in-vitro Ag-Ab reactions.

Any Any foreign substancesforeign substances which when which when introduced into an animal, can introduced into an animal, can stimulate a stimulate a specific immune responsespecific immune response, , in the form of in the form of production of production of antibodiesantibodies and and specific reactive specific reactive T-lymphycytes.T-lymphycytes.

Antigens have the ability to combine Antigens have the ability to combine

specifically with the antibodies produced or specifically with the antibodies produced or sensitized T-cells induced.sensitized T-cells induced.

Glycoproteins that bind specifically to the Glycoproteins that bind specifically to the antigen that induced their formation.antigen that induced their formation.

AgAg AbAb Ag Ab Ag Ab AgAb

AgAg

1- Diagnosis of infectious diseases: known antigen preparations are used to detect

circulating antibodies in patient's serum as evidence of a current or previous infection with that agent

OR known antibodies are used to detect antigens

associated with an infectious agent directly in body fluids.

2- Identification of unknown cultures: known antibodies are used to detect their

homologous antigens in cultures.

1.1. Precipitation reactionsPrecipitation reactions ((AgAg is is solublesoluble) ) precipitation.precipitation.

2.2. Agglutination reactionsAgglutination reactions ((AgAg is is particlesparticles) clumping.) clumping.

3.3. Complement fixation reactions.Complement fixation reactions.

4.4. Labelling methods:Labelling methods:

a-Immuno-fluorescence reactions.a-Immuno-fluorescence reactions.

b- ELISA.b- ELISA.

This is an Ag-Ab reaction in which the Ag This is an Ag-Ab reaction in which the Ag is soluble (eg: Protein ; Bacterial toxin).is soluble (eg: Protein ; Bacterial toxin).

When antigens and antibody mixed in the When antigens and antibody mixed in the proper proportion, they form large proper proportion, they form large macromolecular complexes called macromolecular complexes called precipitatesprecipitates

One of the easiest of serologic testsOne of the easiest of serologic tests

Agar Gel diffusion method:

1- Double diffusion: a- Elek’s Toxigenicity Test: b- Ouchterlony method:

2- Single radial immunodiffusion.

Elek’s Toxigenicity Test:Elek’s Toxigenicity Test:

Principle:Principle:

To determined the toxigenic strain of To determined the toxigenic strain of C. diphtheriaeC. diphtheriae

Toxin production by Toxin production by C. diphtheriaeC. diphtheriae can be can be demonstrated by a precipitation reaction between demonstrated by a precipitation reaction between exotoxin and diphtheria antitoxin.exotoxin and diphtheria antitoxin.

Procedure:1. Place a strip of filter paper saturated with 1. Place a strip of filter paper saturated with diphtheria antitoxin on a serum agar plate.diphtheria antitoxin on a serum agar plate.

3. Incubate the plate at 353. Incubate the plate at 35ooC for 24 hrs.C for 24 hrs.

2. Streak the test organism across the plate . Streak the test organism across the plate at right angle to the filter paper.at right angle to the filter paper.

Results:

Positive test:Positive test: formation of four radiating lines resulting formation of four radiating lines resulting from the precipitation reaction between exotoxin and from the precipitation reaction between exotoxin and diphtheria antitoxin.diphtheria antitoxin.

Ouchterlony method:Ouchterlony method:

Wells are punched in the agar. The antigen Wells are punched in the agar. The antigen

in one well and the antibody is placed in in one well and the antibody is placed in another.another.

Both will diffuse in the agar, and Both will diffuse in the agar, and precipitation bands are formed where they precipitation bands are formed where they meet at optimal proportions.meet at optimal proportions.

