2. practical No. 3: Agglutination test2

3. 3 INTRODUCTION: In this reaction the antigen is part of the surface of some particulate material such as a red cell, a bacterium or perhaps an inorganic particle (e.g. polystyrene latex), which has been coated with antigen. Antibodies added to a suspension of these particles combine with the surface antigens and link them together to form visible aggregate or clumps.

4. Figure 18.4 4practical No. 3: Agglutination test Definition: It is an antigen antibody interaction, in which the antigen is found in suspended form (particulate antigens e.g., bacterial cells) and the antigen-antibody immune complexes appear in a form of clumps.

5. Agglutination/Haemagglutination : When the antigen is an erythrocyte or carried on erythrocytes the term heamagglutination is used. The general term agglutinin is used to describe antibodies that agglutinate particulate antigens. All antibodies can theoretically agglutinate particulate antigens but IgM, due to its high valence, is particularly good agglutinin and one sometimes infers that an antibody may be of the IgM class if it is a good agglutinating antibody. 5practical No. 3: Agglutination test

6. 6 Interaction of antibodies with particulate antigens is called Direct agglutination”. However, interaction of antibodies with soluble antigens (e.g. viral Ag, polysaccharide Ag or a hapten) carried on carrier particles,e.g., latix is called Indirect or passive agglutination test”. The test is performed just like the agglutination test. Direct agglutination Indirect agglutination

7. practical No. 3: Agglutination test7

8. 8 A- Qualitative agglutination tests: 1- Rapid Slide or plate agglutination test : Exercise No. 1 : Blood grouping slide agglutination, Exercise No. 2 : Pullorum test (avian salmonellosis), Exercise No. 3 : Salmonella typhi and S. paratyphi slide agglutination test Exercise No. 4 : Rose Bengal plate test (bovine brucellosis). 2- Milk ring test:(bovine brucellosis) B- Quantitative agglutination tests: 1- Tube agglutination test: Exercise No. 5 : (bovine brucellosis). Exercise No. 6 : Widal test (human salmonellosis). 2- Microplate agglutination (Microagglutination) Test: Exercise No. 7: Application of microagglutination for diagnosis of bovine brucellosis. 3- Microscopical Agglutination test (MAT):(leptospira infection)

9. practical No. 3: Agglutination test9

10. Blood group A If you belong to the blood group A, you have A antigens on the surface of your RBCs and B antibodies in your blood plasma. Blood group B If you belong to the blood group B, you have B antigens on the surface of your RBCs and A antibodies in your blood plasma. AB0 blood grouping system Blood group AB If you belong to the blood group AB, you have both A and B antigens on the surface of your RBCs and no A or B antibodies at all in your blood plasma. Blood group O If you belong to the blood group O (null), you have neither A or Bantigens on the surface of your RBCs but you have both A and B antibodies in your blood plasma.

11. EXCERCISE NO.1:Blood grouping slide agglutination practical No. 3: Agglutination test 11 Material: Lancets Cotton and ethyl alcohol 70% Monoclonal typing antisera ; anti- A, anti- B and anti-D Clean glass slide Procedure: Clean your finger with alcohol. Stab fingers with lancet and places a drop of blood on each of 3 slides, labeled A, B and D. Add a drop of anti A to one slide, a drop of anti-B sera to the slide labeled as B and a drop of anti-D to the slide D. Using applicator stick mix the blood with the antisera on each slide. Use a clean applicator stick for each slide. Record the results Blood groupAnti -AAnti-B A+- B-+ AB++ O-- A patient's red blood cells can be mixed with antibody to a blood group antigen ( anti-A,anti-B anti -D ) to determine a person's blood type.

