Antigen-antibody - PowerPoint Presentation

Slide1

Antigen-antibody reactionsSlide2

Let’s startThe immune system is a system of biological structures and processes within an organism that protects against disease.

To function properly, an immune system must detect a wide variety of agents, from viruses to parasitic worms, and distinguish them from the organism’s own healthy tissue. Slide3

AntigensAntigens are any substances that are capable, under appropriate conditions, of inducing the formation of antibodies and reacting specifically with the antibodies so produced.

These antigenic molecules may have several antigenic determinants, called epitopes, and each epitope can bind with a specific antibody. Thus, a single antigen can bind to many different antibodies with different binding sitesSlide4

Chemical Nature of Antigens ((Immunogens

A

. Proteins

The vast majority of

immunogens

are proteins. These may be pure proteins or they may be glycoproteins or lipoproteins. In general, proteins are usually very good immunogens

.

B. Polysaccharides

Pure polysaccharides and lipopolysaccharides are good immunogens

.

C. Nucleic Acids

Nucleic acids are usually poorly immunogenic. However, they may become immunogenic when single stranded or when complexed with proteins

.

D. Lipids

In general lipids are non-immunogenic, although they may be

haptens

.Slide5

Antibodies

An antibody is a protein produced by the body’s immune cells “ activated B-lymphocytes” when it detects a foreign antigen.

Classes of antibodies:

IgG

:

The main antibody in blood(70%), made in 2ry immune response and has a long half

life”up

to 20 years”.

IgM

: accounts for(10%) of

Igs

, has a key role in 1ry immune response.

IgA

: (20%) of

Igs

present in serum and secretions.

IgE

:

(0.001%), involved in allergy and parasitic infections.

IgD

: (1%) present on the surface of B cells, so that it plays a role in induction of

Ab

productionSlide6
Slide7

Nature of antigen-antibody reactionsA. Lock and Key Concept 

The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains

B. Non-covalent Bonds 

The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds.

C. Reversibility

Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible.Slide8

Affinity and avidity

Affinity

Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.

Avidity

Avidity

is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. Slide9
Slide10

Specificity and cross reactivity

Specificity

Specificity refers to the ability of an individual antibody combining site to react with only one antigenic

Cross reactivity

Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant

Sensitivity

Sensitivity

refers

to the ability of an individual antibody to locate antigen even when it is greatly diluted.Slide11

Factors affecting measurement of antigen-antibody reactions  The only way that one knows that an antigen-antibody reaction has occurred is to have some means of directly or indirectly detecting the complexes formed between the antigen and antibody.

The ease with which one can detect antigen-antibody reactions will depend on a number of factors.

1. Affinity

The higher the affinity of the antibody for the antigen, the more stable will be the interaction. Thus, the ease with which one can detect the interaction is enhanced.

2

. Avidity 

Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect

.Slide12

Factors affecting measurement of antigen-antibody reactions3. Antigen to antibody ratio 

The ratio between the antigen and antibody influences the detection of antigen-antibody complexes because the size of the complexes formed is related to the concentration of the antigen and antibody.

4. Physical form of the antigen

The physical form of the antigen influences how one detects its reaction with an antibody. If the antigen is a particulate, one generally looks for agglutination of the antigen by the antibody. If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble antigen-antibody complexes.

 Slide13

Types of Antigen – Antibody Reaction

The

types of antigen – antibody reactions are

:

Precipitation

Reaction

.

Agglutination Reaction

.

Complement Fixation

.ELISA – Enzyme Linked ImmunoSorbent Assay

Immunofluorescence

.Slide14

AgglutinationAgglutination is the clumping of particles. Agglutination occurs if an antigen is mixed with its corresponding antibody called

isoagglutinin

.

This

term is commonly used in blood grouping

.Slide15

AgglutinationThis occurs in biology in several examples:The clumping of cells such as bacteria or red blood cells in the presence of an antibody or complement. The antibody or other molecule binds multiple particles and joins them, creating a large complex. This increases the efficacy of microbial elimination by phagocytosis as large clumps of bacteria can be eliminated in one pass, versus the elimination of single microbial antigens.

Another example occurs when people are given blood transfusions of the wrong blood group.Slide16

CharacteristicsIs 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

Antigens may be:

On a cell (direct agglutination)

Attached to latex spheres (indirect or

passive agglutination

)

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. Slide17

Steps in Agglutination1- Sensitization

Involves

antigen-antibody combination through single antigenic determinants on the particle surface

Antibody molecules attach to their corresponding Antigenic site (epitope) on the red blood cell membrane. There is no visible clumping.Slide18

2- Aggregative Stage (lattice formation)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

Antibody molecules crosslink RBCs forming a lattice that results in visible clumping or agglutination.Slide19

Uses of Agglutination Reactions Aid in the identification, by means of known antisera (serum containing antibodies specific for a given antigen), microorganisms cultured from clinical specimens.

