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ELISA.pptx
1.
2. ELISA(Enzyme linked immunosorbent assay)
Is one of the immunoassay method used to
detection of 1-ANTIBODIES
2 PROTEINS
3 PEPTIDES
4 BIOMOLECULES
3. The term “immunoassay” is a
combined term of
“immuno”=(practically antigen-
antibody reaction) and
“Assay”=(Determination of purity of
substance or the amount of any
constituent of a mixture.
4. Enzyme Linked ImmunosorbentAssay
1.Antigen/Antibody of interest is absorbed on to plastic
surface (‘Sorbent’)
2.Antigen is recognised by specific antibody (‘Immuno’)
3.This antibody recognised by second antibody
(‘immuno’) which has enzyme attached(‘enzyme
linked’)
4.Substrate react with enzyme to produce product usually
coloured.
5. Radioimmunoassay was first described in a scientific
paper by Rosalyn Sussman Yalow and Solomon
Berson Published in 1960.
In 1971, Peter perlmann and Eva Engvall stockhlom
University in sweeden,and anton schuurs and Bauke
van Wccmen in the Netherlands independently
published papers that synthesized this knowledge into
methods to perform EIA/ELISA.
7. Antibody:proteins produced by the immune system
which help defend against antigens
Symbol for
antibody
The variable regions are thoughTo be the place for
recognition and binding with antigen.
8. • Any molecules that induces production of antibodies
when introduced in the body is called antigen,
OR
• Any “thing” foreign to the immune system eg.bacteria
viruses,(or their parts), pollen..etc.
9. in immunoassay, it is necessary to use any marker to
know the antigen-antibody binding.for such purpose,
we label either antigen or antibody with some
materials that do not interfere with the binding.
eg.Horse radish peroxidase
ezyme Substrate
:trimethylbenzidine
12. use an enzyme to detect the binding of antigen(Ag)
antibody(Ab)
The enzyme convert a colourless substrate
(chromogen)to coloured product,indicating the
presence of Ag-Ab binding.
An elisa can be used to detect either the presence of
antigen or antibodies in a sample depending how the
test is designed
Elisa was developed in 1970 and become rapidly
accepted
13.
14. Qualitative
determines antigen or antibody is present or absent
Quantitative
o determine the quantity of antibody
o titer
o the highest dilution of the specimen usually
serum which gives positive reaction in the test
15.
16. Reagents are relatively cheap and have long shelf life
ELISA is highly specific and sensitive
No radiations hazards occur during labelling or
disposal of waste.
Easy to perform and quick procedures
Equipment can be inexpensive and widely available.
ELISA can be used to variety of infections.
17. Measurement of enzyme activity can be more
complex than measurement of activity of some type of
radioisotopes.
Enzyme activity may be affected by plasma
constituent.
Kits are commercially available ,but not cheap
Very specific to a particular antigen. Won’t recognize
any other antigen.
False positive/negative possible, especially with
mutated/altered antigen
19. Initially the substrate should be colourless
After degradation by enzyme it should be strongly
coloured or flurescent.
ENZYME SUBSTRATE CHROMOGEN STOPPING
Alkaline phosphatase P-NPP P-
NPP+diethandamine+
MgCl2
1M NaOH
Horse radish peroxide H2O2 Tetramethylbenzidine
+phosphate citrate
buffer
1M H2SO4
Horse radish peroxide H2O2 O-phenylenediamine+
HCL
1M HCL
20. 1.Collection and processing of serum
2.Coating of wells antibodies/antigens
3.Washing
4.Incubation with the test sample
5.Washing
6.Incubation with enzyme conjugated antibodies
7.Washing again
8.Colour development by adding chromogenic substrate
9.Stopping the colour development
10.Reading of results
21.
22. Collect blood in a tubes that does not
contain any chemicals or
anticoagulants.
Collect 5ml of whole blood(for every small
children collect 1ml).
Place tube upright 30-60 minutes then
when firm clot has formed centrifuge tube
for 20 minutes at 2500rpm.
Remove serum with the pipette and place
in a plastic storage tube(2-3,microtube or
cryovial).
