Download Indirect ELISA

Practical Immunology
Tel:8462530； 8462468(lab)
E-mail:[email protected]
Weifang Medical University
Test Seven
Enzyme-linked Immunosorbent Assay
Immediate hypersensitivity induced in guinea pig
ELISA
• Enzyme-linked immunosorbent assay, commonly known as ELISA, is a method
which labels antigen or antibody with enzyme depends on an enzyme label. An
enzyme conjugated with an antibody reacts with a colorless substrate to
generate a colored reaction product. Such a substrate is called a chromogenic
substrate.
• A number of enzymes have been employed for ELISA including alkaline
phosphatase, horseradish peroxidase, -galactosidase.
• This method have the advantage of being safe and less costly.
ELISA
• ELISA allowing qualitative detection or quantitative measurement
of either antigen or antibody, each type of ELISA can be used
qualitatively to detect the presence of antibody or antigen.
• Alternatively, a standard curve based on known concentrations of
antibody or antigen is prepared, from which the unknown
concentration of a sample can be determined.
ELISA:
Enzyme-Linked ImmunoSorbent Assay
96-well microtiter plate
Run multiple tests on one plate
Small volumes
Use less of reagents
Can use bound Ab to quantitate soluble antigen
Can use bound antigen to quantitate serum Ab
An ELISA plate
• A number of variations of ELISA have been eveloped
• Sandwich ELISA
• competitive ELISA
• ELISPOT assay
• indirect ELISA
Sandwich ELISA
• Antigen can be detected by a sandwich ELISA.
• In this method, the antibody is immobilized on a microtiter well. A sample
containing antigen is added and allowed to react with the immobilized
antibody.After the well is washed, a second enzyme-linked antibody
specific for a different epitope on the antigen is added and allowed to react
with the bound antigen. After any free second antibody is removed by
washing, substrate is added, and the colored reaction product is measured.
Competitive ELISA
• Antibody is first incubated in solution with a sample containing antigen.
The antigen and antibody mixture is then added to an antigencoated
microtiter well. The more antigen present in the sample, the less free
antibody will be available to bind to the antigen-coated well.
• The enzyme conjugated secondary antibody (Ab2) specific for the isotype
of the primary antibody can be used to determine the amount of primary
antibody bound to the well
• In the competitive assay, the higher the concentration of antigen in
the original sample, the lower the absorbance.
ELISPOT ASSAY
• ELISPOT assay allows the quantitative determination of the number of
cells in a population that are producing cytokines.
• In this method, the plates are coated with the antibody (capture antigen)
• A suspension of the cell population under investigation is then added to the
coated plates and incubated. The cells settle onto the surface of the plate,
and secreted cytokines reactive with the antibodys
• The plate is then washed and an enzyme-linked antibody specific for the
secreted cytokines is added and allowed to bind, subsequent development
of the assay by addition of a suitable chromogenic substrate reveals the
position of each antigen-producing cell as a point of color .
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a well is coated with antibody
against the antigen, then a
suspension of a cell population
secreting the cytokine are layered
onto the bottom of the well and
incubated.
Most of the cytokine secreted by a
particular cell react with nearby
well-bound antibodies.
After the incubation period, the
well is washed and an enzymelabeled anti-cytokine antibody is
added. After washing away
unbound antibody, substrate is
added. The colored product
precipitates and forms a spot only
on the areas of the well where
cytokine-secreting cells had been
deposited.
Indirect ELISA
• Humoral as well as tissue constituents can be measured using
indirect ELISA. In this method , antibody can be detected or
quantitatively determined.
Principle:
• Serum or some other sample containing primary antibody (Ab1) is added to an
antigen-coated microtiter well and allowed to react with the antigen attached
to the well. After any free Ab1 is washed away, the presence of antibody
bound to the antigen is detected by adding an enzyme-conjugated secondary
antibody (Ab2), which binds to the primary antibody. Any free Ab2 then is
washed away, and a substrate for the enzyme is added.
• The result is measured by specialized spectrophotometric plate readers, which
can measure the absorbance of all of the wells of a 96-well plate in seconds.
Indirect ELISA
Materials
• Sheep erythrocytes（SRBC）, rabbit antibody （Ab1）, anti-rabbit
IgG （labeled with enzyme, Ab2）.
• Washing fluid （PBS）, substrate（OPD） for enzyme, 2mol / L
H2SO4（stopping fluid）.
• 96 well assay plate, humidified chamber, icropipetter, tips.
Method
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To coat the plate with the appropriate antigen.
