serological techniques. antigen-antibody reactions

8/3/2019 Serological Techniques. Antigen-Antibody Reactions

1/53

1


2/53

2


3/53

I. Agglutination Reactions:

3


4/53

+

Agglutinins


5/53

1. Slide agglutination:i. Identification and typing of isolated

bacteria (Serotyping):ii. Blood grouping

2. Tube agglutination

3. Haemagglutination (Passive & Active)i. Passive haemagglutinationii. Active haemagglutination

4. Latex agglutination

5. Coagglutination test6. Coomb' s test:

i. Direct Coomb' s testii. Indirect Coomb' s test 5


6/53

1. Slide Agglutination:

i. Identification and typing ofisolated bacteria (Serotyping)

6


7/53

ii. Blood Grouping

7

This is an example of active haemagglutination.


8/53

2. Tube Agglutination

It is a semiquantitative test

Used mainly to detect Abs in patients' sera

Widaltest for the diagnosis of entericfever (typhoid and paratyphoid)

8


9/53

Widal test

Serial dilution of pt serum + Ags

1/20 4080 160 320 640

THAH

BH

CH

O

M


10/53

3. Haemagglutination (Passive &Active)

i. Passive Haemagglutination

Red cells sensitized with the Ag can be used todetect antibodies

Example TPHA for detection of treponemalantibodies.

10


11/53

ii. Active Haemagglutination:

This character can be used in ahaemagglutination inhibition test to detectviral antibodies.

11


12/53

Influenza virus


13/53

Haemagglutination inhibition test


14/53

4. latex agglutination:

latex beads could be conjugated to antibodies and used

for agglutination of antigens in solutions.

Rapid identification of pathogenic bacteria and fungisuch as N.meningitidisand Cryptococcus neoformans.

It is also possible to conjugate latex particles with the

Ags and use it for detecting antibodies in patients' seraor other body fluids.

14


15/53

5. COAGGLUTINATION TEST:

This test takes advantage of this protein A

Uses Staph aureus as an antibody-carrying

particleIf we add the appropriate bacteria or Ag to

be tested, it well attach to the specific Fabsite and the reaction will then be visualizedby clumping of staphylococci

15


16/53


17/53

6. Coomb' S Test

Incomplete or blocking antibodies are able to bindto specific antigen without producing visible effect

They can be looked for by Coombs test

17


18/53

i. Direct Coomb' S Test: It detects Abs already present on red cells.

In erythroblastosis foetalis (E.F.), baby' s redcells are coated with anti- Rh Abs.

18
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.png


19/53


20/53

II. Precipitation Reactions:

Requirements:

1. The Ag has to be soluble (in solution).

2. The Ag and the Ab have to be in optimalconcentration.

20
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.png


21/53

PRECIPITATION IN SOLUTION

21
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.png


22/53

i. Slide precipitation:as RPR and VDRL fordiagnosis of syphilis.

ii. Tube precipitation :as Lancefield test forgrouping streptococci ( Ring test).

iii.Agar ( Gel) Diffusion tests:

A. Double immunodiffusion

B. Single (Radial) immunodiffusion

22
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.png


23/53

i. Slide Precipitation

RPR and VDRL for diagnosis of syphilis.

23
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.png


24/53

VDRL

Heat inactivated patient serum +Cardiolipinlecithin cholesterol antigen---->

Examined microscopically .
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.png


25/53

RPR

Carbon containing cardiolipinantigen+unheated patient serum ---}rotate

Aggregates of carbon particles asblackclumps ,read by the naked eye.
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.png


26/53

ii. Tube Precipitation

Lancefield test for grouping streptococci ( Ringtest).

26
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.png


27/53


28/53

b. Toxin-antitoxin Reaction As In Elek's Test

28
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.png


29/53

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 tomeasure the concentration of theunknown Ag.

This technique is used for estimatingthe amount of different Ig classes inthe serum for example IgG

29
http://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://upload.wikimedia.org/wikipedia/commons/1/1c/Coombs_test_schematic.pnghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


30/53

SINGLERADIALIMMUNODIFFUSION
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


31/53

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

This is an antigen antibody reaction thatoccurs in the presence of a third componentknown as the complement.

The antigen reacts with its specific antibodyand the resulting complex fixes thecomplement.

The C.F.T. is used in the diagnosis of manydiseases by detecting C.F. antibodies in theserum of patients, as in syphilis, whoopingcough, chronic gonorrhoea, typhus and otherdiseases as well as many viral infections.

