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Enzyme
Linked Immuno Sorbent Assay [ELISA]
 What
will the assay tell us?
The assay quantifies the
amount of antibody to a specific antigen in a sample (serum, nasal
secretion, etc.).
It can also be used to quantify the amount of a class of antibody (IgA,
E, G
or
M) to a specific antigen.
Necessary
equipment
 2
plastic test tubes
serum
(or other sample)
enzyme
labeled antibody specific for the Ig being identified
(in
class example: goat antibody against rat serum)
colorless
electron receptor
How
the assay works
The antibodies bind to an
antigen placed on the sides of a test tube or on a latex bead that
is placed into the test
tube. Antibodies against the antibodies to the antigen then bind
to
them. The quantity of antibodies
against the antigen is determined indirectly by quantifying
the antibodies that bind
to them. This quantification is possible because the antibodies against
the antigens are labeled
with an enzyme that generates a color reaction or with light emit by a
chemiluminescence reaction.
The
enzyme labeled antibody is specific for the Ig being identified.
Hence, to quantify IgA, you
would use anti-IgA antibody;
for IgM, you would use an anti-IgM antibody, etc.
The
antibodies bind to an antigen placed on the sides of a test tube or on
a latex bead that is
placed into the test tube.
Antibodies against the antibodies to the antigen then bind to them.
The quantity of antibodies
against the antigen is determined indirectly by quantifying the antibodies
that bind to them.
This quantification is possible because the antibodies against the antigens
are
labeled with an enzyme that
generates a color reaction or with light emit by a chemiluminescence
reaction.
Steps
in conducting the assay
In
the class example, such antibody against the antibody for the antigen was
collected by injecting
a goat with rat (animal
under study) Ig. The goat formed anti-rat antibody Ig in serum.
A serum
sample is then collected.
The sample contains antibodies against rat antibodies.
The
antibodies against antibodies serum is then labeled with an enzyme
that is capable of
generating a color reaction
when it comes in contact with a colorless enzyme receptor. The serum
is then added to the test
tubes. The antibodies against rat-antibodies then bind to the rat
antibodies
that are bound to the antigen.
Form
of quantitative results
Usually reported as optical
density (OD), a measure of how much color product is formed.
The amount of colored product
is determined by the amount of enzyme in the tube which is
related to the amount of
antibody bound to the antigen.
How
results are interpreted
Normal sera is used to get
a normal range. The test sera being evaluated must be greater
(usually 2 times greater
OD than the normal control) to be considered clinically significant.
Potential
problems with reliability of the assay
Sometimes the antigen wouldn't
bind to the plastic tube. Careful control of incubation times
and incubation temperatures
is required.
Fluorescent Activated
Flow Cytometry
What
will the assay tell us?
Percent of cells (e.g.,
lymphocytes, monocytes, neutrophils, T, B, helpers, suppressors/cytotoxic).
Count of cells that have
a surface marker identifiable by a monoclonal antibody.
How
the assay works
Monoclonal antibodies with
fluorescent dyes bind to specific cell types. The cytometer can count
cells that are labeled by
the fluorescent dye and cells that are not labeled.
Necessary
equipment and materials
Flow
cytometer (cell sorter)
Labeled
monoclonal antibodies to cell being counted
Steps
in conducting the assay
The
machine software differentiates granulated from non-granulated cells. Lymphocytes
are not
granulated. The instrument
will give the percentage of lymphocytes labeled by the particular
monoclonal antibody used.
More than 1 monoclonal antibody can be used at the same time. For
example, a T lymphocyte
can be simultaneously labeled with an antibody to the CD3 marker and
the CD8 marker.
How
results are interpreted
About
70% of human peripheral blood lymphocytes should be T cells and react
with
the monoclonal antibody that binds to the CD3 marker.
20-25%
B
Of
T cells:
About
25% cytotoxic CD8
45%
helpers CD4
Normal
Helper/Suppressor ratio is between 1 and 2.
Number
of each cell type per ml of blood can be calculated by multiplying % of
cell type X
number of leucocytes/ml
of blood (as determined by separate counting procedure).
Potential
problems with reliability of the assay
This assay does not tell
you if cells are functional, only if they are present.
Immunofluorescence
Called
direct immunofluorescence when using patient tissue and indirect when using
serum to
detect antibodies to an
antigen placed onto a glass microscope slide.
What
will the assay tell us?
Indirect immunofluorescence
can be used to detect antibodies or antibodies against
infectious agent.
How
the assay works
Fluorescein labeled anti-antibodies
(goat anti-human immunoglobulin serum) bind to antibodies
you want to detect.
Fluorescent microscope can be used to detect those antibodies to which
fluorescent anti-antibodies
are bound.
