Transcript Rapid Plasma Reagin

Precipitation tests
Precipitation tests
Flocculation tests
1- VDRL.
2-RPR
3-One step H.pylori
Test device
Immunodiffusion
Assay (ID)
Countercurrent
Immuno-electrophoreis
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 .
+
=
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 .
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.
The principle of the test :
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
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.
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.
One Step H.pylori test device :
Helicobacter pylori lives in the surface of the stomach and causes
stomach ulcers and problems .
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.
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.
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
3- Immunodiffusion Assay (ID)
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.
that’s it
Thank you