This document provides an overview of ELISA (enzyme-linked immunosorbent assay), including its principle, evolution, types, basic steps, and applications. ELISA is an immunoassay technique that detects and quantifies substances like proteins, antibodies, and hormones. It relies on antibodies and antigens binding specifically together, and uses enzyme labeling to identify these bindings colorimetrically. The document discusses various ELISA techniques like direct ELISA, indirect ELISA, and sandwich ELISA.

1. By,
Dr. Anunoy Samanta
MBBS, MHA, DNB (Patho) Resident, JLNH & RC, Bhilai

2.  Elementary Lessons on Immunoassay
 What is ELISA?
 Evolution to ELISA
 Principle of ELISA
 Is ELISA Sensitive?
 We can use ELISA in…
 Basic Steps of ELISA
 Types of ELISA Test
 ELISA Test requires…
 A typical Kit contains…
 Procedure Flow Chart
 Calculation of Results
 Quality Control
 External Control
 E Ratio
 Levvy Jennings Chart
 Advantages & Disadvantages of ELISA

3.  The term ‘Immunoassay’ is a combination of ‘Immuno’
(any antigen-antibody reaction) and ‘Assay’
(determination of purity of substance or the amount of
any constituent of a mixture).
 Immunoassays are biochemical tests that detect the
presence of a given substance (or measure its
concentration) in a solution through the use of an Ab
(usually), or an Ag (sometimes).
 Most important components of any immunoassay
technique are antigen (Ag), antibody (Ab) & labeling
materials.

5.  Any ‘thing’ foreign to our immune system (such as
protein molecule, carbohydrate molecule, allergens,
microorganisms, viruses etc) can be called an Ag.
 Proteins produced by our immune system which help to
defend against Ag is called an Ab.
 The “variable regions” in an Ab molecule are thought
to be site for recognition & binding with Ag.
 In immunoassay, it’s necessary to use any marker to
know the Ag-Ab binding. So, we label either Ag, or Ab
with some material that doesn’t interfere with the
binding (e.g. Horseradishperoxidase enzyme)

7. There’re several methods of conducting immunoassays:
 Precipitin Test – Use precipitin (Ab) that precipitates
out of a solution upon binding to an Ag.
 Agglutination Test – Clumping of cells on exposure to
specific Ab, as used in diagnosis of enteric fever etc
 Complement Fixation Test – Complement fixing Ab in
serum are formed in response to some bacterial Ag.
 Radio-immunoassay (RIA) – Radio labelled molecules
are used for the detection of hormones, serum
proteins, infectious agents, vitamins etc.
 Enzyme-linked Immunosorbent Assay (ELISA) – We’ll
talk about in upcoming slides.

8.  ELISA (Enzyme-linked Immunosorbent Assay) is an
immunoassay technique designed for detecting &
quantifying substances (peptides, proteins, antibodies,
hormones) in which highly specific Ag-Ab reaction is
monitored by enzyme measurements. It’s self
explanatory:
 Enzyme-linked: Enzymes are attached with either the
Ag or the Ab.
 Immuno: Ag is recognized by specific Ab & sometimes
the Ab is recognized by another Ab.
 Sorbent: Ag/Ab of interest is absorbed onto the
plastic surface.
 Assay: Substrate reacts with enzyme to produce
coloured product.

10.  The only option for conducting an immunoassay was RIA
which was described by Rosalyn & Solomon back in 1960.
But, radioactivity posed a potential health threat. A safer
alternative was sought, such as an enzyme.
 But the enzyme had to be linked to an appropriate Ab.
This linking process was developed by Avrameas & Pierce.
 The Ab/Ag had to be fixed to the surface of the
container. This immunosorbent technique was designed by
Wide & Porath in 1966.
 Perlmann & Engvall were the first to coin the term
ELISA and publish the complete technique in 1971.

11.  Based on “Lock and
Key” concept.
 Some Ag & Ab can
get attached to
plastic surfaces.
 Ag & Ab can be
bonded to enzymes.
 The enzyme
detects the binding
of Ag with an Ab.

12.  The enzyme
converts a colourless
substrate
(chromogen) to a
coloured product.
 This test can be
used to detect either
the presence of Ag,
or Ab in a sample
depending on how the
test is designed.

