Enzyme linked immunosorbent assay

-DR.NISHIGANDHA
 Introduction
❑ ELISA commonly used analytical biochemistry 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 first 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 specific 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 perform EIA/ELISA.
 PRINCIPLE OF ELISA
• ELISA Is so named because of
1. Immunosorbent
2. Enzyme
• Substrate – chromogen system: Added at the final step of ELISA
(Ag- Ab complex)-enzyme + substrate = activates the chromogen colour change detected
by spectrophotometry
• Performed on a microtiter plate containing 96 wells (micro-ELISA) or less commonly in tubes
(macro-ELISA)
• ELISA kits- contain all the necessary reagents ( such as enzyme conjugate, dilution buffer,
substrate/chromogen etc.)
• The 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:
The process of adding an antigen /antibody, diluted in buffer,
so it attaches to the solid phase on incubation.
• WASHING:
The simple flooding and emptying of wells with a buffered
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:
The 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 peroxidase Hydrogen peroxide TMB (tetramethyl 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
❑ Primary 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 primary
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.
❑ Differs from direct ELISA – secondary antibody(an antibody targeted
to Fc region of any human Ig) is labelled with enzyme instead of
Primary antibody.
❖ Indirect ELISA for antibody detection:
Step 1.- wells precoated with antigen
Step 2. - Test serum ( containing primary antibody specific to the
antigen) is added
Step 3. – After washing , enzyme labelled secondary 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. Primary antibody raised in rabbits (reagent) is added, Ag
binds to primary Ab
Step 3. After washing , enzyme labelled secondary 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 primary
antibody or secondary 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 primary “detector antibody”
specific for antigen is added
Step 4: After washing , substrate-chromogen system is added and colour
is developed
 SANDWICH ELISA
❑ Indirect sandwich ELISA:
Primary antibody and capture antibody belongs to different
species
Wells coated with capture Ab + Ag (test serum) + primary Ab +
secondary Ab-enzyme + substrate-chromogen = colour
More specific than Direct sandwich ELISA
 COMPETITIVE ELISA
❑ Antigen in test serum competes with another antigen
of same type coated on well to bind to primary
antibody
❑ Step 1: Primary antibody and test antigen (first
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 secondary
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
DIRECT ELISA ❑ Short protocol , saves time and reagents. ❑ Potential high background ,all proteins in
❑ No cross reactivity from secondary antibody the sample bind to the surface
❑ No signal amplification
❑ Low flexibility ,the primary antibody must
be conjugated

INDIRECT ELISA ❑ Signal amplification, several secondary antibodies will bind ❑ Long protocol if compared to direct elisa
to the primary antibody ❑ Potential cross reactivity from secondary
❑ High flexibility , same secondary may be used as several antibody
primary antibodies
❑ high specificity ,involves two Abs detecting different ❑ Demanding designs , finding two antibodies
SANDWICH ELISA epitopes on the same antigen against the same target that recognise
❑ High flexibility & sensitivity, both direct & indirect different epitopes and work well together
methods can be used 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 classified 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. The assays may use a
combination of recombinant and synthetic peptides as antigens.
 TROUBLESHOOTING IN ELISA
Possible cause Corrective action

Dispensing error Calibrate micropipettes. Check

other dispensing equipments.

Improper washing Check all eight ports/ manifold

for uniform flow of wash buffer.
If there are blockages , clean the
ports.
 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 CONTROL HIGH OD VALUES OF “NEGATIVE CONTROLS
Possible cause Corrective action
Possible cause Corrective action

Plate terminated after Follow the protocol meticulously

Stop solution was Follow the protocol 10 mins.
added before 10 meticulously Same pipette used for Change micropipette tips while
mins. positive and negative addition of negative / positive
Reaction terminated controls. controls
before 10 mins.
Nonspecific If plates get scratches /
OD taken at Read OD values at 450 nm
attachment / binding aberrations during washing, non
incorrect wavelength
of other reagent specific proteins may bind while
addition of next step
 High OD reading in most of the wells
Possible cause Corrective action

Liquid substrate not properly protected from light Incubate the plate in dark after addition of
substrate
Insufficient washing Follow wash protocol meticulously

Poor quality of water used for diluting wash Glass distilled water is preferred
buffer concentrate
 ADVANTAGES AND DISADVANTAGES OF ELISA
ADVANTAGES:
• High sensitivity & specificity
• Easy to perform
• Protocols are easy to follow
• Quantitative & Qualitative
• Possibility to test various sample types
DISADVANTAGES
• Temporary readouts:
• Detection is based on enzyme/substrate
reaction
• So readout must be obtained in a short time
span
• Limited antigen information , limited to
amount or presence of antigen in sample
 ELISA CV
❑ The CV(%) or coefficient 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 surface 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