Help

estimate the titer of antibacterial agglutinins in the serum of patients with unknown diseases. Slide20

Types of Agglutination ReactionsDirect Agglutination Passive AgglutinationReverse

Passive Agglutination

Agglutination

inhibition

Hemagglutination

-inhibition

Coagglutination

/Conglutination Slide21

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

agglutinationSlide22

HemagglutinationHemagglutination, is agglutination that involves red blood cells (RBCs). It has two common uses in the laboratory:

Blood

typing

Q

uantification

of virus dilutions in a 

Haemagglutination

assay

.Slide23

Blood typingBlood type can be determined by using antibodies

 that bind to the A or B blood group antigens in a sample of blood

.

For example, if antibodies that bind the A blood group are added and agglutination occurs, the blood is either type A or type AB.

To

determine between type A or type AB, antibodies that bind the B group are added and if agglutination does not occur, the blood is type A.

If

agglutination does not occur with either antibodies that bind to type A or type B antigens, then neither antigen is present on the blood cells, which means the blood is type 

O.Slide24

Blood typingSlide25

Viral hemagglutination assayHemagglutination phenomenon is almost commonly used for diagnosis of infection produced by some viruses.

The presence of virus in infected cell cultures can be detected by

hemagglutination

The

identity of the virus or of antibodies in a patient’s serum can be determined by specific inhibition of that

hemagglutination

. Slide26

Viral hemagglutination assayThe basis of the HAI assay is that antibodies to that particular virus (for example-influenza virus) will prevent attachment of the virus to RBC. Therefore

hemagglutination

is inhibited when antibodies are present.Slide27

Hemagglutination and hemagglutination inhibitionSlide28

Passive agglutination Employs particles that are coated with antigens not normally found on their surfaces. Inert materials commonly used:

Bentonite

Colloidion

Latex

particles

Colloidal charcoal

Passive

agglutination tests have been used to detect rheumatoid factor and antinuclear antibody.

Slide29

Reverse Passive agglutinationReverse Passive agglutination - antibody rather than antigen is attached to a carrier particle. Several kits are available today for rapid identification of such antigens from such infectious agents as group A and B streptococci, Staph. , Neisseria, and others. Slide30

Passive and reverse passiveSlide31

Agglutination inhibitionAgglutination 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. Slide32

The classic example of agglutination inhibition is the early types of home pregnancy test kits included latex particles coated with human chorionic gonadotropin (HCG) and antibody to HCG The addition of urine from a pregnant woman, which contained HCG, inhibited agglutination of the latex particles when the anti-HCG antibody was added; thus the absence of agglutination indicated pregnancy. Slide33
Slide34

Coagglutination/ConglutinationCoagglutination/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. Slide35

Antiglobulin mediated agglutinationThe antiglobulin test (

Coomb’s

test) can be used to detect red cells sensitized with IgG antibodies

In order for agglutination to occur an additional of anti-antibody

, which

reacts with the Fc portion of the IgG

antibody

This

will form a "bridge" between the antibodies or complement coating the red cells, causing agglutination

.

Slide36

InstrumentationSeveral systems were developed to increase sensitivity of results reading, many of these utilize turbidimetry

Particle Enhanced

Turbidimetric

Inhibition Assay (PETINA

)

Particle counting immunoassay (PACIA)Slide37

Particle Enhanced Turbidimetric Inhibition Assay (PETINA)This

method uses the creation of light scattering particles to measure drug levels.

The

latex particle-bound drug binds to the drug-specific antibody, forming insoluble light- scattering aggregates.

This causes an increase in the turbidity of the reaction mixture.

Non-particle-

bound drug in the patient sample competes with the particle-bound drug for antibody binding sites, inhibiting the formation of insoluble aggregates.

Therefore

the rate of increase of absorbance (hence the rate of the increase in turbidity,) is inversely proportional to the concentration of the drug.Slide38
Slide39

Particle counting immunoassay (PACIA)Involves measurement of the number of residual non-agglutinating particles in a specimen.Latex particles are coated with whole antibody

molecule, if antigen is present complexes will form and will screened out by counter .

An inverse relationship exists between the number of

unagglutinated

particles counted and the amount of unknown in the patient specimen. Slide40

40Qualitative Agglutination TestAgglutination 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. Slide41

41Quantitative 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

and 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.Slide42

42

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

4

Patient

1

2

3

4

5

6

7

8Slide43

43

Determining Antibody titer

Titer

is the quantity of a substance required to produce a reaction with a given volume of another substance.

Antibody titer

is the highest dilution of the biological sample that still results in agglutination, with no agglutination being observed at any higher dilution.