Ouchterlony method:Ouchterlony method:

21

C 3

Ag

21

C 3

AgIncubate for

1h at 37°c

Single radial immunodiffusion:Single radial immunodiffusion:

The antibody is mixed with the agar before pouring The antibody is mixed with the agar before pouring it in the plate, while the antigen is placed in a well it in the plate, while the antigen is placed in a well punched in the agar. punched in the agar.

the Ag diffuses in all directions, and where its the Ag diffuses in all directions, and where its concentration is optimal in relation to the antibody, a concentration is optimal in relation to the antibody, a precipitation ring will form around the well. The precipitation ring will form around the well. The diameter of the well depends on the Ag diameter of the well depends on the Ag concentration.concentration.

e.g., quantitation of various Ig classes in human e.g., quantitation of various Ig classes in human serum samples. serum samples.

When Ag is in form of particles, it will When Ag is in form of particles, it will become clump if react with specific Ab.become clump if react with specific Ab.

Example of Slide agglutination: Example of Slide agglutination:

Blood grouping (ABO Blood grouping (ABO grouping)grouping)

There are 4 blood groups There are 4 blood groups depending on the presence depending on the presence or absence of either or both or absence of either or both two types of antigens A and B two types of antigens A and B on the surface of RBCs.on the surface of RBCs.

Blood groupBlood group

AA

BB

ABAB

OO

RBC surface AgRBC surface Ag AA

BB

A & BA & B

None None

Serum AbSerum Ab

Anti-BAnti-B

Anti-AAnti-A

NoneNone

Anti-A & Anti-BAnti-A & Anti-B

Universal AcceptorUniversal AcceptorUniversalUniversal donordonor

              

Anti-AAnti-A Anti-BAnti-B

Drop of bloodDrop of blood

• Agglutination in 1 only gp AAgglutination in 1 only gp A

• Agglutination in 2 only gp BAgglutination in 2 only gp B

•Agglutination in 1 and 2 gp ABAgglutination in 1 and 2 gp AB

• NoNo Agglutination in 1 or 2 gp O Agglutination in 1 or 2 gp O

1 2

Rhesus blood groupRhesus blood group

Rh or D is clinically and medically important.Rh or D is clinically and medically important. According to presence or absence of Rh antigen According to presence or absence of Rh antigen

on the RBCs surface, the individuals classify to on the RBCs surface, the individuals classify to Rh+ve (if present) 0r Rh-ve ( if absent).Rh+ve (if present) 0r Rh-ve ( if absent).

Drop of bloodDrop of blood

Anti-Rh (anti-D)Anti-Rh (anti-D)

• If agglutination occures Rh+veIf agglutination occures Rh+ve

• If No agglutination Rh-veIf No agglutination Rh-ve

The complement is a group o heat-The complement is a group o heat-labile proteins normally found in blood labile proteins normally found in blood and tissue fluids (except urine & CSF).and tissue fluids (except urine & CSF).

CF is an Ag-Ab reaction that occurs in CF is an Ag-Ab reaction that occurs in the presence of the the presence of the complement.complement.

The Ag unites with its specific Ab and The Ag unites with its specific Ab and the resulting complex fixes (consumes) the resulting complex fixes (consumes) the complementthe complement

Two systems are used in CFT:Two systems are used in CFT:1- Test system:1- Test system: The serum sample (heated to 56The serum sample (heated to 56°)°) Measured amount of Ag.Measured amount of Ag. Complement (Guinea pig serum).Complement (Guinea pig serum). if the serum contains the specific Abif the serum contains the specific Ab→ Ag-Ab → Ag-Ab

complexes→ will fix all the complement. complexes→ will fix all the complement. Ag + Ab + C fixationAg + Ab + C fixation

Ag + C No fixation (free complement)Ag + C No fixation (free complement)

2- Indicator system2- Indicator system e.g;e.g; sensitized sheep RBCs.sensitized sheep RBCs. (Sheep RBCs coated with their specific Abs).(Sheep RBCs coated with their specific Abs).

Result:Result: ++ve reaction No lysis

-ve reaction Haemolysis

These are Ag-Ab reactions in which Ab These are Ag-Ab reactions in which Ab is labelled with fluorescein.is labelled with fluorescein.

Fluorescein is a dye which emits Fluorescein is a dye which emits greenish fluorescence under UV light.greenish fluorescence under UV light.

