Antigens, Antibodies and Their Interactions

in-vitro Ag-Ab reactions.

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

Antigens have the ability to combine

specifically with the antibodies produced or 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. Precipitation reactions (Ag is soluble) precipitation.

2. Agglutination reactions (Ag is particles) clumping.

3. Complement fixation reactions.

4. Labelling methods: a-Immuno-fluorescence reactions. b- ELISA.

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

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

• One 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:

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: Wells are punched in the agar. The antigen

in one well and the antibody is placed in another.

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

• Ouchterlony method:

21

C 3

Ag

21

C 3

AgIncubate for

1h at 37°c

• Single radial immunodiffusion: The antibody is mixed with the agar before pouring

it in the plate, while the antigen is placed in a well punched in the agar.

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

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

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

• Example of Slide agglutination:

• Blood grouping (ABO grouping)

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

• No Agglutination in 1 or 2 gp ONo Agglutination in 1 or 2 gp O

1 2

Rhesus blood group• Rh or D is clinically and medically important.• According to presence or absence of Rh antigen

on the RBCs surface, the individuals classify to 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-labile proteins normally found in blood and tissue fluids (except urine & CSF).

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

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

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

complexes→ will fix all the complement. Ag + Ab + C fixation Ag + C No fixation (free complement)

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

Result:Result: ++ve reaction No Lysis

-ve reaction Haemolysis

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

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

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

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

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

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

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

• The test is used to detect Ab in patients’ sera.

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

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

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

• examine under UV.

• positive test for rabies • negative test for rabies

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

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

• Direct ELISA (double Ab technique): used for detection of Ags.

– Known specific Ab is immobilized by adsorption onto a plastic surface.

– Clinical sample is added (if Ag present it will bind to the immobilized Ab)

– enzyme-labelled specific Ab is addad(attach to the fixed Ag if present)– wash the excess – add the substrate

– If Ab specific to Ag change the color– If Not specific No color change

– Dark yellow highly +ve– Yellow moderate +ve– Color less --ve

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

AntigensAntigens

++

Antibody labelled Antibody labelled with enzymewith enzyme

washwash

++

substratesubstrate

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

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

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

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

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

measure for the degree of color change.

AntigensAntigens

++

AntibodyAntibody

==washwash

++

Ig enzyme labelled Ig enzyme labelled

== ++

substratesubstrate

==