Description about ELISA and types of ELISA

1. SUBMITTED BY :SUMEENA KARKI
DATE : 4th MARCH,2016
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2.  ELISA stands for Enzyme Linked Immunosorbent
Assay
 Also k/a SPIA(Solid Phase Immuno Assay)
 Substrate: O-phenyl diamine dihydrogen chloride (for
enzyme Horseradish peroxidase ) and P-nitrophenol
phosphate (for Alkaline phosphate) and enzyme β
galactosidase
 Chromogeneic substrate : enzyme+Ab+colorless
substrate=colored product
 Enzyme is linked with appropriate antibody to impart
signal for either antigen or antibody( Stratis
Avrameas and G. B. Pierce)
 Immunosorbent: washing doesnot remove Ag/Ab
fixed to surface of container(Wide and Jerker,1966)
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3.  Result: special spectrophotometric plates readers
 Absorbance of all 96 well plates
 Standard curve: unknown concentration of sample
determined based on known concentration of Ag/Ab
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5. 1.Qualitative:positive or negative results
2.Quntitative:optical density is interpreted by
standard curve
ELISA METHODS
1.Direct ELISA
2.Indirect ELISA
3.Sandwich ELISA
4.Competitive ELISA
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6.  Most commonly used for detection of serum antibodies
especially in case of HIV
 Used if primary Ab is not conjugated
 Multiple binding of secondary Ab leads to signal
amplification
 Diadv: Non specific binding may be observed
Procedure:
a. Known antigen is applied to the surface of the well and coating buffer is then
added
b. Washed with washing buffer
c. Specific antibody to be measured is added and incubated
d. Again washed with buffer
e. Enzyme linked secondary antibody is added
f. Again washed to remove unbound secondary antibody
g. Substrate is added and chromogenic/ fluorescent signal is observed
h. Result viewed using spectrophotometer
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7.  Detection of protenaceous antigens
 Performed when desired antibody
is present in pre-conjugated state
Adv: Fast,Eliminates possible non specific binding
of antigens
Diadv: May result to reduced conjugation of primary
antibody leading to low signal
 Measures amount of antigen between two
layers of antibody hence called sandwich model.
 Antigens to be measured must consist at least
two antigenic sites capable of binding to
at least two antibodies.
 Also k/a direct ELISA
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8. Sandwich ELISA diagram 8

9.  Measures amounts of antigens
 Principle: more antigen present
in sample ,less free antibody available
to bind to the antigen
 Also k/a Inhibitory Type Assay as in this case the concentration of
antigen is inversely proportional to the color product
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11.  Measurement of light produced by chemiluminescence during
certain chemical reactions
 Convenient and highly sensitive alternative to absorbance
 Luxogenic (light-generating) in place of the chromogenic
substrate in conventional ELISA
 Oxidation of the compound luminol by H2O2 and the enzyme
horseradish peroxidase (HRP) produces light
 As little as 5 ×10^̂-18 moles (5 attomoles) of target antigen
have been detected.
 Enhanced sensitivity at least 10 times
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12.  Quantitative determination of Ab specific for a given Ag OR Ag
for a specific Ab
 Plates coated with Ag (capture Ag) recognized by the Ab of
interest OR with the Ab (capture Ab) specific for the Ag whose
production is being assayed
 Cell suspension on investigation is added to the coated plates
and incubated cells settle onto the surface of plate
 Secreted molecules are bound by the capture molecules
producing a ring of Ag-Ab complexes around each cell that is
producing the molecule of interest
 The plate is then washed and an enzyme-linked Ab specific for
the secreted antigen is added and allowed to bind
 Subsequent development of the assay by addition of a suitable
chromogenic or chemiluminescence-producing substrate reveals
the position of each Ag/Ab producing cell as a point of color or
light.
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13. cytokine molecules secreted by a particular
cell react with nearby well-bound antibodies
suspension of a cell population thought to
contain some members synthesizing
and secreting the cytokine are layered onto the
bottom of the well
*highly sensitive but
less specific
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14.  Reagents and equipments have longer shelf life
 No radiation hazards
 Quick procedures
 Huge no. of samples can be handled at once
 Broad menu of over 800 targets
 Optimized for sensitive, accurate, and consistent
performance
 Thorough instructions to complete protocol in
2.5 to 4 hours (varies by kit)
 Validated for typical sample types (e.g., serum,
plasma, supernatant, lysates)
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15.  Measurement of enzyme activity can be more
complex than measurement of radiolabelled
isotopes
 Enzyme activity may be influenced by plasma
constituents
 Very sensitive to particular
antigen;confirmation by Western Blotting
 Expensive kit
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16.  Kuby . J, Osborne B.A, Goldsby
R.A,”Immunology”, 5th edition.Pg.no 148-150
 Lequin, R. M. (2005). "Enzyme Immunoassay
(EIA)/Enzyme-Linked Immunosorbent Assay
(ELISA)". ClinicalChemistry
https://en.wikipedia.org/wiki/ELISA
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