Dump out the fluid into the sink and bang the plate upside down on some paper. Wash the
unbound antigen with 200ul (PBS), Wash 3 times.
100ul negative fluid is added into the first well, 100ul rabbit antibody (anti-SRBC,Ab1) is
added into the second well, then incubated at 37℃ for 45 minutes.
Wash plate as above (3 times).
100ul enzyme conjugated IgG (E-Ab2) is added into two wells, then incubated at 37℃
for 30 minutes.
Repeat washing step.100ul substrate for enzyme is added into two wells and then
incubated for 20-30 minutes.
One drop of stopping fluid is added into each well to cease the reaction, and the color
reaction is estimated through naked eyes or spectrophotometer.
Steps in the ELISA
SRBC antigens pre-coated onto
an ELISA plate
ELISA plate wells already coated with SRBC antigen
Steps in the ELISA
Primary antibody (Ab1)
serum added which contains antibodies to SRBC
antigen
Steps in the ELISA
Primary antibody
Variable domain binds antigen;
constant domain free
Unbound antibodies washed away
Steps in the ELISA
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Secondary antibody
Variable domain of 2 antibody binds constant
domain of 1 antibody
Steps in the ELISA
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Secondary (2) antibody
Unbound antibodies washed away
Steps in the ELISA
Chromagen or substrate added
Addition of chromagenic substrate results in color reaction
Color intensity increases with concentration of antibodies
Darker color = higher titer
Steps in the ELISA
Primary (1) antibody
Negative reaction:
There are no antibodies present that bind SRBC
antigen
Steps in the ELISA
Negative reaction :
All primary antibodies washed away
Steps in the ELISA
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Secondary (2) antibody
Negative reaction :
There are no primary antibodies for the
secondary antibodies to bind
Steps in the ELISA
Negative reaction :
All secondary antibodies washed away
Steps in the ELISA
Substrate added
Negative reaction
There is no enzyme to carry out the color reaction
NO COLOR CHANGE
Negative well
Positive well
Immediate hypersensitivity induced in guinea pig
Principle
• I hypersensitive reaction is induced by certain types of antigens referred
to as allergens, However, an allergen induces a humoral antibody
response by the same mechanisms as other soluble antigens, resulting in
the generation of antibody secreting plasma cells and memory cells.
• What distinguishes a type I hypersensitive response from a normal
humoral response is that the plasma cells secrete IgE.
• IgE binds with high affinity to Fc receptors on the surface of mast cells
and basophils. Mast cells and basophils coated by IgE are said to be
sensitized. A later exposure to the same allergen cross-links the embrane
bound IgE on the sensitized mast cells and basophils, causing
degranulation of these cells. The pharmacologically active mediators
released from these granules act on the surrounding tissues.
• These granules dissolve and release histamine serotonin and heparin and
other mediators,they act on the smooth muscle and vascular endothelial
cells,lead to allergic reaction,even shok and death.
• We inject heterogenous protein (allergens ) to guinea pig, after 10-14 days,
the IgE level in guinea pig rised and fix to the surface of mast cells and
basophils, when the animal is exposed to the same antigen for the second
time,the antigen seek out those cell-fixed IgE and react with them at the
cell surface, an antigen moleculer combines with two antibody moleculers
to form a bridge.
• This bridging bring together two IgE receptor moleculers which can
trigger an enzymy cascade reaction, this cascade reaction can cause mast
cells and basophils release granules and lead to allergic reaction. However,
the symptom of some guinea pig are quite slightly for the reason of
individual difference, this is called desensitization.
Crosslinking of FcεRI on the surface of mast cells by antigen and
IgE causes mast-cell activation and degranulation.
Materials
• Guinea pig:three young healthy guinea pigs each weighs
about 150g.
• Antigen: horse serum
• sterile injection syringes, needles, alcohol tampon.
Method
• Inject antigen for the first time: inject 0.5ml horse serum to in
abdomen(two weeks ago)
• Inject antigen for the second time: inject 1ml horse serum into
guinea pig’s heart.
• Observe the result after 1-5 minutes.
Result
• guinea pig appears excitement, scratching nose, singultus,
spasm, hair standing, urinary and fecal incontinence,even
death. Emphysema can be found after been dissected.
Announcements
• Heart-injection of guinea pig:. Antigen should be injected into
guinea pig’s heart on the condition that countercurrent blood
appears in the injection syringes.
• One grasp and fix , expose the breast , the other one touch the
4,5,6 intercostal space of left hand side.select the site where
heartbeat is most obviously.
Test report
• Principle, Method , Result
• Indirect ELISA
• Immediate hypersensitivity induced in guinea pig.