/8

1/16

1/32

1/64

1/12

8

1/25

6

1/51

2

1/10

24

Pos

.

Neg

.

Sample

Sample 1 Dil.

Sample 1

Sample 2 Dil.

Sample 2

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Interpretation of Results

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

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