Enzyme linked immunosorbent assay
Introduction
ELISA commonly used analytical biochemist assay
Assay uses solid – phase enzyme immunoassay to detect presence of Ligand in
liquid sample using antibodies directed against the protein to be measured
Diagnostic tool in medicine, biotechnology, and plant pathology as well as quality
control check in various industries
Was rst screening test widely used for HIV because of its high sensitivity
detect various kind of diseases such as dengue, malaria, Chagas disease,& others
Detection of Mycobacterium Ab in TB, Rotavirus in feces, hepatitis B,C marker in
serum, enterotoxin of E.coli in feces, HIV ab in blood samples
HISTORY
WHAT WAS THERE BEFORE ELISA??????
RADIOIMMUNOASSAY
First described by Rosalyn Sussman Yalow
and Solomon Berson in 1960.
A technique using radioactively labelled
antigens / antibodies
the radioactivity provides the signal which
indicates whether a speci c antigen or
antibody is present in the sample
As radioactivity poses a potential health
threat a safer alternative to RIA would
substitute a non-radioactive signal in place
of radioactive signal
In 1971,Peter Perlmann and Eva Engvall independently published papers that
synthesized this knowledge into methods to pe orm EIA/ELISA.
PRINCIPLE OF ELISA
•ELISA Is so named because of
1. Immunosorbent
2. Enzyme
•Substrate – chromogen system: Added at the nal step of ELISA
(Ag- Ab complex)-enzyme + substrate = activates the chromogen colour change
detected by spectrophotomet
•Pe ormed on a microtiter plate containing 96 wells (micro-ELISA) or less
commonly in tubes (macro-ELISA)
•ELISA kits- contain all the necessa reagents ( such as enzyme conjugate, dilution
bu er, substrate/chromogen etc.)
• e procedure involves a series of steps
done sequentially
•At each step, a reagent is being added,
and then incubated followed by washing
of the wells (manually or by an
automated ELISA washer) .
BASIC TERMS:
SOLID PHASE:
Usually a microtiter plate well, having 96 wells,
8/ 12 wells format.
•ADSORPTION:
e process of adding an antigen /antibody, diluted in
bu er, so it attaches to the solid phase on incubation.
•WASHING:
e simple ooding and emptying of wells with a
bu ered solution to separate bound form un-bound
reagents in ELISA.
.
BASIC TERMS
•ENZYME CONJUGATE:
Any molecule that attached irreversibly to an antibody.
e.g. Horseradish peroxidase(HRP)
•CHROMOGEN:
A chemical that alters colour as a result of an enzyme interaction with substrate (colour
reaction used as signal) e.g. Trimethyl benzidine(TMB).
•STOPPING:
e process of stopping the action of an enzyme on a substrate
•READING:
•Spectrophotometric measurement of colour developed in ELISA.
EQUIPMENTS
1. MICROTITER PLATE 2.MULTIPIPETTE
Flat bottom polystyrene plate, contains
8 *12 wells holding 350 microliter each.
EQUIPMENTS
3.WASHING DEVICE: 4.ELISA PLATE READER:
MICROPLATE WASHER:
ENZYMES USED AND THEIR SUBSTRATE-
CHROMOGEN SYSTEM
ENZYMES SUBSTRATE CHROMOGEN
Horseradish Hydrogen peroxide TMB (tetramethyl
peroxidase benzidine)
urease urea Bromocresol
Beta- galactosidase ONPG ONPG
Alkaline phosphatase pNPP pNPP
Types of ELISA
1.Direct ELISA
2.Indirect ELISA
3.Sandwich ELISA
4.Competitive ELISA
1. DIRECT ELISA
Used for detection of antigen
Prima antibody (targeted against the
serum antigen) – labelled with the enzyme,
directly conjugated to HRP or other
detection molecules
Step 1 - Well(not precoated )
Step 2 - antigen (test serum) added into
wells,(passive adsorption)
Step 3 - After washing, Enzyme labelled
prima antibody (raised in rabbit) are added
Step 4 - After washing , a substrate-
chromogen system is added & colour is
measured
2. INDIRECT ELISA
Used for detection of antibody or less commonly antigen in
serum.
Di ers from direct ELISA – seconda antibody(an antibody
targeted to Fc region of any human Ig) is labelled with enzyme
instead of Prima antibody.
Indirect ELISA for antibody detection:
Step 1.- wells precoated with antigen
Step 2. - Test serum ( containing prima antibody speci c to
the antigen) is added
Step 3. – After washing , enzyme labelled seconda antibody (
anti-human immunoglobulin)added.