13.  ELISA is one of the most
sensitive (True Positive Rate)
immunoassays available presently.
 It has a typical detection range
between 0.01 & 0.1 ng
 Sensitivity relies on the specific
characteristics of the interaction
between the Ab and the Ag.
 A number of substrates, like
the ones yielding fluorescent
signal or enhanced chemi-
luminecscent, can be utilized to
enhance results

14.  Detection of Infectious Agents – Ag in HBV, Malaria
 Detection of Serum Proteins - Ferritin
 Detection of Hormones - hCG
 Detection of Immunoglobulin – IgG for H. Pylori
 Detection of Tumour Markers – CA125 for Ovarian Ca
 Detection of Drug Markers – studying
pharmacokinetic, illegal drug detection
 In Newborn Screening – Cystic fibrosis, PKU
 In Vaccine Quality Control – to quantify Ag
 In Clinical Research
 Detecting allergens in food such e.g. milk, peanuts,
walnuts, almonds, eggs
 Identification of genetically modified protein in
transgenic produce/GMO

15.  The sample (Ag/Ab), which
may or may not be labelled,
is added to polystyrene
micro-titer wells which
already has a reagent
(Ag/Ab) bound to its
surface.
 The sample is incubated
for a suitable time and at a
suitable temperature to
allow binding of the two
reagents.

16.  Excess or unbound reagent
is washed off with a
washing buffer solution.
 If the first reagent wasn’t
tagged, then a second
enzyme-linked Ab (called
antihuman Ab/Anti Ab) is
also added to bind with the
first one, and allowed to
incubate.
 A substrate is added for
the enzyme.

17.  The enzyme converts the substrate (chromogen) into
a coloured substance, or any other type of photo signal.
 Measure (assay) of the Ag present is directly
proportional to the intensity of the coloured product.

18. Based on how the analytes & Ab are bonded and used,
ELISA Test has been divided into following types:-
 Non-competitive ELISA
* Direct ELISA
* Indirect ELISA
* Sandwich ELISA
 Competitive ELISA
 Modified ELISA (avidin-biotin ELISA)

19. In Direct ELISA, a labelled primary Ab (conjugated
with an enzyme) is used to detect the presence of an
Ag. It involves the following steps:-
 Coating surface with Ag
 Adhesion to the plastic
 Blocking unoccupied sites
 Washing off excess Ag
 Addition of primary Ab
 Washing off excess Ab
 Addition of substrate
 Colour change
 Quantification by spectrometry

20. In Indirect ELISA, a primary unlabelled Ab binds with
immobilized Ag, and is in turn bound to a secondary
enzyme-linked Ab, which produces colour change on
addition of a suitable substrate. It is used to detect
the presence of an Ab. It involves the following steps:-
 Coating surface with Ag
 Washing off excess Ag
 Addition of primary Ab
 Binding of Ag-Primary Ab
 Washing off excess Ab
 Addition of secondary Ab
 Washing off excess Ab
 Addition of substrate
 Colour change & quantification by spectrometry

21. In Sandwich ELISA, the Ag is sandwiched between a
capture Ab (bound to the well) & a primary Ab (added
later). A secondary enzyme-linked Ab binds with the
primary Ab, and after the addition of a substrate, the
Ag-Ab binding is detected via colour appearance.,
which produces colour change on addition of a suitable
substrate. It involves the following steps:-
 A known quantity of “Capture Ab” (meant to trap a
specific Ag) is bound to the surface
 Blocking unoccupied sites – any nonspecific binding
site on the surface is blocked
 Addition of sample containing Ag onto the plate & its
capture by the Ab.
 Washing off unbound Ag

22.  Addition of primary Ab – a specific Ab is added which
binds to the Ag. Now the Ag is stuck between two Ab
like a sandwich
 Addition of secondary Ab – Enzyme linked secondary
Ab are applied as detection Ab that binds specifically
to the primary Ab’s nonspecific region
 Washing off unbound Ab-Enzyme conjugates
 Addition of substrate to be converted by the enzyme
into a colour signal
 Quantification by spectrometry – the absorbance of
the plate wells is measured to determine the presence
& quantity of Ag

24.  Advantages of Sandwich ELISA are:-
* High Specificity – since two Ab are detecting
different epitopes on the same Ag
* High Sensitivity & Flexibility – both Direct &
Indirect methods can be used
* It is suitable for complex samples
* Use of the purified specific capture Ab eliminates
the need to purify the Ag from complicated mixtures
before the measurement
 Its only disadvantage is its demanding design, i.e.
finding two Ab against the same target that recognizes
different epitopes & function together can be
challenging