12. 12 practical No. 3: Agglutination test Microscopical appearance of agglutination Macroscopical appearance of agglutination EXCERCISE NO.1:Blood grouping slide agglutination

13. It is a rapid plate agglutination test. Used for diagnosis of bovine brucellosis. The brucella antigen is stained with rose bengal stain (rose colour). It is a screening test and positive cases should be confirmed with tube agglutination or CFT tests practical No. 3: Agglutination test13

14. Principal: The RBP-test is a rapid agglutination test that is effective in the diagnosis of bovine brucellosis when used as a screening test. The antigen for RBPT is a dense (8%) suspension of inactivated Brucella- organisms stained with Rose Bengal and adjusted to pH 3.65. Material; 1.Rose-Bengal stained antigen. 2.Positive and negative control sera. 3.Tested serum sample. 4.Micropipette. 5.Plates of white enamel or microscopic slides. 6.Applicator sticks. Antigen, control sera and test sera should be removed from the refrigerator 1/2-1 hour before test. Sera and antigen if used straight from the refrigerator will react poorly 1- Place one drop (30 µ1) of antigen on each square of the plate/ microscopic slide. 2- Place one drop (30 µl) of the serum alongside (but not into) the antigen. 3- With the applicator stick, mix the antigen and serum thoroughly. 4- When all sera have been mixed, shake gently or place the plate/slide on the rocking machine and rock for 4 min. 5- Read the results immediately after 4 minutes by examining for agglutination in a good light. Agglutination takes place almost immediately after the serum and antigen have been mixed, whereas, in other cases, agglutination is delayed until the end of the 4 minute period. 6- Recording: The result may be judged as positive or negative according to the presence or absence of any degree of agglutination. Interpretation of the result: Any degree of agglutination is taken as a positive result. This may be supported by the confirmatory test. practical No. 3: Agglutination test14

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16. Widal test The Widal test is a serological test for Salmonella. It is a demonstration of salmonella agglutinating antibodies against antigens O-somatic and H-flagellar in the blood. It is used as a presumptive diagnostic test for Enteric fever.serologicalSalmonella Clinical signs: Typhoid fever and Salmonellosis are endemic in many part of development world. the disease characteristic with: 1-Prolonged fever 2-Bacteremia 3-Involvement of the Reticuloenothelial system, particularly liver, spleen, intestines, and mesentery 4-Dissemination to multiple organs 5-Occurs more often in tropical and subtropical countries practical No. 3: Agglutination test16

17. Compositions *Febrile antigen consist of : Salmonella typhi O Salmonella typhi H SalmonellaO paratyphi a SalmonellaH paratyphi a SalmonellaO paratyphi b SalmonellaH paratyphi b SalmonellaO paratyphi c SalmonellaH paratyphi c *Control positive (control antibody +) *Control negative (control antibody -) Specimen and sample preparation Use fresh serum obtained by centrifugation of clotted blood. practical No. 3: Agglutination test17

18. PROCEDURE 1- Allow reagents and serum samples to reach room temperature for testing. 2- Shake the antigen bottle gently to insure a uniform suspension. 3- Place one drop sample serum onto the selected ring of the slide. 4- Place one drop of the stained antigen suspensions onto serum sample. 5- Mix serum sample with stained antigen suspensions using stick and rotate the slide for one minute. 6- Repeat these steps using the positive and negative controls instead of serum sample. practical No. 3: Agglutination test18

19. Application of for the diagnosis of bovine brucellosis. practical No. 3: Agglutination test19

20. 20 practical No. 3: Agglutination test

21. 21practical No. 3: Agglutination test Material 1.Test tubes (8 x 100 mm) 2.Dropper (30µl) 3.Water bath 37˚C 4.Stained ring test antigen 5.Positive and negative controls Procedure: 1)-Mix the milk thoroughly to disperse the cream evenly and transfer 1 ml to a narrow test tube. 2)-Add 1 drop of ring test antigen (30 µl) and mix by gentle shaking. 3)-Incubate in the water bath at 37˚C for 1 hour. I In the case of brucellosis, the lipophylic antibodies, attached to the milk fat globules, will fix the blue stained brucella ring test antigen. During incubation, the milk fat globules, surrounded by stained antigen-antibody immuno-complexes will float upward, thus forming a blue cream layer. The test is not effective if the milk has been pasteurized! 4)-Recording: If the intensity of blue colour in the cream layer is equal to or deeper than in the skim portion, the test is considered positive. If the intensity of the colour in the cream layer is less than in the skim portion, the test is considered negative. The milk ring test has not proved effective with the milk of sheep and goats.

22. Practical No.122