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I.

Agglutination Reactions:

Agglutinins

1. Slide agglutination: i. Identification and typing of isolated bacteria (Serotyping): ii. Blood grouping 2. Tube agglutination 3. Haemagglutination (Passive & Active) i. Passive haemagglutination ii. Active haemagglutination 4. Latex agglutination 5. Coagglutination test 6. Coomb' s test: i. Direct Coomb' s test ii. Indirect Coomb' s test

1. Slide Agglutination:
i. Identification and typing of isolated bacteria (Serotyping)

ii. Blood Grouping


This is an example of active haemagglutination.

2. Tube Agglutination It is a semiquantitative test


Used mainly to detect Abs in patients' sera Widal test for the diagnosis of enteric

fever (typhoid and paratyphoid)

Widal test

Serial dilution of pt serum + Ags 1/20

40 80 160 320 640

TH
AH BH CH O

3. Haemagglutination (Passive & Active)

i. Passive Haemagglutination
Red cells sensitized with the Ag can be used to detect antibodies Example TPHA for detection of treponemal antibodies.

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ii. Active Haemagglutination:


This

character can be used in a haemagglutination inhibition test to detect viral antibodies.

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Influenza virus

Haemagglutination inhibition test

4. latex agglutination:
latex beads could be conjugated to antibodies and used for agglutination of antigens in solutions. Rapid identification of pathogenic bacteria and fungi such as N.meningitidis and Cryptococcus neoformans. It is also possible to conjugate latex particles with the Ags and use it for detecting antibodies in patients' sera or other body fluids.

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5. COAGGLUTINATION TEST: This test takes advantage of this protein A Uses Staph aureus as an antibody-carrying particle If we add the appropriate bacteria or Ag to be tested, it well attach to the specific Fab site and the reaction will then be visualized by clumping of staphylococci
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6. Coomb' S Test
Incomplete or blocking antibodies are able to bind to specific antigen without producing visible effect They can be looked for by Coombs test

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i. Direct Coomb' S Test:


It detects Abs already present on red cells. In erythroblastosis foetalis (E.F.), baby' s red cells are coated with anti- Rh Abs.

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ii. Indirect Coomb' s Test:


It detects anti-Rh Abs in mother's serum.

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II. Precipitation Reactions:


Requirements: 1. The Ag has to be soluble (in solution). 2. The Ag and the Ab have to be in optimal concentration.

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PRECIPITATION IN SOLUTION

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i. Slide precipitation: as RPR and VDRL for diagnosis of syphilis. ii. Tube precipitation :as Lancefield test for grouping streptococci ( Ring test). iii. Agar ( Gel) Diffusion tests: A. Double immunodiffusion B. Single (Radial) immunodiffusion

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i.

Slide Precipitation

RPR and VDRL for diagnosis of syphilis.

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VDRL

Heat inactivated patient serum +Cardiolipin lecithin cholesterol antigen---->

Examined microscopically .

RPR
Carbon containing cardiolipin antigen+unheated patient serum ---}rotate Aggregates of carbon particles as blackclumps ,read by the naked eye.

ii. Tube Precipitation


Lancefield test for grouping streptococci ( Ring test).

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iii. Agar ( Gel) Diffusion Tests:


A. Double immunodiffusion a. Both the Ag and Ab will diffuse, and at the site of optimum proportion a precipitation band will occur

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b.

Toxin-antitoxin Reaction As In Elek's Test

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B. Single (Radial) Immunodiffusion:


The Ab is incorporated in the agarose gel plate. The Ag is placed in a well punched in the agarose. This is a quantitative method to measure the concentration of the unknown Ag. This technique is used for estimating the amount of different Ig classes in the serum for example IgG

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SINGLE RADIAL IMMUNODIFFUSION

III. Complement Fixation Test (C.F.T):


This is an antigen antibody reaction that occurs in the presence of a third component known as the complement. The antigen reacts with its specific antibody and the resulting complex fixes the complement. The C.F.T. is used in the diagnosis of many diseases by detecting C.F. antibodies in the serum of patients, as in syphilis, whooping cough, chronic gonorrhoea, typhus and other diseases as well as many viral infections.
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Principle Of C.F.T:
The heated serum of a patient (unknown antibody) is added to the known antigen, then the standardized complement is added no visible reaction A second step should be done in which an indicator system is added made of sheep red cells coated with their antibody. If the complement is free (as with negative sera), it will cause haemolysis of red cells. Thus, haemolysis means negative serum. If the complement is fixed (as with positive sera); no haemolysis occurs. No haemolysis means positive

serum.