31
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


32/53

Principle Of C.F.T:

The heated serum of a patient

(unknown antibody) is added to theknown antigen, then thestandardized complement is added

no visible reactionA second step should be done in

which anindicator systemis

added made ofsheep red cellscoated with their antibody.If the complement is free (as with

negative sera), it will causehaemolysis of red cells.

Thus, haemolysis means negativeserum.

If the complement is fixed (as withpositive sera); no haemolysisoccurs.

No haemolysis means positiveserum. 32
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


33/53

Complement Fixation Assay

33
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


34/53

Complement Fixation Assay

34
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


35/53

IV. Antibody (AB) Labeled Assays:

35

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) fordetection and measurement of unknown Ag

B. Indirect ELISA for detection and measurement ofunknown antibody

3. Radioimmuno assay (RIA)A. Liquid phase RIA

B. Solid phase RIA

4. Chemiluminescence

Fluorescent Antibody
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


36/53

Fluorescent AntibodyTechniques

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 ultravioletmicroscope (UV)

36
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


37/53

37

Principal steps in direct immunofluorescence


38/53

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) labeledwith fluorescein is added

Examined by the UV microscope.

38
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


39/53

B. Indirect Immunofluorescence ( I I F)

ii. For detection of antigens

Specimen suspected to contain theantigen is fixed on a slide,

Specific antibody is added,

Antispecies antibody conjugated withfluorescent dye is added,

Examined by U.V microscope.

39
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


40/53

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 solidphase (plastic surface),

Either antigen orantibody can be linked toan enzyme such as

alkaline phosphatase orperoxidases and thecomplex retain bothimmunological andenzymatic activity.

40
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


41/53

2. Enzyme Linked Immunosorbent Assay(ELISA)

A. ELISA for detection ofunknown Ag (Sandwichmethod )

1. The wells are coated with specificantibody

2. The test solutions thought tocontain antigen are incubated inthe sensitized wells.

3. The conjugate consisting ofenzyme- labeled specific antibodyis then incubated in each well.

4. The enzyme substrate is added.

5. A color change upon degradationcan be assessed visually ormeasured in a spectrophotometer(ELISA reader).

41

2 E Li k d I b t A
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


42/53

2. Enzyme Linked Immunosorbent Assay(ELISA)

B. Indirect ELISA fordetection of unknownantibody

1. Wells are sensitized bypassive adsorption with therelevant antigen

2. The test samples areincubated in the sensitizedwells

3. Enzyme-labeled anti-humanIg conjugate is incubated in

the wells,4. Enzyme substrate is added

5. The rate of degradation ofthe substrate is indicated bya color change 42
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


43/53

3. Radioimmuno Assay (RIA)

Principle:

It isbased onlabelling either the

Ag or the Ab withradioactive

substance such asiodine or tritiatedthymidine.

The amount of

radioactivity ismeasured bygamma or betacounter 43
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


44/53


A.Liquid phase RIA

44
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


45/53


B.Solid phase RIAAg is coated

The test Ab isadded.

Radioactive labeledanti-species Ab isadded,

The radioactivity ismeasured.

45
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://www.answers.com/main/Record2?a=NR&url=http://commons.wikimedia.org/wiki/Image:Luminol2006.jpg


46/53

4. Chemiluminescence

Principle:It depends on labelling the Ab with

acetylaminofluorene ; a luminescent

substance detected by luminometer

46
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://www.answers.com/main/Record2?a=NR&url=http://commons.wikimedia.org/wiki/Image:Luminol2006.jpg


47/53

Intradermal Skin Tests

I. Neutralization tests:

These are in- vivomanifestations ofAg-Ab reactions.

47
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


48/53

A. Schick Test

To test the susceptibility of a person to diphtheria.

48


49/53

B. DICK TEST

Test the susceptibility of a person to get scarletfever.

Using the erythrogenic toxin ofStreptococcus

pyogenes.

49
http://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpghttp://atlas.arabslab.com/data/24/M33992-027-f004.jpg


50/53

C. Schultz-Charlton Reaction

Used for the diagnosis of scarlet fever.

50


51/53

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.

51


52/53

b. Delayed Type Skin Test:

The Ag is injected intradermally, and the test isread after 48-72 hours.

Delayed type hypersensitivity tests areinduced by sensitized lymphoid cells(cellularreaction).

Examples: tuberculin, lepromin, brucellin andDucrey tests.

52


53/53

53

serological techniques. antigen-antibody reactions

Documents