If 5ml of blood was collected it will result in
23. Antigen: Chicken gamma globulin
Primary antibody (PA): Polyclonal anti-chicken
antibody made by rabbits
Secondary antibody (enzyme-linked) SA: Polyclonal
anti-rabbit antibody made by goats linked
(conjugated) to horseradish peroxidase (HRP)
Enzyme substrate (SUB): 3,3’,5,5’ –
tetramethylbenzidine (TMB) – a colorless solution
that when oxidized by HRP turns blue
24. Using a clean pipette, add 100microliter of diluted serum sample
(diluted the sample to be tested 1:100 in sample diluent)to each
well.
Incubate 1hr at 37 degree celcius
After incubation empty out contents of wells into waste
container.
Using pipette ,fill wells with the washing buffer the empty out.
Tap wells upside down o paper towel.
Wash the well 5 times. At the end of washing process the well
must be entirely dry after the last wash.
Distribute 100microliter of antihuman Immunoglobuline
conjugate in each well. incubate 30 minutes at 37 degree celcius.
25. Primary Antibody Secondary Antibody
Raised against specific antigen.
Typically unconjugated (Unlabelled)
Bind to primary antibody.
Conjugated to probes (Labelled)
Recognize and bind with high affinity
and specificity to unique epitopes .
Available as monoclonal and/or
polyclonal antibody.
Specificity for whole Ig molecules or
antibody fragments. Available as
monoclonal or ployclonal antibody.
Specific to protein of interest Specific to primary antibody and no
affinity to protein of interest
Anti-bovine Tubulin antibody is a
primary antibody that will recognize
the tubulin protein that is generated
in the cow (bovine).
Goat anti-mouse IgG antibody is a
secondary antibody developed in the
goat that has an affinity for the
mouse IgG protein.
26.
27. Monoclonal
Antibody
Polyclonal
Antibody
Consists of one antibody
class/subclass which is selective for a
single epitope on the antigen
Contains a mixture of antibodies
(mainly IgGs), often recognizing
multiple epitopes on the antigen
Usually generated in mice, other
rodents, or isolated from a phage
display library
Generated in a variety of species
including rabbit, goat, sheep, and
donkey
Always identical, as they are produced
from the same hybridoma (or
transfected cell line, or bacterial
clone)
Prone to batch to batch variability
Their homogeneous nature ensures
better reproducibility between tests,
where conditions are kept constant.
Useful for quantification experiments
like flow cytometry
Their heterogeneity means they are
more tolerant to slight changes to
the antigen e.g. denaturation,
polymorphism or differences
in glycosylation state
28. Monoclonal
Antibody
Polyclonal
Antibody
Because of their specificity, they
are less likely to cross-react with
other proteins, giving
lower background than polyclonal
antibodies
May contain non-specific
antibodies resulting in
background staining
Specificity makes them ideal as the
primary antibody in an assay, for
detecting antigens in tissue, or for
affinity purification of antigens
Useful as secondary antibodies
or for immunoprecipitation, as
they target multiple epitopes
providing a more robust
detection
34. Blood >> processed Serum >> applied to ELISA plate containing antigen >>
Wash >> enzyme >> colour change.
Accuracy :False positive for person not HIV infected. It is possible in peoples
having antibodies for Human leukocyte Antigen (HLA). Observed in
women who possess multiple pregnancies.
After 4-8 weeks of exposure to the HIV virus, the body will have produced
a detectable level antibodies (immune response) against HIV
35.
36. 0 5 10 15 20
Plant pathology
Autoimmune
Food safety/tolerance
Enzymes
Allergy
Cancer
Inflammation
Protein:Protein interactions
Other
Endocrinology
% Responding
% Responding
Main application areas for respondent’s ELISA
37. 0 20 40 60 80
Serum
Plasma
Cultured cells
Whole blood
Cell lysate
Tissue homogenate
Urine
Cancer/tumour cells
Saliva
Other bodily fluids
Other
Plant material
Biopsy
Faeces
Foodstuff
Environmental sample
Dried Blood Spot
Water
No Responding (All
Respondents)
Sample sources analysed By ELISA
