elisa2 Flashcards
elisa
Enzyme linked immunosorbent assay (ELISA) tests were originally developed
for antibody measurement. These immunoassays have also been
adapted to successfully detect samples that contain antigens.
This HIV
ELISA simulation experiment has been designed to
detect a hypothetical
patient’s circulating IgG directed towards the viral (HIV) antigen.
ELISAs are done in
microtiter plates which are generally made of polystyrene
or polyvinyl chloride. The plates are somewhat transparent and
contain many small wells, in which liquid samples are deposited.
step 1
Antigens are added to the wells where some
remain adsorbed to the wells by hydrophobic
bonds.
- There is no specificity involved with
the adsorption process to the wells, although
some substances may exhibit differential
binding. In certain cases, the antigens can be
covalently cross-linked to the plastic using UV
light.
antiens can b
Antigens can be from whole HIV
lysates, specific HIV proteins, or a mixture of
the two.
step 2
Wells are washed to remove unadsorbed
antigens.
step 3
Block the unoccupied sites on the walls of the
plastic wells with proteins, typically gelatin or
bovine serum albumin.
step 4
2ndary antibody
Infection by HIV-1 causes the individual to
mount an antibody response which eventually
results in plasma IgG molecules that bind to
different HIV proteins (or different areas or the
same protein).
2ndary antbodies
Secondary antibodies are usually raised in rabbits and goats immunized
with human IgG tractions. Secondary antibodies (anti - HIV - IgG) are
purified and covalently cross linked to horseradish peroxidase. This
modification does not usually affect the binding specificity and affinity of
the antibody or the enzymatic activity of the peroxidase.
if antibodies present
If these antibodies are present, as in the plasma sample of an HIV postive
patient, they will bind to the adsorbed antigens in the well and remain
there after washing.
if antibodies not present
If the patient is negative for HIV, there will be no
primary andtibody to bind to the antigen and in turn, no secondary
antibody binding.
step 5
Wells are washed to remove unbound secondary antibody.
step 6
After washing, a solution containing hydrogen peroxide and
aminosalicylate is added to each well.
peroxidase
- possesses a high
catalytic activity and can exceed turnover rates of 106
per second.
Consequently, amplification of an HIV positive sample can occur over
several orders of magnitude. - Many hydrogen donor co-substrates can be
used by peroxidase. these co-substrates develop color upon oxdation - H2O2 (H peroxide) to H2O + O2
aminosalicylate
Peroxidase converts the peroxide to H2
O + O2
using the salicylate as the
hydrogen donor. The oxidized salicylate is brown and can be easily
observed in wells containing anti - HIV - 1 IgG (positive plasma).
polyclonal antibody preps
It should be noted that polyclonal antibody preparations to a given
antigen can have variable binding affinities due to differences in the
immunological responses between animals. Different immunizations with
the same antigen in the same animal can also produce variable binding
affinities. The use of monoclonal antibodies directed against a single
epitope eliminates this variability.
HIV infects
macrophages by
binding to the cells’ CD4 receptors
macrophages
Macrophages are circulating monocytes and are involved in the nonspecific
engulfment of foreign material and normal cellular debris. These
materials are degraded in the lysosomes of the cells. Peptides from
foreign degraded proteins are transported to the macrophage surface
where they remain bound by specialized receptors.
- Immunologically
inactive TH cells interact with these surface bound antigen-receptor
complexes which enables them to become fully activated.
(the rest of the CD4 receptors are internalized and gp 120
appears on the T cell surface.)
Fully activated TH
cells secrete
several types of protein factors collectively known as lymphokines.
interleukins
several lymphokines are the interleukins which stimulate
antibody secretions from B cells enable macrophage activation, stimulate
general T cell growth and activate cytotoxic T cells.
cytotoxic T cells
Cytotoxic T cells
are involved in the actual destruction of foreign cells and body cells
infected with different viruses.
When a TH cell containing provirus undergoes antigenic activation
the
integrated copy DNA becomes open to the transcription of viral RNA.Viral replication causes the destruction of the TH cells.
syncytia
Infected TH cells
also form syncytia, i.e. fused cells. Syncytia occur since the gp 120 on the
infected T cell surface binds to CD4 receptors on other TH cells. Cell to
cell transmission of virus can occur in syncytia without the need for a free
viral intermediate.
