41. What is an ELISA?
•ELISA = enzyme-linked immunosorbent
assay
• Enzyme-Linked: enzyme amplifies
antigen-antibody interaction
• Immunosorbent: reaction products
adsorbed on container
• Assay: assessment of potential analyte
42. HISTORY
Prior to the development of the EIA/ELISA, the only option for conducting
an immunoassay was radioimmunoassay, a technique using radioactively-labeled
antigens or antibodies.
Avramais (1966, 1969) and Pierce (1967) developed methods to chemically
link antibodies to biological enzymes whose activities produce a measurable
signal with solutions containing appropriate substrates.
This signal has to be associated with the presence of antibody or antigen .
COMPONENTS OF ELISA
Antibody: IgG fraction of serum.
Enzyme: Horse Radish Peroxidase (HRP) MW 44, 000, glycoprotein with 4
lysine residues.
Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme acts as a
catalyst to oxidize substrate in the presence of Hydrogen peroxide to
produce a blue color. Reaction stopped with dilute acid to cause complex to
turn yellow.
43. INTRODUCTION TO ELISA
A 96 - well microtiter plate
being used for ELISA.
A test that uses antibodies and color
change to identify a substance.
ELISA is a popular format of a
"wet-lab" type analytic biochemistry assay.
ELISA involves at least one antibody with
specificity for a particular antigen.
ELISA can perform other forms of ligand
binding assays instead of strictly
"immuno" assays.
44. PRINCIPLE
“Wet lab" analytic biochemistry assay, ELISA involves detection of an
"analyte"
in a liquid sample by a method that continues to use liquid reagents
during the "analysis“.
The basic principle of an ELISA is to use an enzyme to detect the Ag-
Ab binding (antigen- antibody binding). The enzyme converts a
colorless substrate (chromogen) to a colored product, indicating the
presence of Ag:Ab binding.
46. TYPES OF ELISA
INDIRECT ELISA
DIRECT ELISA
SANDWICH ELISA
COMPETETIVE ELISA
NON -COMPETETIVE
ELISA
47. INDIRECT ELISA
Antigen is added to plate.
Added Blocking buffer.
Suitable primary antibody is added.
Secondary antibody- HRPO is then added
which recognizes and binds to primary
antibody.
TMB substrate is added, is converted
to detectable form.
Under standard condition ,the enzyme activity measured is proportional to amount
of specific antibody in the original serum.
48. ADVANTAGES OF INDIRECT DETECTION
Wide variety of labeled secondary antibodies are available
commercially.
Versatile, since many primary antibodies can be made in one
species and the
Same labeled secondary antibody can be used for detection.
Immunoreactivity of the primary antibody is not affected by
labeling.
Sensitivity is increased because each primary antibody contains
several epitopes that can be bound by the labeled secondary
antibody, allowing for signal amplification.
DISADVANTAGES OF INDIRECT
DETECTION
Cross-reactivity may occur with the secondary antibody,
resulting in nonspecific signal.
An extra incubation step is required in the procedure.
49. DIRECT ELISA
1. Apply a sample of known antigen to
a surface.
2. Enzyme linked primary antibody is
applied to the plate.
3. Washed, After this wash, only the
antibody-antigen complexes remain
attached.
4. Apply a substrate which is
converted by the enzyme to elicit a
chromogenic signal.
Under standard condition ,the enzyme
activity measured is proportional to
amount of specific antibody in the
original serum.
50. ADVANTAGES OF DIRECT DETECTION
Quick methodology since only one antibody is used.
Cross-reactivity of secondary antibody is eliminated.
DISADVANTAGES OF DIRECT
DETECTION
Immunoreactivity of the primary antibody may be reduced as
a result of labeling.
Labeling of every primary antibody is time-consuming and
expensive.
No flexibility in choice of primary antibody label from one
experiment to another.
Little signal amplification.
51. SANDWICH ELISA
1. a. Plate is coated with suitable antibody.
b. Blocking buffer is added.
2. Sample is added to plate so antigen is bounded by capture antibody.
3. A suitable biotin labeled detection antibody is added to plate.
4. Enzyme HRPO is added and binds the biotin labeled detection antibody.
5. TMB substrate is added and converted by HRPO to colored product.
Under standard condition ,the enzyme activity measured is proportional to the Amount of
specific antigen in the original serum.