25. In this test, a competition is generated between the
Ag which are already bound to the micro-titre wells
and those Ag which are already bound with the Ab. It
involves the following steps:-
 Ag in the sample is incubated with an unlabelled Ab
 The Ag-Ab complex is added to an Ag coated well
 Competition between Ag for the unbound Ab
 Washing off unbound Ab
 Addition of secondary enzyme-linked Ab specific to
the primary Ab
 Washing off excess secondary Ab
 Addition of substrate
 Colour change & quantification by spectrometry

27.  The biotin-avidin
system (BAS) can be
incorporated into ELISA
whereby an Ab is
conjugated to biotin &
then detected with
avidin or streptavidin
conjugated to a variety
of flurochromes &
enzymes.
 Biotin can be
conjugated to a variety
of biological molecules
including Ab.

28.  Avidin is a basic GlyProt
which has 4 identical
subunits & a strong affinity
for the water soluble vitamin
biotin, thereby capable of
binding with upto 4 biotin
molecules.
 The multistage
amplification effect of BAS
makes it highly sensitive,
specific & improves the
precision of detection by
reducing the operation error
due to its high stability.

29.  Testing Sample – Serum, CSF, sputum, urine, semen,
supernatant of culture stool etc.
 Polystyrene micro titre plate – The 96 (12x8) well
plates made of polystyrene, coated with either
inactivated Ag or Ab.

30.  Pipettes, micropipettes and pipette tips
 Incubator
 ELISA Reader – It is an instrument used to detect
biological, chemical or physical events of samples in
micro titre plates.

31.  Antibodies - Generally, monoclonal antibodies are used
because they are more specific for a certain epitope
than polyclonal antibodies. They are originally
produced by inducing a humoral immune response in
some animal (such as Rat, mouse) by injecting an
antigen multiple times. Then cells are extracted and
antibodies are purified.

32.  Enzymes - Enzymes are proteins that speed up the
rate of a chemical reaction without being used up and
usually react only to particular substrates. The rate of
this reaction is proportional to the amount of enzyme
present. In ELISA, the enzyme reacts with a colorless
substrate to produce a colored product. The more
binding of the enzyme conjugate to the antigen, the
stronger will be the color development. Myriad
enzymes used in ELISA are-
• Horseradish peroxidase (HRP)
• Alkaline phosphatase (from E. coli)
• Beta-galactosidase (from E. coli)
• Lacto-peroxidase
• Streptavidin etc.

33.  Enzymes Continued…
In case of HRP (most
commonly used enzyme),
the substrate H2O2 is
converted into H2O and [O]
in the presence of electron
donors such as
Diaminobenzidine (DAB).
DAB gets oxidized which
produces dark brown color.
In place of DAB when 4-
Chloronapthol is used,
purple color is obtained.

34.  Substrate - A substrate is a compound that binds to
active sites on the surface of enzymes and undergoes
chemical change, visually appreciable by change in color.
Commonly used substrates in ELISA are:
• Hydrogen peroxide
• 3,3´, 5,5´ Tetra methyl benzidine (TMB)
• p-nitrophenyl phosphate (pNPP)
• Bromochloro-Indolyl phosphate (BCIP)

35.  Blocking Buffer – It is a solution of irrelevant protein,
mixture of proteins, or other compound that passively
adsorbs to all remaining binding surfaces of the plate.
 Stopping Solution – It stops the enzyme substrate
reaction and colour development.
 Washing Buffer – It acts as a buffered solution
containing detergent to wash unbound material from the
plate.
 Microplate Washer
 Computer
 DBMS
 Printer
 Record sheets

39. A Merilisa HIV Gen 4 EIA Kit used for the detection of
HIV p24 Ag & Ab to HIV-1 & HIV-2 contains-
 HIV Ag & Ab coated microplate
 Sample diluent
 Negative control
 HIV Ag positive control
 HIV Ab positive control
 Washing solution
 Conjugate
 Biotinylated anti-HIV p24 conjugate
 Conjugate diluent
 Substrate solution
 Stop solution
 Adhesive sheets

40. It might differ slightly from kit to kit, manufacturer to
manufacturer, but for a rough idea we’ll have a quick look
at the procedural flow chart given in the Instruction
Manual of Merilisa HIV Gen 4 EIA Kit (for the detection
of HIV p24 Ag & Ab to HIV-1 & HIV-2) which is
commonly used in our TTI laboratory-

