Experiment No.

2: Ouchterlony Double
Immunodiffusion

Name: Bethlehem kifle

Reg no: 18BBT0281
Date:17/12/2019
Slot #: L37+L38

Upload your results of the experiment with interpretation.

STUDY QUESTIONS

1.You are provided with 10 X buffer. How do you prepare 1X

ANSWER: 10x means 10 times concentrated
So, to prepare 1x buffer we need to dilute 10x 10 times or we can
take 1 part of 10x and mix with 9 part of water (1+9=10) to make
10x buffer.
Or use formula
M1V1=M2V2
M1=10X, V1=Z ml, M2=1X, V2=?
10X*Z ml=1x*10ml
Z=1 ml of 10x we need in 10 ml of water
NOTE:Do not add 1 ml in to 10 ml which would be 11 ml and the
concentration will be wrong.

2.Write the composition & pH of PBS

ANSWER: PBS: Is abbreviation form of “ PHOSPHATE - BUFFERED
SALINE”
PH is approximately 7.4.
It is a buffer solution that is particularly valuable because it mimic the ion
concentration, osmolarity , and pH of human body fluids. .

The most common composition of PBS are:

Salt Concentration(g/L)
Concentration(mmol/L)
NaCl 137 8.0
KCl 2.7 0.2
Na2HPO4 10 1.44
KH2PO4 1.8 0.24

3.You are provided with antigen A & B, how do you determine the antigenic
similarity?
Answer :
Ouchterlony method can be used to determine the antigenic similarity.
The Ouchterlony method is one method where a concentration of an antibody can
be measured and seen.
A gel is made up and the antigen and antibody diffuse across the gel towards each
other. Where they meet precipitation/cross linking occurs and this can clearly be
seen on the gel.

4.Define precipitation reaction.

ANSWER:
Precipitation reaction is the chemical reaction occurring in aqueous solutions
where two ionic bonds combine forming up insoluble salts.The salts formed are
precipitates. Or It is the creation of a solid from a solution. Or It is a type of
chemical reaction in which two soluble salts in aqueous solution combine and
one of the product is an insoluble salt called precipitate. The precipitate may stay
in the solution as a suspension, fall out of solution on its own, or can be separated
from the liquid using centrifugation, decantation, or filtration. The liquid that
remains when a precipitate form is called the supernate.
Eg. The reaction between silver nitrate and potassium chloride is a precipitation
reaction because solid silver chloride is formed as a product.
AgNO3(aq) + KCl(aq) → AgCl(s) + KNO3(aq) 5. State the principle of the ODD
test The key

5.State the principle of the ODD test.

ANSWER:
Ouchterlony double diffusion(ODD) test, in this test both antigen and antibody
are allowed to diffuse into the gel.This technique can be used to test the
similarity between antigens.Antigens from different species are loaded into two
wells and the known antibody is loaded in a third well located between and
slightly below the antigen wells to form a triangle.Depending on the similarity
between the antigens , different geometrical patterns are produced between the
antigen and antiserum wells.The pattern of lines that form can be interpreted to
determine whether the antigens are same or different.

6.What is the percentage of agarose used in this assay.

ANSWER:
1.2% agarose (0.3 g/25 ml)

7.Mention the applications of ODD

ANSWER: To determine relative concentration of antibodies /antigens.
To compare antigens.
To determine the relative purity of an antigen preparation .
For disease diagnosis.
Serological surveys.
RESULT
INTERPRETATION
AS it is seen in the above image,
A shows the pattern of identity(the antibodies in the antiserum react with both
the antigens).
B shows the pattern of partial identity(the antibodies in the antiserum react
more with one of the antigens than the other).
C shows the pattern of non identity(none of the antibodies in the antiserum
reacts with antigenic determinants that may be present in both the antigens).