Immunology lab sheet # 1 - Thana’a ALhaDiD

بسم الله الرحمن الرحيم

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http://www.mediafire.com/?4n5poqemq6o6t53
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First of all, this lab is about Immunology not microbiology.

In this lab we will talk about Agglutination and Precipitation Tests, both have the same principle the Antibody-Antigen RXN and both detect the presence of the antibody in our body, but they differ in the lab techniques and preparations.

1. Precipitation Test:
it depends on the Antibody-Antigen RXN.
As we all know that the antibodies are bodies which are produced by the body against an antigen and it’s one of the defense mechanisms in our body.
To detect the presence of an antibody (such as Immunoglobulin Ig) we add a body which is an antibody for the antibody (that we want to detect it) this body is called
Anti-antibody then a RXN will take place between the antibody and the Anti-antibody.
Double Diffusion Test:
From its name “Double” it’s composed of two things the Antigen and the Antibody.
This test requires a proper Media to observe this RXN well. Just like Bac. Those require a medium to observe it well. The medium that we use here are the Agarose medium (remember that the Agarose is a gel like substance and we use it in the Bac media to solidify it) here we use 1% of Agarose, we add it to distilled water, then we dissolve it and then we boil it.
Note: in this kind of media we don’t care if the media get contaminated and require no autoclaving or anything else.
Then we pour it on pitry dish or any piece of glass. Then we put it in the refrigerator to solidify.
After that we make holes in the glass (which are called wells) by using a Cutter which is a hollow tube. Then we use needle to empty these holes. Each hole has specific diameter and specific volume in order to be able to put a specific quantity of any substance.
Then by a capillary which is a very thin tube by which we can withdraw specific quantity of any substance.
Now, this glass is organized in rows each row should contain either the antigen or the antibody .we add the antigen to a series of holes of a fixed antibody.
I.e. we bring a SPECIFIC person’s serum (which will present the fixed antigen) and then on the adjacent row we add VARIOUS antibodies (e.g. IgA IgG IgE…etc)
OR we bring a SPECIFIC antibody (e.g. IgM) on one row and then on the adjacent row we put a VARIOUS antigen (different samples of sera/from different people).
Then, we leave it @ room temperature for 48 hrs why? To allow diffusion to take place. What will happen next is that the antibody will diffuse into the corresponding antigen; the antibody and the antigen form a diffused gradient that crosses each other in a line of Immunoprecipitation, which is so-called Precipitation line. E.g. if a precipitation line was formed between the serum row and the IgA row this means that this serum contains IgA  IgA +




Ab= antibody Ag= antigen
Gel diffusion by antibody and single antigen.  Antibody +

To avoid the gel to get dried we put wet cotton on the top and on the bottom of the glass and we put it in a closed box.
48 hrs is a long period of time if we want to decrease it we apply a current to accelerate the RXN and the diffusion process this period will be decreased into 1 hour only and we use it
When immediate results are required, this is accomplished by a device which conducts a current, and that replaces the room temperature. This process is called Current Electrophoresis, it is the same principle of double diffusion but it is more rapid because of the use of electricity.
Note: this is a Qualitative method which means that the consistency is well known but the quantity or the concentration is unknown. It's Just to detect If the specimen is antibody + or - .
Other method is used which is the Quantitative method: if we want to know the concentration to diagnose if there is some kind of Deficiency or anything else.
But how can this be accomplished?
By labeling 3 wells with a standard concentration and the remaining wells are filled with different sera (unknown concentration). E.g. if we have a plate with 16 wells the first 3 wells are the standard and the remaining 13 are the unknown, each standard well has its own capillary.
After the RXN takes place a zone/ring will be formed and we can measure this radial ring from the centre of the ring to the boundary of the ring. Same as what we've done in the sensitivity test. Then we plot the relationship between the concentration of antigen and the diameter of the precipitation ring in graph an this relation is directly proportional 
The relation between the concentration of the antigen and the Diameter squared will be linear
What is the importance of standard wells?
It allows us to draw this linear relation only.
These standard wells should give you the same data every time you use them under the same conditions that are indicated in the plate (by the company itself).


To understand it well
We measure the diameters of the standard wells and we are already known their concentrations then we plot this info in a graph -conc. Vs d^2 – which will be linear-.
Then we measure the diameter of the unknown well and then we search for it in the graph.
E.g. if we measure the diameter of unknown and it was 2 then d^2=4 then we search for the concentration in the graph.
Simply, like we are doing some kind of comparison between the diameter of the precipitation ring of the unknown and the diameter of the standard rings.
This called is single radial immunodiffusion.





The points A, B and C represent the known concentration of antigen (standard).
This process requires 48 hrs but if we want to accelerate it we can also apply a current but
In this case no radial rings will be formed a rocket like shape will be formed instead. Then we measure the height of the rocket then we plot it on a graph (also we have here a 3 standard wells)





2. Agglutination:
It has the same principle of the precipitation diffusion but the resultant shape of the RXN is granular (small particles)

2 tests:
1. Pregnancy test
2. ABO test / blood group test.


1. The pregnancy test:
This involves the detection of HCG hormone Human Chorionic Gonadotropin in the urine of the pregnant woman. This test can be done by the first day because once the woman is got pregnant this hormone will be secreted immediately.
Procedure:
In a piece of a glass we put a sample of urine (in this lab we use +/- control
That comes from the Manufacturer itself/ in the same kit, and we assumed that it is the urine sample) and then we add anti-HCG but the problem is that the particles of the anti-HCG is very very small that it cannot be observed so what we will do here is that we will add a material that has high molecular weight to hold the small particles of the anti-HCG to observe this RXN well. This material is Latex
If the agglutination occurs pregnant :S
If not  the woman is not pregnant.






2. ABO
If we want to know the blood type of a blood sample we put 3 drops of the blood on a piece of a glass then we add anti-A, anti-B and anti-Rh on each drop if the agglutination occurs then this blood has its antigen
e.g. if we have 3 drops of a blood then we add anti-A and anti-B then the agglutination occurs in the A then it means that this blood sample has antigen A  so it is A type.
Also that applies on the Rh if the agglutination occurs +
If not  - .

Microbiology lab the 5th&6th.Dec.2010
The last session
A big HELLOoOoOoOo la kol banat el dof3a especially: Halah & Lana, Sura, Amirah, Thara2, Tamara, Abrar, Karmel, Tara, Asma’a , Abeer, Saja, Hanan, Fatimeh, Muna, Lama, Amal , and ofcourse Farah and Noura o yalla Safa’a …



Done by: Thana’a ALhaDiD


Best wishes <3

it's immuno lab #1
it's the 1st and last one