1. Precipitation tests

2. Flocculation tests Countercurrent Immuno-electrophoreis Immunodiffusion Assay (ID) 1- VDRL. 2-RPR 3-One step H.pylori Test device

3. 1-Flocculation tests : In flocculation tests the precipitin end product forms macroscopically or Microscopically visible precipitate. Patient serum ( antibody or antibody like protein ) + appropriate antigen (in tube or on card ) = particle flocculate. + =

4. Applications : 1 – VDRL The Venereal Disease Research Laboratory Test, is the most widely used Flocculation test. Patient infected with pathogenic treponemas (T.pallidum ) ( syphilis disease ) From antibody-like protein called reagin that bind to the test antigen ( Cardiolipin - lecithin – coated cholesterol particles ) causing the particles To flocculate. Since reagin is not a specific antibody directed against T.pallidum antigens, The test is not highly specific, but is a good screening test detecting more than 99% of cases of secondary syphilis.

5. A blood test for syphilis. a person may a negative VDRL and still have syphilis since, in the early stages of the disease, the VDRL often gives negative results. This is called a false negative VDRL. The VDRL test is sometimes positive in the absence of syphilis. For example, a false positive VDRL can be encountered in infectious mononucleosis, lupus, the antiphospholipid antibody syndrome, hepatitis A, leprosy, malaria and, occasionally, pregnancy.

6. The principle of the test :

7. 2- RPR Rapid Plasma Reagin Test. The RPR appears to be a more specific screening test for syphilis than the VDRL and it is certainly easier to perform (card). 50 micron of antibody (patient serum ) + 50 micron of antigen = flocculation

8. Rapid Plasma Reagin (RPR) refers to a type of test that looks for non-specific antibodies in the blood of the patient that may indicate that the organism (Treponema pallidum) that causes syphilis is present. The term "reagin" means that this test does not look for antibodies against the actual bacterium, but rather for antibodies against substances released by cells when they are damaged by T. pallidum.

9. Rapid Plasma Reagin (RPR) is basically an improved version of the VDRL test and uses the same antigen. A large number of iteration of this test are available but one of the most common, easiest and cheapest are those run on a small test card. In addition to screening for syphilis, once RPR reactivity has been qualitatively established, a quantitative titer can be used to indicate the stage of infection and its response to therapy. The RPR test can be done on unheated plasma or serum, but is not recommended for use on CSF. The term "reagin" refers to the anti-cardiolipin antibodies. In the test, a patient’s serum (or plasma) is mixed with a stabilized VDRL cardiolipin antigen suspension that has cholesterol, lecithin, and carbon added in. The antigen is not attached to these particles, but the carbon is trapped in the lattice formed by the antigen-antibody complex that forms when reactive serum is added. The antigen is lipoidal in nature and because of this the antigen-antibody reaction remains suspended, as a visible flocculation.

10. One Step H.pylori test device : Helicobacter pylori lives in the surface of the stomach and causes stomach ulcers and problems.

13. 2- Countercurrent Immuno-electrophoreis (CIE) : AIM: To check antisera for the presence and specifity of antibodies for a particular antigen by Counter current immunoelectrophoresis. INTRODUCTION Counter current immunoelectrophoresis (CCIEP) is a rapid version of Ouchterlony double diffusion (ODD) technique. The technique is used to check antisera for the presence and specificity of antibodies for a particular antigen.

14. PROCEDURE Prepare10 ml of 1.5% agarose(0.15g/10ml) in 1X reservoir buffer by adding dry agarose to the buffer and heating slowly to dissolve the agarose completely. Mark the end of the slide that will be towards positive electrode during the electrophoresis. Place the slide on a leveled tabletop and quickly pipette 7ml agarose onto 50X75mm slide, spreading while releasing the agarose. Allow solidifying for 15min. Place the gel plate on the template holder and fix it for CCIEP. Punch 3mm wells with the gel punch at position indicated for CCIEP. Place the slide in electrophoresis tank and fill the tank with buffer. Add 10 l of antigen in the four wells towards –ve electrode and 10 l of positive control antiserum and three test antisera in wells towards positive electrode. Apply 50v and allow the electophoresis to continue for about 45 min. Observe for precipitin line between the antigen and antisera wells.

15. Observation: Here, pattern shows precipitin line between antigen and antisera wells. INTERPRETATION Precipitin line indicates the presence of antibody the antigen in the test sera. The presence of more than one precipitin lines indicate the heterogenicity of the antibody for the antigen in the test sera. The absence of the precipitin line indicates the absence of any antibody for the antigen in the test sera

16. About The Immunodiffusion Test Immunodiffusion is a qualitative or semi-quantitative test based on the principles of double diffusion described by Oudin and Ouchterlony. An antibody and its homologous soluble antigen are placed in separate wells cut in a suitable diffusion medium (agarose or CleargelTM) and allowed to diffuse outward into the medium. Between the two wells, a concentration gradient of each of the reaction components is established ranging from antigen excess closest to the antigen well, to antibody excess closest to the antibody well. A visible line of precipitate forms at the point of equivalence. 3- Immunodiffusion Assay (ID)

19. that’s it Thank you