Thursday, May 30, 2013
Immunodectection test: deals with antigens. Antigens
generate antibodies. Know it is a bacterial virus, take antibodies and they
will bind to the antigens on the virus and you will know you have a virus.
You have to look at the proteins. Proteins are different. Take hamburger
extract, then look for hamburger proteins. It will have: bovine albumin, goat
anti-horse albumin, goat anti-bovine albumin, goat anti-swine albumin.
In our bodies the outside of our cells are lined with a
specific set of proteins and carbohydrates that identify our cells as belonging
to our body. When the immune system
finds a foreign or harmful cell within the body it creates antibodies to
destroy the potential threat. When
injected with foreign cells all organisms with immune systems produce the same
kind of antibodies to fight against them.
This is good for humans because we can collect and purify antibodies
from other animals and use them to assist our own immune systems and to test
for what kind of cells may be in our food or other organic substance.
In this experiment we are going to test how different
anti-bodies will detect certain antigens (the special markers which cover the
outside and label cells) react in the agar.
We first create a set of holes in the agar and place certain
anti-body solutions within them.
1.
Bovine Albumin
2.
Goat Anti-horse Albumin
3.
Goat Anti-bovine Albumin
4.
Goat Anti-swine Albumin
The Anti-bodies are labeled “Goat” because these particular
anti-bodies came from goats. The term
“Anti-“ tells us what the antibodies are produced to find and mark. In the case of Anti-swine, it will create
antibodies to mark the antigens of swine cells.
When you inject the goat with anti-horse albumin, it will
produce antibodies against the horse albumin.
Same idea with Rh factor. Make an antigen to block the Rh
factor in pregnant mothers. The Antibodies
bind to the stray cells from the baby which have different proteins than the
mother’s cells, and then mark the cells with antigens so that the mother’s
antibodies will not find the cells, mark them as foreign and then create
anti-bodies against the baby. The
injected anti-bodies are only inserted into the mother’s bloodstream and not in
a high enough dose to actually harm the baby, where as if the mother’s immune
system were to find the baby’s different cells, it would mark them as foreign
and create antibodies to eradicate the foreign cells and therefore harm the
baby.
Inject a snake bite victim with horse anti-snake antibodies
because these will cover the snake venom antigens faster than the human’s
antibodies against the snake venom and therefore not allow the venom to hurt
the body.
Exercise 2: ANTIBODY-ANTIGEN REACTION IN AGAR
We took a petri dish and made four wells in it and filled
them with the following albumin: 1-bovine albumin, 2-goat anti-horse albumin,
3-goat anti-bovine albumin, and 4-goat anti-swine albumin. The function of this
experiment is to show you which antibodies will target which antigens.
Serum conversion - Searching for Antigens to determine
whether a person has been exposed to the pathogen. For example: a physician might not be able to
find HIV virus within the patient if it is latent. But if the patient has been exposed to HIV,
then the body will have produced antibodies for the virus which should still be
in the blood.
First add the purified antigen. This antigen will bind to antigens produced
by the immune system to fight the disease we are testing for (i.e. HIV). Next add a serum sample which represents the
blood sample extracted from the patient.
If the specific antigen of our disease is present within the serum, then
our antibodies cultivated to bind to the pathogen antigen will bind to the
pathogen antigen. Next add an enzyme. This enzyme will bind to the primary antibody
if it is connected to the pathogen. Then
enzyme will produce a specific color when it binds, indicating that the
pathogen is present.
We were given a mystery serum, which we are to test for the
presence of a specific pathogen antigen.
We will test it with a Positive control, a negative control, a secondary
antibody that has the enzyme attached and then the substrate.
Procedure:
1.
Add purified disease antigen to the wells of a
microplate strip. Inoculate for 5 minutes
to allow the proteins to bind to the plastic wells via hydrophobic
interaction. This is called an immunosorbent assay because proteins absorb or bind to the plastic wells.
2.
Wash out the wells to remove anything that has
not bound to the plastic walls of the wells.
3.
Add the serum sample and control samples to
wells and incubate. The Serum contains
millions of different types of antibodies, but only if your serum contains
antibodies that were produced in response to the disease will antibodies bind
to the antigen in the wells.
4.
Detect the serum or primary antibodies with
HRP-labeled secondary antibody. If serum
antibodies have bound to the antigen, the secondary antibodies will bind
tightly to the serum antibodies.
5.
Add enzyme substrate to the wells, wait 5
minutes and then evaluated the assay results.
If the primary antibody was present in your serum sample, the wells will
turn blue. This is a positive diagnosis. If the wells remain colorless, the primary
antibody was not present in your serum sample, and the diagnosis is negative.
Our sample had a negative diagnosis, indicating that the
primary antibody was not present in it.
Bacterial concoction
At the end of lab today, Dr. Pathakamuri took bacteria from
every person in micro lab and mixed them up in a beaker of water. He brought
out a UV radiation device that blasts bacteria with UV rays and kills them
after a certain amount of time. To show this, he took samples of before and
after radiation and spread them onto plates and incubated them. We will see
next time if the radiation really worked!
Yummy yogurt
Dr. Pathakamuri took regular milk, boiled it, and placed a
small amount of yogurt in it and incubated it. This was to demonstrate how a
live culture (in the yogurt) would colonize the milk and produce more yogurt.
Also, we had to examine samples and measure the zone of inhibition in the Kirby-Bauer test.
·
Penicillin: not sensitive
·
Neomycin: 20mm
·
Tetracycline: not sensitive
·
Erythromycin: sensitive, intermediate
·
Chloramphenicol: sensitive, intermediate
Staph aureus does not use mannitol.
Ours grew in the mannitol and no color change.
Nose swab: right now all negative. Maybe positive for
Elizabeth
On blood agar unknown bacteria: no hemolysis, therefore no
staph
Blood agar throat: not strep
Eosin Methylene Blue (EMB) Agar – to isolate and differentiate
gram-negative enteric bacilli. The EMB
agar plate contains eosin and methylene dyes and sugars lactose and
sucrose. The dyes will inhibit growth of
many gram-positive, which makes this a selective medium. EMB is also a differential medium that will
distinguish between the bacteria that ferment lactose and/or sugar. If bacteria ferment lactose and/or sucrose
then they will produce acid. The lower
pH will cause the dyes to precipitate on the colonies. If there is a significant amount of acid produced
then the bacteria will be a dark bluish color with a green-metallic sheen. Low acid production results in pink colored
colonies. No fermentation will result in
colorless colonies on the agar surface.
To prepare the petri dish we inoculated an EMB agar with our
bacteria and let it grow overnight in the incubator at 24 degrees Celsius.
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