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Agglutination

Agglutination. It is an Ag-Ab reaction in which the antigen is bound to a surface. when the Ab react with the Ag that Bound to its original surface we call it Direct agglutination . when the Ab react with the Ag that Bound to artificial surface we call it Indirect agglutination .

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Agglutination

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  1. Agglutination It is an Ag-Ab reaction in which the antigen is bound to a surface when the Ab react with the Ag that Bound to its original surface we call it Direct agglutination when the Ab react with the Ag that Bound to artificial surface we call it Indirect agglutination

  2. Agglutination +VE -VE

  3. Coomb’s test (Antiglobulin test or AGT) • identify antibodies present in a patient's serum prior before blood transfusion. • Blood transfusion preparation. • Antenatal antibody screening. immune-mediated hemolytic anemia. Hemolytic disease of the newborn (ABO, Rh)

  4. +VE Agglutination Latex agglutination

  5. Agglutination Latex agglutination: Lancefield grouping for streptococci

  6. Agglutination  Aggregation of “particulate antigens” bound with agglutinins of more than one specificity. Example: coagglutinationstaphylococcus aureusprotein A for serotyping strains of Streptococcus pneumoniae, Co-agglutination

  7. Agglutination Haemagglutination

  8. Haemagglutination inhibition Example: Influenza virus

  9. Heterophiles antibodies tests (Monospot test, Paul – Bunnell test): •  agglutination of the horse RBCs by heterophile antibodies in patient's serum for rapid diagnosis of EBV infectious mononucleosis. • Cold agglutnintest: • Cold agglutinins are antibodies made by the immune system in response to infection having the ability to agglutinate RBC in Low temperature. E.g. Mycoplasma pneumoniae • Weil felixtest: • it is agglutination test for the diagnosis of rickettsial infections. • based on the presence of antigenic “cross-reaction” between Rickettsiaand certain serotypes of  Proteus • VDRL: screening for syphilis caused by treponemapallidum. • Widal test: standard tube agglutination test for salmonella typhi.

  10. Precipitation Ag Ab • It is a reaction in which the Ab interact with a free soluble Ag . • At the optimum concentration ,positive reaction form a precipitin line

  11. Precipitation: Ouchterlony, agar diffusion test Complete identity Double immunodiffusion • This is precipitation reaction to test identity between two antigens or between two antibodies Partial identity Non identity

  12. Precipitation: Ouchterlony, agar diffusion test Complete identity Double immunodiffusion • This is precipitation reaction to test identity between two antigens or between two antibodies Partial identity Non identity

  13. Negative control (non Toxogenic) Test organism Positive control (Toxogenic) antitoxin strip Precipitin lines Elek's Test

  14. an Antigen (toxin), antibody • (antitoxin) reaction. • +ve reaction produce • a precipitin lines. (precipitation reaction) • Used to identify • the toxogenic • corynebacteriumdiphtherae Elek's Test

  15. Precipitation Sample concentration Ag Mg/ml Sample Diameter Diameter /mm • This is precipitation reaction to test presence of entire subclass of immunoglobulin by applying specific antiglobulin forming a precipitation rings. Single radial immunodiffusion

  16. Precipitation • This is method to separate the serum by electrophoresis. • Create a trench containing specific anti serum. • Diffusion of both serum and anti serum forming a precipitin lines Counter immuno-electrophoresis

  17. +VE -VE IMMUNOELECTROPHORESIS

  18. Western blot technique used to detect specific proteins in the given sample. It uses ”gel electrophoresis” to separate proteins (or polypeptide) and are then transferred to a membrane. (nitrocellulose)where they are stained with labled antibodies specific to the target protein.

  19. E E E E E E +VE -VE Sample AB Test Ag Enzym linked Ab Enzyme substrate

  20. Western blot

  21. Immunofluorescence • Antibodies containing fluorescent dye react with serum (either Ag or Specific Ab).. • Positive reaction is shown under fluorescent microscope.

  22. Toxin antitoxin Neutralization • Testing the presence of the antitoxin in the serum. • If they are present the toxin will be neutralized .RBCs not lysed. • If there is no antitoxin the toxin will lyse the RBCs sample (serum) Ab The toxin( hemolysin) RBC lysed with the toxin

  23. +VE -VE RBC lysed with the toxin The toxin( hemolysin) sample (serum) Ab

  24. Toxin antitoxin Neutralization invitro example Anti-streptolysin O ASO +VE -VE

  25. Schick’s test: Testing immunity of the patient against diphtheria. 0.2 ml of the diphtheria toxin in the test forearm and 0.2 ml of inactivated diphtheria toxin (by heat ) in the control forearm.

  26. Complement fixation test (CFT) • Preparing sensitized complement & two systems: • System1: • Containg serum sample & prepared antigen • System 2 (indicator system): • containing sensitized sheep RBCs & Ab to sheep RBCs. • In +ve reaction the serum containing Ab will react with the Ag the complement will be consumed by system 1 (RBCs will not be lysed).. • In –ve reaction where there is no Ab in the serum so the complement will consumed by system 2 (RBCs will belysed).

  27. +VE -VE Test Ag Sample AB complement Sheep RBC Ab to sheep RBC

  28. Radioimmunoassay

  29. Enzyme Linked Immuno-Sorbent Assay (E.L.I.S.A) • This a solid phase reaction through which the serum antibody will be captured to the wells using capture Ag. • An Antihuman globulin conjugate containing chromogenic enzyme will react with the sample Ab. • Adding enzyme substrate will produce colour on the well. • So +ve test is manifested by presenece of colour , in –ve test there is no colour. Indirect E.l.I.S.A

  30. E E E INDIRECTE.L.I.S.A +VE -VE Ag Sample AB Enzym linked Ab Enzyme substrate

  31. Enzyme Linked Immuno-Sorbent Assay (E.L.I.S.A) • This a solid phase reaction through which the serum antibody will be captured to the wells using capture Ag. • The conjugate here is directed to the Ag i.e. the sample and conjugate will compete each other. • Adding enzyme substrate will not produce colour on the well. • So +ve reaction is manifested by no colour , -ve reaction there is a colour. Competitive E.l.I.S.A

  32. E E E COMPETITIVEE.L.I.S.A +VE -VE Ag Sample AB Enzym linked Ab Enzyme substrate

  33. Enzyme Linked Immuno-Sorbent Assay (E.L.I.S.A) • This a solid phase reaction through which the serum antigen will be captured to the wells using capture Ab. • The conjugate here is directed to the Ag i.e. the Ag will be sandwiched between capture & test Abs • Adding enzyme substrate will produce colour on the well. • So +ve test is manifested by presenece of colour , in –ve test there is no colour. Sandwich E.l.I.S.A

  34. E E SANDWICHELISA Sample Ag AB Enzym linked Ab Enzyme substrate

  35. Enzyme Linked Immuno-Sorbent Assay (E.L.I.S.A) Spectrophotometer

  36. Flowcytometry  is a biophysical laserbased technology employed in cell counting, cell markerdetection, allowing synchronizedanalysis of the physical and chemical characteristics of up to thousands of particles per second. Example: used in the diagnosis  blood cancers, and CD4 cell counting in AIDS patients

  37. Immunochromatography rapid and easy. Antigen-antibody immune complexes flow through a region and encounter antibody against them, resulting in a visible colouredline E.g Used in rapid diagnosis of Malaria

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