The term is used in serological reactions and is determined by preparing serial dilutions of antibody to which a constant amount of antigen is added. Slide44

44

Determining Antibody titer

Prozone

Equivalence Zone

Post

Zone

Serum Dilution

1:10

1:20

1:40

1:80

1:160

1:320

1:640

1:1280

1:2560

1:5120

Antigen Conc.

X

X

X

X

X

X

X

X

X

X

Agglutination

–

–

+

++

+++

++++

+++

++

+

–Slide45

Advantages and disadvantages

Advantages of agglutination tests:

Low

individual

test cost.

Ability to obtain

semi quantitative

results.

Short time to obtain result.

Don’t need expensive instrument.

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

If the sample contain micro-organisms, it does not need to be viableSlide46

Agglutination Requirements Availability of stable cell or particle suspension Presence of one or more antigens close to the surface

Knowledge

that ‘incomplete’ or

nonagglutinating

antibodies are not detectable without modifications, e.g.

antiglobulin

(

Coomb’s

) technic Slide47

Advantages and disadvantages

Disadvantages

of agglutination tests

:

It must kept in mind that agglutination reaction are screening tests only, and negative result doesn't rule out disease; the quantity of antigen or antibody may be below the sensitivity of the test system

May give false positive or negative resultsSlide48

False positive results in agglutination reactionsOvercentrifugationButton is packed too tight and is difficult to resuspend

.

Contaminated Equipment

Dust, dirt or fingerprints may cause cells to clump.

Autoagglutination

Test cells clump without specific AB present, mainly problematic with RBCs

.

Delay in Reading Tests

Dried out

Ag

may look like agglutination

Saline Stored in Glass Bottles

Colloidal Silica may leach out and cause agglutinationUsing plasma instead of serum, hemolysed

or

lipemic

sample.

Cross reactionSlide49

False negativeUndercentrifugationCells may not be close enough to interact

Inactive

Reagents

May cause improper

storage

Incorrect Incubation Temperature

May result in the lack of association for AGs and ABs

Insufficient

Incubation Time

AGs and ABs may not have time for association

Prozone

PhenomenonToo much patient AB for amount of testingDelay in Testing ProcedureAB may be eluted from RBCsSlide50

Experiment 1 Determination of the titer of blood group cold antibodySlide51

Isotonic saline.Volumetric pipettes, or equivalent: 0.1- to 0.5-mL delivery, with disposable tips.

Red cells with the antigen to be tested.

The tested serum.

MaterialsSlide52

Using 0.5-mL volumes, prepare serial twofold dilutions of serum in saline. The initial tube should contain undiluted serum and the doubling dilution range should be from 1 in 2 to 1 in 2048 (total of 12 tubes). Place 0.1

mL

of each dilution into appropriately labeled test tubes.

Add 0.1 mL of the 2% suspension of red cells to each dilution.

Gently agitate the contents of each tube; incubate for 15 min.

Centrifuge for 15 sec.

Examine the red cells macroscopically; grade and record the reactions.

ProcedureSlide53

Preparation of serial dilution

8

7

6

5

4

3

2

1

Tube #

1/256

1/128

1/64

1/32

1/16

1/8

1/4

1/2

Dilution

500

500

500

500

500

500

500

500

Serum

500

500

500

500

500

500

500

Saline

Results Slide54

The titer is reported as the reciprocal of the highest dilution of serum at which 1+ agglutination is observed. InterpretationSlide55

Exercise Preparation of serial dilutionSlide56

DilutionsDilution is decreasing the concentration of a solution by a calculated factor using an approved diluent.

As well, dilution is used to prepare samples, buffers, and controls.

In serology tests it is Used to detect the titer of a specific Ab.

When a strong positive reaction is encountered, dilution should be made to detect the titer.

Volumes used in dilutions mustn't be below 10ul or more than 1000ul .Slide57

for example: (1:4) 1 express the volume of sample4 express the total volume( sample + reagent)

This is done by mixing 100 ul of sample with 300 ul of reagent.

Serial dilution :

1:4

300ul

saline

100ul

From 1

1:16

=1/4 * 1/4

100ul

From 2

300ul

saline

1:64

=1/4 * 1/16

sample

100ul

sample

300ul

saline

Total Dillution

1

2

3

Dillution of tube 1

Dillution of tube 2Slide58

Another example on serial dillution :

sample

1:10

900ul

saline

100ul

From 1

1:100

= 1/10 * 1/10

100ul

From 2

900ul

saline

1:1000

=1/10 * 1/100

100ul

sample

900ul

saline

Total Dillution

1

2

3

Dillution of tube 1

Dillution of tube 2Slide59

9:250??It can be done only by serial dilution…9:25 * 1:10

sample

9:25

900ul

100ul

From 1

90ul

160ul

Total Dillution

9 : 250

9:25 * 1:10

1

2

1:10

9:250