Agglutination - PowerPoint Presentation

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AgglutinationSlide2

Agglutination

The interaction between antibody and a particulate antigen results in visible clumping called

agglutinationParticulate antigen include: bacteria, white blood cells, red blood cells, latex particles Antibodies that produce such reactions are called agglutininsIf 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 environmentAgglutinogenIt is an antigen on the surface of particles such as red blood cells that react with the antibody known as agglutinin to produce agglutinationThe 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 diseases measure levels of certain therapeutic drugs, hormones, and plasma proteins4Slide5

Testing Abs &

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 testedWhen testing for antigen, the antibody concentration is constant for each dilution being tested5Slide6

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 antigensPecipitation reactions represent a phase change, while the agglutination reactions manifest as clumping of antigen/ antibody complexesAgglutination is more sensitive than precipitation6Slide7

Advantages of Agglutination Techniques

The agglutination reaction has wide spread use in the clinical

laboratory due to the following reasons:they are simpleinexpensivereliableThe visible manifestation of the agglutination reaction eliminates the need for complex procedures and expensive instrumentationNumerous 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

same7Slide8

Qualitative and Quantitative Techniques

Qualitative agglutination testSemi-quantitative agglutination test8Slide9

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 antigenFor example, a patient’s red blood cells can be mixed with antibody to a blood group antigen to determine a person’s blood typeIn 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 serum9Slide10

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 and then adds 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 titerThe results are reported as the reciprocal of the maximal dilution that gives visible agglutinationThis

can be done using a microtiter plate10Slide11

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

32

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Patient

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Steps in Agglutination

Agglutination is a two-step process that results in the formation of a stable lattice network

SensitizationThe first reaction involves antigen-antibody combination through single antigenic determinants on the particle surface and is often called sensitization Lattice formationThe second step is the formation of cross-links that form the visible aggregatesThis represents the stabilization of antigen–antibody complexes with the binding together of multiple antigenic determinantsEach 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 reactions13Slide14

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 agglutinationCrosslinking Abs

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Factors that Affect Agglutination

Buffer

pHThe relative concentration of Antibody and AntigenLocation and concentration of Antigenic Determinants on the ParticleElectrostatic Interactions between ParticlesElectrolyte ConcentrationAntibody IsotypeTemperature16Slide17

Hemagglutination

The agglutination of red blood cells by either

direct agglutination or indirect agglutinationDirect agg. Ag is an intrinsic component of RBCIndirect agg. Soluble Ags are adsorbed to the RBCThere are 3 ways in which Ags can be bound to RBCs:Spontaneous adsorption of

Ags by RBCsCovalent 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 diameterWhen 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 phase18Slide19

The Latex

Particles

Black ball chains represents polystyrene with sulfate free radicals (shaded balls)Blue balls denote the sulfonic acid group of the SDSTail represent the hydrocarbon tailThe 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

Positive20Slide21

Viral

Hemagglutination

Many viruses have nonserological hemagglutinating propertiesThey 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 membranesHemagglutination (HA) can be used todetermine titers of certain viruses

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Types of Agglutination

Direct Agglutination

Indirect or Passive AgglutinationReverse Passive AgglutinationAgglutination Inhibition Coagglutination 22Slide23

Direct

Agglutination

In this reaction the antigen is an intrinsic component of the particleThe agglutination test is used to determine whether antibody, specific for the antigen is present in the biological fluidsserumurine or CSFDirect 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 surfacesAntigen has been affixed or adsorbed to the particle surfaceA variety of particles, including erythrocytes, latex and others are used for this purposeThe 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 particleThe antibody must still be reactive and is joined in such a manner that the active sites are facing outwardThis type of testing is often used to detect microbial antigens

Latex particle coated with Ab (known) + serum looking for particular AgIf 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 agentsSuch 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 immunoassaysBut 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 useful26Slide27

Agglutination

Inhibition

Agglutination inhibition reactions are based on competition between particulate and soluble antigens for limited antibody-combining sitesThe lack of agglutination is an indicator of a positive reactionThe technique is called hemagglutination inhibition if the particle in the reaction is a RBC 27Slide28

Agglutination

Inhibition

- PositiveTube containing free known Ab specific for the Ag to be detectedPatient has Ag and will combine with Ab No visible agglutinationLatex beads coated with same Ag to be detected is addedIt has nothing to attach toNo visible reaction

Therefore agglutination inhibition is positive28Slide29

Agglutination inhibition -

Negative

Tube containing free known AbPatient serum does not contain Agtherefore no combinationLatex beads coated with same Ag to be detected is addedVisible agglutination, Therefore agglutination inhibition is negative29Slide30

Positive

Negative

Hemagglutination

inhibition for detection of

influenza

antibodies

Hemagglutination

InhibitionAntibodies to the virus in the patient serum bind to the virus; blocks binding sites on the viral surfacesprevents the virus from agglutinating the red cells Exampledetecting antibodies to influenzaSlide31

Coagglutination

The

name given to systems using bacteria as the inert particles to which antibody is attachedStaphylococcus 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 moleculesThe 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 infectionIn 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 vaccination33Slide34

Principle

When used by recommended technique, reagent will agglutinate in presence of Abs to HBV

No agglutination generally indicates absence of AbsTest cells are preserved avian erythrocytes coated with Ags of HBAny non-specific reaction should be detected using the control cellsAvian erythrocytes not coated with Ags of HB34Slide35

Procedure for Qualitative Screening Technique

Each specimen requires 3 wells of a

microtiter plateTo well 1 add 190 µl of diluent and 10 µl of serum, -ve or +ve controlsUsing a pipette, mix contents of well 1 and transfer 25 µl to wells 2 & 3Resuspend both test and control cells and then add 75 µl of control cells to well 2 & 75 µl of test cells to well 3Tap the plate gently to mix the contents making sure to avoid cross contaminationIncubate the plate for 45-60 minutes at RT keeping the plate away from heat, direct sunlight & any source of vibrationRead and record the results, the results are stable for 24 hours if the plate is covered

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Interpretation of Results

Results

Test cellsControl cellsStrong PositiveFull cell pattern covering the bottom of the wellNo agglutination, tight buttonWeak PositiveCell pattern coves 1/3 of well bottom

No agglutination, tight buttonIntermediate

Cell pattern shows distinctly open center

No agglutination, tight button

NegativeCells settled to a compact buttonNo agglutination, tight buttonNon-SpecificPositive reactionPositive reaction36

Reference ValuesHepatitis B Surface AntibodyUnvaccinated: negativeVaccinated: positiveSlide37

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