Necessary
equipment
Fluorescence
microscope (uses ultraviolet light)
Slide
with several circular areas to hold different dilutions of serum and saline.
Patient
serum (fluid from clotted blood is serum, fluid from unclotted blood is
plasma--contains
fibrin)
Goat
antihuman immunoglobulin (IgG) serum with fluorescent label
Steps
in conducting the assay
Form
of quantitative results
The antibody titer
is the reciprocal of the highest dilution showing a positive reaction.
For example, if the highest
dilution is 1:8 (one part serum to seven parts of saline), the titer is
8.
Sera known to be positive
or negative on the assay are always included in order to make certain
that the test is working
properly.
How
results are interpreted
The highest dilution showing
a positive reaction is reported as the titer. Assays for different
antigens have different
titers that are clinically significant.
Potential
problems with reliability of the assay
What is the cutoff for when
a reaction is present or not? Considerable experience is required
to
interpret the assay.
Mitogen Stimulation
(also known as Lymphocyte Proliferation)
What
will the assay tell us?
Extent of mitosis by a class
of lymphocytes stimulated by a non-specific mitogen (molecule
that nonspecifically stimulates
cell division in a broad group of cells). In short, how readily do
particular classes of lymphocytes
reproduce when excited by a mitogen? Specific antigen can
also be used and this will
provide information on whether some lymphocytes had been previously
activated by the antigen
being used in the assay.
How
the assay works
Either T or B lymphocytes
(depending on the mitogen) are activated to mitosis (division) by a
nonspecific mitogen or specific
antigen. Dividing cells incorporate a radioactive isotope into the
newly synthesized DNA which
can be quantified. The greater the radioactivity, the greater the
proliferation.
Necessary
equipment
Culture
dish
Radioactive
marker
Beta
counter (scintillation counter)
Mitogen
Steps
in conducting the assay
Form
of quantitative results
Results may be expressed
as counts per minute (cpm) or as a stimulation index (SI).
SI = cpm experimental/cpm
control. The cpm control is determined by quantifying the
radioactivity of lymphocytes
incubated without mitogen.
How
results are interpreted
Results are evaluated based
on mean increase or decrease in proliferative response of
experimental subject compared
to control subjects.
Potential
problems with reliability of the assay
The mitogens stimulate a
broad range of cell types: TH, TC, TS or B memory, B nonactivated,
and it is difficult to correlate
an altered response with one specific cell type.
The
assay is highly variable within any one group of experimental subjects
and can be affected
by batch of mitogen (which
often is not highly purified), time of day (circadian rhythms of
experimental subject), person
performing assay, etc.
Natural Killer
Cell Activity
What
will the assay tell us?
The ability of natural killer
(NK) cells to kill tumor cells or virally infected cells.
To
test the functional ability of the NK cells, white blood cells are obtained
from subjects and
tested for their ability
to lyse or kill tumor cells (targets) in vitro.
How
the assay works
Target cells that contain
radioactivity are combined with effector cells. When NK cells lyse
target
cells, radioactivity is
released. The radioactivity is quantified and compared to spontaneous
release
of radioactivity by target
cells and to the maximum radioactivity a target cell could release.
Necessary
equipment
radioactive
marker
gamma
counter
tumor
cell line
Steps
in conducting the assay
Form
of quantitative results
The data is converted to
% cytotoxicity using the following formula:
% Cytotoxicity = [experimental
(cpm) - spontaneous (cpm)]
X 100
maximum (cpm) - spontaneous (cpm)
% Cytotoxicity can be calculated
for each dilution. Lytic units is a complex calculation that
collapses over all dilutions.
Lytic units are usually (but not always) the number of effectors
required to kill 20% of
107 targets.
How
results are interpreted
Results are expressed as
a % cytotoxicity at 1 and 2 effector:target ratios or as LU.
The maximum cpm is determined
by putting target cells in with a lysing agent. An example of a
maximum cpm would be 8000,
and spontaneous cpm 754. In the presence of NK cells,
anywhere from 5-60% of the
maximum cpm may be released. Thus if 4,000 cpm are released
by NK cells with a maximum
release of 8,000, and a spontaneous release of 754, the %
cytotoxicity would be 44.8%.
% cytotoxicity may range
from 2% to 40%. 8 - 12 LU for suppressed, 30-60 for others.
Potential
problems with the assay
It is unclear which dilution
is appropriate or preferred.
The assay only measures percent
cytotoxicity of NK cells in lymphoid tissue or blood. It doesn't
separate number of NK cells,
from the activity of those cells. It is, however, possible to quantify
NK cells and determine if
a change in % cytotoxicity between experimental and control samples
is due to a change in the
number of NK cells or due to a change in the functional abilities of the
NK cells present.
Core-E
MainBiological
Measures Used |
