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Q: elisa/aids ( Answered 5 out of 5 stars,   0 Comments )
Question  
Subject: elisa/aids
Category: Miscellaneous
Asked by: cynnryan-ga
List Price: $30.00
Posted: 21 Jul 2003 07:17 PDT
Expires: 20 Aug 2003 07:17 PDT
Question ID: 233298
Why does the destruction of th cells compromise the entire immune
system and how does HIV target cells? Why are there so many
immunological variants of HIV? If the elimination of several steps in
the ELISA could be accomplished if the primary antibody was made into
an enzyme conjugate.Why is this generally done and what can cause a
false positive in an ELISA?

Clarification of Question by cynnryan-ga on 22 Jul 2003 19:18 PDT
why has this been up for a couple of days and no answer. I need to
understand the questions that I have asked. Please help me
understand...
Answer  
Subject: Re: elisa/aids
Answered By: synarchy-ga on 23 Jul 2003 01:30 PDT
Rated:5 out of 5 stars
 
Hello - I'll tackle this in parts -

1)  T-helper lymphocytes (Th cells) are the 'managers' of the immune
response - they integrate the responses of the immune system cells
which first detect a foreign substance (so-called antigen presenting
cells, or APC's) with the cells that will then mount an immune
response - eg the B-cells (humoral immune system) which will produce
antibodies and the T-cells (cell mediated immune system) which
directly act to destroy invading organisms and infected tissues.  By
destroying the helper cells, HIV destroys the body's ability to mount
a coordinated response, thereby crippling the entire immune response.

A good, but detailed overview (and though tailored towards allergy, it
still covers the basics):
http://www.interna.fk.ui.ac.id/ami-online/ami3303/Review_article1.htm

T helper cells overview:
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Th1_Th2.html

A nice site with animations of the activation of T and B cells by Th
cells:
http://sprojects.mmi.mcgill.ca/immunology/Th_effector.htm

2)  HIV targets cells that express the CD4 protein on their surface in
conjunction with either CCR5 or CXCR4 proteins.  These proteins are
expressed on the surface of T-helper cells (as well as on the surface
of some tissue macrophages throughout the body).  The HIV protein
which binds to CD4 is called gp120 (stands for glycoprotein 120 - the
120 is the size of the molecule in kilodaltons):
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AIDS.html#Infection

Many other less commonly expressed proteins can also work with CD4 to
allow entry of the HIV virus into cells as can be seen in the abstract
for this paper:
http://www.aegis.com/pubs/aidsline/1998/apr/m9840034.html

3) HIV has many immunological variants because the enzyme that
replicates the viral genes (reverse transcriptase) is a very primitive
enzyme that lacks the ability to check it's work (so-called
proofreading activity) - this leads the virus making many errors in
the replication process - each of these errors has the possibility of
making a change in the proteins which are at the surface of the viral
particle - any change in the surface of the virus can lead to the
production of a different variant.  This amount of variability is both
good and bad for the virus.  The downside is that each error also has
the possibility of being a fatal error that will lead to inactive
virus.  The upside, for the virus, is that the more differences that
there are in how the surface is shaped, the less likely the immune
system is to recognize the virus, allowing it to escape clearance by
the immune system.

A nice, detailed, and very long essay on the immunologic variability
of HIV:
http://www.ktl.fi/hiv/mirrors/hmjf/thesis/Thesis_2.html#RTFToC8

4) An enzyme-linked immunoadsorbent assay (ELISA) is an
antibody-mediated detection scheme for detecting particular proteins. 
In the simplest form, it could be as you suggested, with a single
antibody conjugated to a detection enzyme.  You could anchor your
protein sample and detect the amount of protein by reacting it with
your enzyme conjugate to detect the amount of immunoreactivity, which
should correlate with the amount of target protein - ie an
antibody-conjugate to gp120 should tell you how much gp120 is in the
sample.  This simple method is faster and might be more sensitive
(requires less sample), but suffers from a lack of specificity (it is
not good at only detecting what it's supposed to).  The reason for
this is that other proteins may cross-react with either the antibody
or the detection enzyme, thereby binding to the antibody-conjugate and
inflating the amount of immunoreactivity - a false positive.  By using
the more classic ELISA method, in which one antibody to the target is
immobilized, the sample is added to bind the target antigen to the
first antibody, a second antibody to the target is then added to bind
to the target, much higher sensitivity can be acheived.  This is
because the addition of a second antibody to the same target requires
two steps of specificity before immunoreactivity is determined.  This
is sort of like looking through a phone book for someone's name - if
all you have is one name, you will come across alot of numbers - if
you have both their first and last names, there will be a much greater
chance that you will find the right person.

The ELISA tutorial on this page is reasonable:
http://www.hhmi.org/lectures/biointeractive/vlabs/index.htm

an animation of the process:
http://www.biology.arizona.edu/immunology/activities/elisa/technique.html

More than you likely ever wanted to know about the ELISA technique:
http://webmed.unipv.it/immunology/agabint.html



Please let me know if you wish any further explanation.

synarchy



Search for references:
Th cells
T-helper cells
CD4 HIV
ELISA tutorial
cynnryan-ga rated this answer:5 out of 5 stars
Thank you for clearing up my questions. I now understand what i was
reading.I thank you....

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