There are two ways for this test;There are two ways for this test;Direct immunofluorescence,Direct immunofluorescence,Indirect immunofluorescence.Indirect immunofluorescence.

In this test a fluorescein-labelled Ab is added In this test a fluorescein-labelled Ab is added to detect the presence of Ag in tissue section to detect the presence of Ag in tissue section fixed on a microscopic slide. fixed on a microscopic slide.

A drop of the labelled Ab is placed on the A drop of the labelled Ab is placed on the section and left to react for some min.section and left to react for some min.

the excess unattached Ab is washedthe excess unattached Ab is washed Examine under UV rays.Examine under UV rays. If Ag is present fluorescenceIf Ag is present fluorescence If Not No fluorescenceIf Not No fluorescence

Disadvantage:Disadvantage: expensive method (for each expensive method (for each Ag we need specific labelled Ab) Ag we need specific labelled Ab)

TheThe test is used to detect Ab in patients’ test is used to detect Ab in patients’ sera.sera.

Fluorescein labelled anti-human Ig is used .Fluorescein labelled anti-human Ig is used . Known Ag is fixed on a slideKnown Ag is fixed on a slide Add the patient's serum & allow to react for Add the patient's serum & allow to react for

some timesome time the excess is washed, the excess is washed, add Fluorescein labelled anti-human Ig add Fluorescein labelled anti-human Ig

(attach to the Fc portion of the human Ig if (attach to the Fc portion of the human Ig if present). present).

examine under UV.examine under UV.

                                                                                                        

• positive test for rabies • negative test for rabies

This technique is ;This technique is ;Very sensitive Very sensitive Does not require specialized equipmentDoes not require specialized equipment Avoid the hazards of radioactivity.Avoid the hazards of radioactivity.

The method depends on conjugation of The method depends on conjugation of an enzyme to either Ag or Ab, then an enzyme to either Ag or Ab, then substrate is added as a quantitative substrate is added as a quantitative measure of enzyme activity.measure of enzyme activity.

Direct ELISA (double Ab technique):Direct ELISA (double Ab technique):

used for detection of Ags.used for detection of Ags. Known specific Ab is immobilized by Known specific Ab is immobilized by

adsorption onto a plastic surface.adsorption onto a plastic surface.Clinical sample is added (if Ag present it will Clinical sample is added (if Ag present it will

bind to the immobilized Ab) bind to the immobilized Ab) enzyme-labelled specific Ab is addadenzyme-labelled specific Ab is addad

(attach to the fixed Ag if present)(attach to the fixed Ag if present)wash the excess wash the excess add the substrateadd the substrate

If Ab specific to Ag change the colorIf Ab specific to Ag change the color If Not specific No color changeIf Not specific No color change

Dark yellow highly +veDark yellow highly +veYellow moderate +veYellow moderate +veColor less --veColor less --ve

Disadvantages:Disadvantages: expensive method expensive method (for each Ag we need (for each Ag we need specific Ab labelled)specific Ab labelled)

AntigensAntigens

++

Antibody labelled Antibody labelled with enzymewith enzyme

washwash

++

substratesubstrate

Indirect ELISA:Indirect ELISA: In this testIn this test an enzyme- labelled anti-human Ig an enzyme- labelled anti-human Ig

is used to detect the presence of specific is used to detect the presence of specific Abs in patients’ sera. Abs in patients’ sera.

Known Ag is fixed Known Ag is fixed by adsorption onto a by adsorption onto a plastic surface.plastic surface.

The serum sample is added The serum sample is added ( if specific Ab is ( if specific Ab is present, it will bind the fixed Ag).present, it will bind the fixed Ag).

WashWash Add the enzyme-labelled antihuman Ig Add the enzyme-labelled antihuman Ig wash the excess wash the excess add the substrate, then quantitatively add the substrate, then quantitatively

measure for the degree of color change. measure for the degree of color change.

AntigensAntigens

++

AntibodyAntibody

==washwash

++

Ig enzyme labelled Ig enzyme labelled

== ++

substratesubstrate

==