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Practical Immunology: Antigens Preparation

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Roua Jassim
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8 views11 pages

Practical Immunology: Antigens Preparation

Uploaded by

Roua Jassim
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Practical Immunology

Antigens Preparation
{Third Class 2024-2025}
Antigen:-
Antigen is a foreign body (Particulate or soluble) that can able
to sensitize immune system in highly specialized manner and can
react with the product of immune system either antibodies
(related Ag) or highly sensitized T-lymphocytes .
Hapten:-
A small molecule that can not initiate an immune response
un less first bound to an immunogenic carrier molecule .
Immunogen:-
An substance that is capable of inducing an immune
response
{antibody response or cell mediated immunity }.The most
potent immunogen such as proteins and polysaccharides .
Some types of antigens :-
Physically:
1 Particulate Ags :-
Bacteria , viruses , parasites and erythrocytes .
2 Soluble Ags :-
Serum , serum protein ……etc.
Bacterial antigens:
Somatic antigen (O-Ag):-
- Its heat – stable Ag .
-Carbohydrate – Protein - lipids , complex in nature .
- Resistant to weak acids and alkaline .
- Example :Cell wall (LPS) of Gram negative bacteria.
Flagella Ag (H-Ag):
- It is heat labile Ag
.
- Protein in nature .
- Example : Flagella
of Salmonella sp.
Capsular Ag (K –Ag ):
Preparation of antigens (Methods):-
1Purification of bacteria by selective techniques cultured the bacteria
on the suitable media .
2 To obtain massive growth or culturing of bacteria we must culture the
bacteria on agar media for somatic antigen (LPS) , to prepare flagellar Ag
we
must cultured on broth media & examine the motility test .
3Harvesting by preparation of bacteria suspension from culture with
normal saline or PBS do not use distilled water to prevent lysis of
bacteria .
4 Wash three times with PBS by centrifugation 3000 rpm for 20-50 minutes.
5 Heating or boiling in water bath at 100 ⁰ C for 2.5 hours in case of O-Ag
, while H-Ag we used PBS with formalin (0.6 %) over night at 37⁰C
incubation to prevent denaturation of flagellar
proteins. 0.3% for
preservation for both types of Ags.
6- Wash three times by centrifugation .
7- Resuspend in PBS (pH 7.2) .
8 – Sterility test to check purity & contamination on suitable
media for 24-48
hours .
9 – Standardization by using McFarland ʹs tubes (10 capacity
tubes contains
different concentrations of 1% BaCl2 & 1% H2SO4 .
Tube ……..3----------9× 10⁸ cell/ ml .
Tube ……….4 -----------1.2 × 10⁹ cell/ ml .
Or by using haemocytometer chamber , also , in case of H-Ag
we used spectrophotometer .
McFarland Method :-
The method consists of comparing the opacity of the
vaccine with a series of ten standard tubes
containing varying amounts of barium sulfate in
suspension:-
McFarland tube BaCL2 1% ML H2SO4 1% ML No. of Bacteria
1 0.1 9.9 300.000.000
2 0.2 9.8 600.000.000
3 0.3 9.7 900.000.000
4 0.4 9.6 1.200.000.000
5 0.5 9.5 1.500.000.000
6 0.6 9.4 1.800.000.000
7 0.7 9.3 2.100.000.000
8 0.8 9.2 2.400.000.000

9 0.9 9.1 2.700.000.000

10 1.0 9 3.000.000.000
These turbidities correspond to varying concentration of
bacteria in the size range of Staphylococcus , Streptococcus
,and the enteric bacteria .
Preparation of RBCs:-
Collect blood in sterile test tube containing suitable
anticoagulants , Centrifugation and wash RBCs three times
with isotonic saline solution , injection of RBCs in
laboratory animals recording anti RBCs serum .
Preparation of immunoglobulin:-
Collect hyper immune serum , precipitation of
immunoglobulin by using highly concentration of salts like
ammonium sulfate , ligation of Ig with adjuvant , injected
in laboratory animals , recording of anti immunoglobulin
(Antibodies) .

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