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Complement Fixation Assay

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Complement Fixation Assay

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IV. Antibody (AB) Labeled Assays:


1. Fluorescent Antibody techniques
A. Direct immunofluorescence (D I F) B. Indirect immunofluorescence ( I I F)
i. For detection of antibodies ii. For detection of antigens

2. Enzyme Linked Immunosorbent Assay( ELISA)


A. Sandwich method ELISA (Double antibody) for detection and measurement of unknown Ag B. Indirect ELISA for detection and measurement of unknown antibody

3. Radioimmuno assay (RIA)


A. Liquid phase RIA B. Solid phase RIA

4. Chemiluminescence

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Fluorescent Antibody Techniques A. Direct immunofluorescence (D I F)


The Ag to be detected is present in a smear The fluorescein labeled specific Ab is added The slide is then examined by an ultraviolet microscope (UV)

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Principal steps in direct immunofluorescence

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B. Indirect immunofluorescence ( I I F)
i. For detection of antibodies
Placing the Ag on a clean slide, the patient' serum is added, Antispecies (Anti-human gamma globulin) labeled with fluorescein is added Examined by the UV microscope.

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B. Indirect Immunofluorescence ( I I F)
ii. For detection of antigens
Specimen suspected to contain the antigen is fixed on a slide, Specific antibody is added, Antispecies antibody conjugated with fluorescent dye is added, Examined by U.V microscope.

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2. Enzyme Linked Immunosorbent Assay ( ELISA)


Principle of the test:

It is based on two assumptions that :


Antigen or antibody can be attached to a solid phase (plastic surface), Either antigen or antibody can be linked to an enzyme such as alkaline phosphatase or peroxidases and the complex retain both immunological and enzymatic activity.

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2. Enzyme Linked Immunosorbent Assay (ELISA)


A. ELISA for detection of unknown Ag (Sandwich method )
1. The wells are coated with specific antibody 2. The test solutions thought to contain antigen are incubated in the sensitized wells. 3. The conjugate consisting of enzyme- labeled specific antibody is then incubated in each well. 4. The enzyme substrate is added. 5. A color change upon degradation can be assessed visually or measured in a spectrophotometer (ELISA reader).

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2. Enzyme Linked Immunosorbent Assay (ELISA)


B. Indirect ELISA for detection of unknown antibody
1.
2. 3. 4. 5.

Wells are sensitized by passive adsorption with the relevant antigen The test samples are incubated in the sensitized wells Enzyme-labeled anti-human Ig conjugate is incubated in the wells, Enzyme substrate is added The rate of degradation of the substrate is indicated by a color change

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3. Radioimmuno Assay (RIA)


Principle: It is based on labelling either the Ag or the Ab with radioactive substance such as iodine or tritiated thymidine. The amount of radioactivity is measured by gamma or beta counter

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3. Radioimmuno Assay (RIA)


A. Liquid phase RIA

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3. Radioimmuno Assay (RIA)


B. Solid phase RIA
Ag is coated The test Ab is added. Radioactive labeled anti-species Ab is added, The radioactivity is measured.
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4. Chemiluminescence
Principle: It depends on labelling the Ab with acetylaminofluorene ; a luminescent substance detected by luminometer

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Intradermal Skin Tests


I. Neutralization tests: These are in- vivo manifestations of Ag-Ab reactions.

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A. Schick Test

To test the susceptibility of a person to diphtheria.

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B. DICK TEST
Test the susceptibility of a person to get scarlet fever. Using the erythrogenic toxin of Streptococcus

pyogenes.

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C. Schultz-Charlton Reaction
Used for the diagnosis of scarlet fever.

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II. Hypersensitivity TESTS


a. Immediate type skin test:

As testing for the sensitivity to penicillin. An immediate wheal and flare indicates that the person is hypersensitive to penicillin.

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b. Delayed Type Skin Test:


The Ag is injected intradermally, and the test is read after 48-72 hours. Delayed type hypersensitivity tests are induced by sensitized lymphoid cells(cellular reaction). Examples: tuberculin, lepromin, brucellin and Ducrey tests.

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