Step 4. – after washing, substrate-chromogen system is added
and colour is developed
Indirect ELISA for antigen detection:
Step 1. Test antigen (serum) is added to wells(not
precoated with antigen/antibody)
Step 2. Prima antibody raised in rabbits (reagent) is
added, Ag binds to prima Ab
Step 3. After washing , enzyme labelled seconda
antibody (anti rabbit Ab) is added
Step 4. After washing , a substrate-chromogen system is
added and colour is developed
SANDWICH ELISA
Detects antigen in test serum
Antigen get sandwiched between a capture antibody and
detector antibody
Two types : (Depending upon whether the detector antibody is
prima antibody or seconda antibody)
Direct sandwich ELISA:
Step 1: well precoated with capture antibody( monoclonal Ab raised
in rabbit) targeted against test antigen
Step 2: test serum (antigen) is added
Step 3: After washing , enzyme labelled prima “detector antibody”
speci c for antigen is added
Step 4: After washing , substrate-chromogen system is added and
colour is developed
SANDWICH ELISA
Indirect sandwich ELISA:
Prima antibody and capture antibody belongs to
di erent species
Wells coated with capture Ab + Ag (test serum) +
prima Ab + seconda Ab-enzyme + substrate-
chromogen = colour
More speci c than Direct sandwich ELISA
COMPETITIVE ELISA
Antigen in test serum competes with another antigen
of same type coated on well to bind to prima
antibody
Step 1: Prima antibody and test antigen ( rst
incubated)
Step 2: antigen-antibody mixture is then added to
microtiter plate ( precoated with same type of antigen )
Step 3: free antibodies bind to the antigen coated on
the well . more the test antigens present in sample,
lesser free antibodies will be available to bind to
antigens on well
Step 4: After washing, enzyme-conjugated seconda
Ab is added
Step 5: After washing, a substrate-chromogen system
is added & colour is developed
ADVANTAGES &DISADVANTAGES OF DIFFERENT TYPES OF ELISA
ADVANTAGES DISADVANTAGES
Sho protocol , saves time and reagents. Potential high background ,all proteins
DIRECT ELISA No cross reactivity from seconda antibody in the sample bind to the su ace
No signal ampli cation
Low exibility ,the prima antibody
must be conjugated
Signal ampli cation, several seconda antibodies Long protocol if compared to direct
INDIRECT ELISA
will bind to the prima antibody elisa
High exibility , same seconda may be used as Potential cross reactivity from
several prima antibodies seconda antibody
high speci city ,involves two Abs detecting di erent Demanding designs , nding two
SANDWICH ELISA
epitopes on the same antigen antibodies against the same target
High exibility & sensitivity, both direct & indirect that recognise di erent epitopes and
methods can be used work well together can be challenging
at times
Suitable for small antigen Depends on base ELISA selected
COMPETITIVE ELISA
MODIFICATION OF ELISA
ELISPOT TEST:
Allows quantitative
detection of cells
producing antibodies
(plasma cells) or
cytokines(e.g.
macrophages)
Number of coloured
spots = number of
cytokine producing
cells present in the
added suspension
ELISA is also classi ed on the basis of the antigen utilized into:
1st generation: Infected cell lysate is used as the antigen
2nd generation: Glycopeptides ( recombinant antigens) are used as the antigen.
3rd generation: Synthetic peptides are used as the antigen
4th generation : Antigen and antibodies are detected simultaneously. e assays
may use a combination of recombinant and synthetic peptides as antigens.
TROUBLESHOOTING IN ELISA
Possible cause Corrective action
Dispensing Calibrate micropipettes.
error Check other dispensing
equipments.
Improper Check all eight po s/
washing manifold for uniform ow of
wash bu er. If there are
blockages , clean the po s.
Troubleshooting in ELISA
If the negative controls are giving positive results:
Contamination of the substrate solution,
enzyme-labelled antibody, control themselves.
Inadequate rinsing of plates.
Inadequate blocking of plates.
If no colour has developed for the positive controls
or for the samples:
Check all reagents for dating and storage
conditions
Microwell plates not coated properly
Reagents applied in wrong order or step omitted
Enzymes conjugate defective or inhibited by
contaminant.
TROUBLESHOOTING IN OD VALUES
LOW OD VALUE OF “POSITIVE HIGH OD VALUES OF “NEGATIVE
CONTROL CONTROLS
Possible cause Corrective action
Possible cause Corrective action
Plate terminated Follow the protocol
Stop solution was Follow the protocol after 10 mins. meticulously
added before 10 meticulously Same pipette used Change micropipette tips
mins. for positive and while addition of negative /
Reaction negative controls. positive controls
terminated before
10 mins. Nonspeci c If plates get scratches /
attachment / aberrations during washing,
OD taken at Read OD values at 450
binding of other non speci c proteins may
incorrect nm
reagent bind while addition of next
wavelength
step
High OD reading in most of the wells
Possible cause Corrective action
Liquid substrate not properly protected Incubate the plate in dark after addition of
from light substrate
Insu cient washing Follow wash protocol meticulously
Poor quality of water used for diluting wash Glass distilled water is preferred
bu er concentrate
ADVANTAGES AND DISADVANTAGES OF
ELISA
ADVANTAGES: DISADVANTAGES
•High sensitivity & speci city •Tempora readouts:
•Easy to pe orm •Detection is based on enzyme/
substrate reaction
•Protocols are easy to follow
•So readout must be obtained in a sho
•Quantitative & Qualitative time span
•Possibility to test various sample types •Limited antigen information , limited to
amount or presence of antigen in
sample
ELISA CV
e CV(%) or coe cient of variability shows how consistent the assay is.
Generally calculated to evaluate the inter-assay precision or plate to plate
consistency and intra- assay precision or consistency between duplicates run in
same experiment.
Inter-assay % CVs of less than 15 are generally acceptable
intra-assay % CVs should be less than 10.
APPLICATIONS OF ELISA
ELISA can be used both for antigen and antibody detection
For antigen detection : Hepatitis B su ace antigen (HBsAg) and precore antigen
(HBeAg) , NS1 antigen for dengue, etc
For antibody detection : Hepatitis B , Hepatitis C, HIV, Dengue, EBV, HSV,
toxoplasmosis, leishmaniasis etc
