2. in-vitro Ag-Ab reactions.

3. 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. 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. Antigens have the ability to combine specifically with the antibodies produced or sensitized T-cells induced.

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

5. AgAb Ag Ab Ag Ab Ag Ab Ag

6. 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.

8. 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. a-Immuno-fluorescence reactions. b- ELISA. b- ELISA.

10. This is an Ag-Ab reaction in which the Ag is soluble (eg: Protein ; Bacterial toxin). 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 When antigens and antibody mixed in the proper proportion, they form large macromolecular complexes called precipitates One of the easiest of serologic tests One of the easiest of serologic tests

12. Agar Gel diffusion method: 1- Double diffusion: a- Elek’s Toxigenicity Test: b- Ouchterlony method: 2- Single radial immunodiffusion.

13. Elek’s Toxigenicity Test: Elek’s Toxigenicity Test: Principle: To determined the toxigenic strain of C. diphtheriae 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. 3. Incubate the plate at 35 o C for 24 hrs.. Streak the test organism across the plate at right angle to the filter paper. 2. Streak the test organism across the plate at right angle to the filter paper.

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

15. Ouchterlony method: Ouchterlony method: Wells are punched in the agar. The antigen in one well and the antibody is placed in another. 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. Both will diffuse in the agar, and precipitation bands are formed where they meet at optimal proportions.

16. Ouchterlony method: Ouchterlony method: 2 1 C 3 Ag 2 1 C 3 Incubate for 1h at 37°c

18. Single radial immunodiffusion: 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 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. 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. e.g., quantitation of various Ig classes in human serum samples.

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

21. Example of Slide agglutination: Example of Slide agglutination: Blood grouping (ABO grouping) Blood grouping (ABO grouping) There are 4 blood groups 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. on the surface of RBCs.

22. Blood group ABABO RBC surface Ag AB A & B None Serum Ab Anti-BAnti-ANone Anti-A & Anti-B Universal Acceptor Universaldonor Universal donor

24. Anti-AAnti-B Drop of blood Agglutination in 1 only gp A Agglutination in 1 only gp A Agglutination in 2 only gp B Agglutination 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 O No Agglutination in 1 or 2 gp O 12

25. Rhesus 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 on the RBCs surface, the individuals classify to Rh+ve (if present) 0r Rh-ve ( if absent). 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 blood Anti-Rh (anti-D) If agglutination occures Rh+ve If agglutination occures Rh+ve If No agglutination Rh-ve If No agglutination Rh-ve

30. The complement is a group o heat- labile proteins normally found in blood and tissue fluids (except urine & CSF). 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. 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 The Ag unites with its specific Ab and the resulting complex fixes (consumes) the complement

31. Two systems are used in CFT: Two systems are used in CFT: 1- Test system: The serum sample (heated to 56°) The 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 Ab → Ag-Ab complexes→ will fix all the complement. if the serum contains the specific Ab → Ag-Ab complexes→ will fix all the complement. Ag + Ab + C fixation Ag + Ab + C fixation Ag + C No fixation (free complement) Ag + C No fixation (free complement) 2- Indicator system e.g; sensitized sheep RBCs. (Sheep RBCs coated with their specific Abs). (Sheep RBCs coated with their specific Abs).

32.  Result:  +  +ve reaction No lysis  -ve reaction Haemolysis

35. These are Ag-Ab reactions in which Ab is labelled with fluorescein. These are Ag-Ab reactions in which Ab is labelled with fluorescein. Fluorescein is a dye which emits greenish fluorescence under UV light. Fluorescein is a dye which emits 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.

36. In this test a fluorescein-labelled Ab is added to detect the presence of Ag in tissue section fixed on a microscopic slide. 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. A drop of the labelled Ab is placed on the section and left to react for some min. the excess unattached Ab is washed the excess unattached Ab is washed Examine under UV rays. Examine under UV rays. If Ag is present fluorescence If Ag is present fluorescence If Not No fluorescence If Not No fluorescence Disadvantage: expensive method (for each Ag we need specific labelled Ab) Disadvantage: expensive method (for each Ag we need specific labelled Ab)

37. The test is used to detect Ab in patients’ sera. The test is used to detect Ab in patients’ sera. Fluorescein labelled anti-human Ig is used. Fluorescein labelled anti-human Ig is used. Known Ag is fixed on a slide Known Ag is fixed on a slide Add the patient's serum & allow to react for some time Add the patient's serum & allow to react for some time the excess is washed, the excess is washed, add Fluorescein labelled anti-human Ig (attach to the Fc portion of the human Ig if present). add Fluorescein labelled anti-human Ig (attach to the Fc portion of the human Ig if present). examine under UV. examine under UV.

38. positive test for rabies negative test for rabies

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

41. 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 adsorption onto a plastic surface. 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) Clinical sample is added (if Ag present it will bind to the immobilized Ab) enzyme-labelled specific Ab is addad enzyme-labelled specific Ab is addad (attach to the fixed Ag if present) wash the excess wash the excess add the substrate add the substrate

42. If Ab specific to Ag change the color If Ab specific to Ag change the color If Not specific No color change If Not specific No color change Dark yellow highly +ve Dark yellow highly +ve Yellow moderate +ve Yellow moderate +ve Color less --ve Color less --ve Disadvantages: expensive method (for each Ag we need specific Ab labelled) Disadvantages: expensive method (for each Ag we need specific Ab labelled)

43. Antigens + Antibody labelled with enzyme wash + substrate

44. Indirect ELISA: Indirect ELISA: In this test an enzyme- labelled anti-human Ig is used to detect the presence of specific Abs in patients’ sera. 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. 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). The serum sample is added ( if specific Ab is present, it will bind the fixed Ag). Wash Wash Add the enzyme-labelled antihuman Ig Add the enzyme-labelled antihuman Ig wash the excess wash the excess add the substrate, then quantitatively measure for the degree of color change. add the substrate, then quantitatively measure for the degree of color change.

45. Antigens + Antibody = wash + Ig enzyme labelled = + substrate =