52. MATERIALS NEEDED FOR ELISA KIT
ELISA Plate
Positive control
Negative control
Dilution Buffer
Conjugate
TMB Substrate
Stop Solution
53. PROCEDURE OF ELISA
A B C D E
Wash
3X
Wash
4X
Wash
4X
Wash
4X
Alkaline
phosphatase
substrate is added
and developed
colour is read at 405
nm wavelength to
measure plasma
cencentration
Wells are
coated with
0.2 μg
primary
antibody
Diluted
plasma
is added to
coated wells,
which bind
to antibodies
0.1 μg of
biotinylated
(biotin = – )
antihuman
secondary
antibody
Incubated
overnight at 4˚C
Add 1.2000
dilution of
streptavidin
conjugate to
alkaline
phosphatase
( E)
2h 2h 1h
Incubated at room temperature (24˚C)
55. COMPETETIVE ELISA
COMPETETIVE ELISA
Solid phase coated with
antibody
Add unknown amount of
unlabeled antigen and known
amount of labeled antigen
Free and labeled antigen are
captured
Color formation by oxidation of
substrate
into a colored compound
Under standard condition ,the enzyme activity measured is proportional to the
proportion of labeled antigen in the mixture of labeled and unlabled antigen.
56. ADVANTAGES:
Suitable for complex (crude or
impure) samples, since the antigen
does not require purification prior to
measurement.
DISADVANTAGES
Each antigen may require a different
method to couple it to the enzyme.
57. COMPARISON BETWEEN VARIOUS TYPES OF
ELISA
Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA
58. ELISA RESULTS
The ELISA assay yields three different types of data output:
Quantitative: ELISA data can be interpreted in comparison to a
standard curve in order to precisely
calculate the concentrations of antigen
in various samples.
Qualitative: ELISAs can also be used to achieve a yes or no
answer indicating whether a
particular antigen is present in a sample,
as compared to a blank well containing no antigen
or an
unrelated control antigen.
Semi-quantitative: ELISAs can be used to compare the relative levels
of antigen
in assay samples, since the intensity of signal will
vary
directly with antigen concentration.
59. ELISA data is typically graphed with Optical density Vs Log
concentration to produce a sigmoidal curve.
Known concentrations of antigen are used to produce a standard curve
and then this data is used to measure the concentration of unknown
samples by comparison to the linear portion of the standard curve.
This can be done directly on the graph or with curve fitting software
which is typically found on ELISA plate readers.
61. APPLICATIONS
Screening donated blood for evidence of viral contamination
by
HIV-1 and HIV-2 (presence of anti-HIV antibodies)
Hepatitis C (presence of antibodies)
Hepatitis B (testing for both antibodies and a viral antigen)
Measuring hormone levels
HCG (as a test for pregnancy)
LH (determining the time of ovulation)
TSH, T3 and T4 (for thyroid function)
Detecting infections
Sexually-transmitted agents like HIV, syphilis and chlamydia
Hepatitis B and C
Toxoplasma gondii , H.pylori
Detecting illicit drugs.
Detecting allergens in food and house dust
62. Diagnostic Applications (Home)
•Pregnancy test
• hCG hormone in pregnant woman’s
urine
• 1-10 min: (+) two lines, (-) one line
HIV test
• Oral fluid: OraSure, OraQuick,
Calypte AWARE
• Blood: Home Access
63. Industrial Applications
•Allergen detection
• 8 major food allergens
• Detection at low levels (ppm)
• Alerts for contamination
•Vaccine quality control
• Licensing
• Validate consistency of production
65. SENSITIVITY
ELISAs are one of the most sensitive immunoassays
available. The typical detection range for an ELISA is 0.1 to 1 fmole
or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular
characteristics of the antibody – antigen interaction.
In addition, some substrates such as those yielding
enhanced chemiluminescent or fluorescent signal, can be used to
improve results.
As mentioned earlier, indirect detection will produce
higher levels of signal and should therefore be more sensitive.
However, it can also cause higher background signal thus reducing
net specific signal levels.
66. Sensitivity and Specificity
•Sensitivity
• Probability: positive test for a patient with disease
• Takes false negatives into account
• More important for ELISA
•Specificity
• Probability: negative test for a patient without disease
• Takes false positives into account
•ELISA has high sensitivity (>99%) and high specificity
(>99%)
• Not perfect: false positives/negatives can occur
Chard, T. (1992). Review: pregnancy tests: a review. Human Reproduction, 7(5), 701-710.
Dipiro, J.T., Talbert, R.L., Yee, G.C., Matzke, G.R., Wells, B.G., & Posey, L.M. (2008): Pharmacotherapy A Pathophysiologic Approach Seventh Edition. New York: McGraw-Hill Companies Inc.
Picture 1: http://newdadtobe.com/post-2/
Picture 2: http://www.oraquick.com/Taking-the-Test/Understanding-Your-Results
Le, C. T., Grambsch, P. M., & Giebink, G.S. (2003). Quality control and the identification of vaccine responders using ELISA-derived antibody data. Statistics in Medicine, 22(18), 2935-2942.
Stephan, O., & Vieths, S. (2004). Development of a real-time PCR and a sandwich ELISA for detection of potentially allergenic trace amounts of peanut (Arachis hypogaea) in processed foods. Journal of Agricultural and Food Chemistry, 52(12), 3754-3760.
Picture 1: http://www.ifood.tv/blog/nut-free-snacks
Picture 2: http://www.wisegeek.com/what-is-needle-phobia.htm