41.  50uL of sample diluent is added to each well except
the Blank well
 100uL of negative controls/ positive controls/ samples
are added to their designated wells
 Covered and incubated at 37 deg C for 30 min
 The wells are washed with 350uL of wash buffer 5
times
 100uL of Biotinylated HIV p24 Ab conjugate is added

42. to all wells except Blank well
 Plate is covered & incubated for 15 min at 37 Deg C
 Content of the microwell is discarded
 100uL of working conjugate solution is added to all
wells except Blank well
 Plate is covered & incubated for 15 min at 37 Deg C

43.  The wells are washed with 350uL of wash buffer 5
times
 100uL of substrate is added to each well
 Plate is covered & incubated for 30 min at room
temperature
 50uL of stop solution is added to each well
 Within 15 min absorbance of each is read at 450nm
using 630nm as reference wavelength

44.  Results should be recorded by reading the optical
density (OD) of each plate in an ELISA Reader at
450nm using 630nm as reference wavelength.
 Specific software is used for calculating the titers.
Software calculates the result by comparing OD of
unknown serum to that of reference serum.
 Mean absorbance of 3 negative controls is calculated.
 Cut-off value is calculated by adding 0.20 to the
mean of NC.

45.  Interpretation:
Samples giving abs < Cut-off value~ Non Reactive
Sample giving abs = or > Cut-off value~ Initially Reactive
Then the sample is retested in duplicate. If found
Reactive in at least one of the duplicates, then the
positivity is reaffirmed & further investigations (e.g.
PCR) are suggested.
Sample with abs within 10% below Cut-off value~
Suspicious ~ Retested in duplicate.

47. Results of our assay are valid if:
 The abs of the Blank < 0.05
 Mean abs of 3 Negative controls < 0.10
 Each of the Positive controls should have abs > 1.0
If the above criteria is not met, Assays need to be
repeated.

48. EC refers to the control included from outside (not
provided in the ELISA kit). It helps in detecting minor
error in the assay performance. We prepare the EC in-
house by following these steps:-
 Sero-reactive serum is selected
 The sample serum is Retested with another kit
 The sample serum is Heat Inactivated at 56 Deg C
for 30 min
 Serial dilution of the sample is done with either
normal saline or sero-negative serum

49.  After Test Run, ELISA Ratio (ER) for each dilution is
calculated. ER = Sample OD / Cut-off OD
 The dilution with ER between 1.5-2.0 is selected
 EC aliquots of dilution selected above are prepared
 Stored at -20 Deg C for 1 Yr. Once thawed it can be
kept at 2-8 Deg C for a Wk.

50.  Cut-off value of run depends on the principle of the
test, manufacturer guidelines and recommended
protocols for the calculation
 There can be some degree of variation in the internal
controls
 Other factors can also influence variation in the cut-
off values like- Incubation period, Preparation of
reagents, plate to plate and well to well variation etc
 OD of the controls would accordingly influence the
OD of the test sample in its same direction.

51.  Relative reactivity of the given sample and the cut-
off would not vary. This relative reactivity in a
particular run is termed as E ratio.
ER = OD value of EC/ OD value of Cut-off
 ER are plotted on the Y-axis in Levvy Jennings Chart
and consecutive dates of runs are plotted on X-axis.
 Limit of 2SD on either side of the Mean are drawn.
 Mean & +/- 2SD are plotted on the graph

54.  LJ Chart helps in the detection of – Systemic
Variation (Trend / Shift), Random Variation (due to
transcription errors, sample mix up, poor pipette
precision, poor mixing of samples, inconsistent washing,
un-calibrated reader), Lot to lot Variation, Day to day
Variation.
 Highlights the outliers (values outside +/- 2SD)
 Reveal batch to batch variation
 Reveal operator to operator variation
 Detects changes in assay performamce even when
test runs are valid

55.  Reasonably Sensitive – detects even < 1 ng protein
 No radiation hazard unlike its predecessor RIA
 Reagents are comparatively cheaper with long self
life. Detection kits easily available commercially.
 Can handle a large number of samples at a time
 Comparably simple technique
 Adaptable to automation and high speed
 Can be both- Qualitative as well as Quantitative

56.  Can only test for a specific Ag or Ab
 Highly skilled technician is required for quality test
 Generally requires incubation, hence it can not be
taken outside the laboratory for field test
 Can give false positive result if washed inadequately
 Time consuming test