SEROLOGIC

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SEROLOGIC REACTIONS
Antigens, Antibodies and Their Interactions
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• in-vitro Ag-Ab reactions.

Serologic reactions
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Antigens

• 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.

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• Antibodies

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

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Ab
Ag
Ab
Ag
Ag
Ag
Ag
Ab
Ab
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Diagnostic applications of serologic reactions

• 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.

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Methods for Detecting an Ag-Ab Reaction
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• Precipitation reactions
  (Ag is soluble)
  precipitation.
• Agglutination reactions
  (Ag is particles)
  clumping.
• Complement fixation reactions.
• Labelling methods
• a-Immuno-fluorescence reactions.
• b- ELISA.

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Precipitation Reactions
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Precipitation Reactions

• 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

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Precipitation Reactions
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Example of Precipitation Reactions

• Agar Gel diffusion method
• 1- Double diffusion
• a- Eleks Toxigenicity Test
• b- Ouchterlony method
• 2- Single radial immunodiffusion.

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Example of Precipitation Reactions

• Eleks Toxigenicity Test

To determined the toxigenic strain of C.
diphtheriae
Principle
Toxin production by C. diphtheriae can be
demonstrated by a precipitation reaction between
exotoxin and diphtheria antitoxin.
Procedure
1. Place a strip of filter paper saturated with
diphtheria antitoxin on a serum agar plate.
2. Streak the test organism across the plate at
right angle to the filter paper.
3. Incubate the plate at 35oC for 24 hrs.
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Example of Precipitation Reactions
Results
Positive test formation of four radiating lines
resulting from the precipitation reaction between
exotoxin and diphtheria antitoxin.
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Example of Precipitation Reactions

• 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.

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Example of Precipitation Reactions

• Ouchterlony method

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1
Incubate for 1h at 37c
Ag
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C
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Example of Precipitation Reactions

• 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.

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Agglutination Reactions
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Agglutination Reactions

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

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Agglutination Reactions

• 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.

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Blood grouping
Blood group A B AB O
RBC surface Ag A B A B None
Serum Ab Anti-B Anti-A None Anti-A Anti-B
Universal donor
Universal Acceptor
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Anti-A
Anti-B
Drop of blood
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2

• Agglutination in 1 only
  gp A
• Agglutination in 2 only
  gp B
• Agglutination in 1 and 2
  gp AB
• No Agglutination in 1 or 2
  gp O

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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
  Rhve (if present) 0r Rh-ve ( if absent).

Anti-Rh (anti-D)

• If agglutination occures Rhve
• If No agglutination Rh-ve

Drop of blood
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Complement Fixation Reactions
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Complement Fixation Reactions

• 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

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Complement Fixation Reactions

• 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).

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• Result
• ve reaction No lysis
• -ve reaction Haemolysis

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Labeling methods
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Immuno-fluorescence

• 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.

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Direct 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)

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Indirect immunofluorescence

• 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.

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• positive test for rabies

•  negative test for rabies

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ELISA
Enzyme-Linked Immuno Sorbant Assay

• 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.

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ELISA

• 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

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• 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)

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wash

Antigens
Antibody labelled with enzyme

substrate
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ELISA

• 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.

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wash


Antigens
Antibody




substrate
Ig enzyme labelled
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Thank You