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Ouchterlony Double Diffusion (for Antibody Titeration Ration)

Ouchterlony Double Diffusion (for Antibody Titeration Ration)

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Published by kiedd_04
Principle:
Interaction between antigen (Ag) and antibody (Ab) at the molecular level forms the basis for several techniques that are useful in modern day scientific studies and in routine clinical diagnosis. Ouchterlony double diffusion (ODD) or immunodiffusion technique is one of the simplest techniques extensively used to check antisera for the presence of antibodies for a particular Ag and to determine its titer.

Principle:
Interaction between antigen (Ag) and antibody (Ab) at the molecular level forms the basis for several techniques that are useful in modern day scientific studies and in routine clinical diagnosis. Ouchterlony double diffusion (ODD) or immunodiffusion technique is one of the simplest techniques extensively used to check antisera for the presence of antibodies for a particular Ag and to determine its titer.

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Published by: kiedd_04 on Oct 24, 2008
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02/06/2013

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[PRACTICAL 3]MTEB2404
OUCHTERLONY DOUBLE DIFFUSION (FOR ANTIBODY TITRATION)Objectives
 To learn the technique of ouchterlony double diffusion
Principle
Interaction between antigen (Ag) and antibody (Ab) at themolecular level forms the basis for several techniques that are useful inmodern day scientific studies and in routine clinical diagnosis.Ouchterlony double diffusion (ODD) or immunodiffusion technique is oneof the simplest techniques extensively used to check antisera for thepresence of antibodies for a particular Ag and to determine its titer.
Procedure
1.
100mg of agarose was boiled to dissolve in 10ml of 1X assaybuffer. Cool to 55°C.
2.
5ml of the gel solution was poured onto a clean glass plate placedon a horizontal surface. The gel was allowed to set it tookapproximately 20-30 minutes.
3.
 The gel plate was placed on the template provided. Wells in the gelwas punched with the help of a gel punch corresponding to themarkings on the template. Gentle suction was used to avoid formingrugged wells.4.The test antiserum was serially diluted up to 1:32 dilution asfollows:
 Take 20ul of 1x assay buffer in each of the five vials.
Add 20ul of test antiserum into the first vial and mix well. Thedilution of antiserum in this vial is 1:2.
 Transfer 20ul of 1:2 diluted antiserums from the first vial into thesecond vial. The dilution in this vial is 1:4.
 The dilution was repeated up to fifth vial.
5.
10ul of the antigen was added to the center well and 10pl each of neat (undiluted), 1:2, 1:4, 1:16, 1:32 dilutions of antiserum into thesurrounding wells as shown in figure 3.
6.
 The plate was placed in a moist chamber and incubated at roomtemperature, overnight.
7.
After incubation, opaque precipitin line between the antigen andantisera wells was observed.
8.
 The observation was recorded.
1 |Page
 
[PRACTICAL 3]MTEB2404
Figure 3: Pattern of addition of antigen and antiserum to thewells.
Result
Precipitin line was formed at the well of 1:16 antiserum.
2 |Page
1:2A1:41:81:161:32Neat1:2ANeat1:321:161:81:4
 
[PRACTICAL 3]MTEB2404
Discussion
 The Ouchterlony wasa test used for detectingantigens and specificantibodies which it placed inadjacent wells in a platethat containing agar gel. The Anti-sera will be placedin the central well, andantigens will be added intothe wells around the centralwell. Then, the antibodyand antigen molecules willdiffuse through the agaroselawn. When antibody meetswith its specific antigen attheir equivalent zone, theprecipitation reactionoccurs. Antibody-antigen precipitates in agarose appear as a lightwhite band between the antibody and the antigen wells. The serological relationship between antigens can be moreprecisely determined with this method. However, this test was lesssensitive serology because the formation of precipitation lines isdepends on high and equivalent concentrations of specific antigensand antibodies. The Ouchterlony test also can be used to estimate the relativeconcentration of antigens. When an antigen has a relatively higherconcentration, the equivalent zone will be formed a little bit away fromthe antigen well. When an antigen has a relatively lowerconcentration, the equivalent zone will be formed a little bit closer theantigen well. There are some possible error could be encounter during thisexperiment done, which are:1.Contaminating of antibodies which may develop a precipitinlines between antiserum wells on double diffusion.2.Sample that contains lipimic which produces non-specificprecipitin of protein gel.3.Hemolysed sample were also given similar characteristic tolipimic sample.
4.
Others non-antibody such as C-Reactive protein,
3 |Page

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