2. What is ELISA????
• An enzyme-linked immunosorbent assay, also called ELISA or EIA, is a test that
detects and measures antibodies in your blood. This test can be used to determine if
you have antibodies related to certain infectious conditions. Antibodies are proteins
that your body produces in response to harmful substances called antigens.
• An ELISA test may be used to diagnose:- HIV, which causes AIDS,Lyme
disease,pernicious anemia,Rocky Mountain spotted fever,rotavirus,squamous cell
carcinoma syphilis toxoplasmosis varicella-zoster virus, which
causes chickenpox and shingles,Zika virus
5. Required reagents
• 1. Bicarbonate/carbonate coating buffer (100 mM) Antigen
or antibody should be diluted in coating buffer to immobilize
them to the wells:
3.03 g Na2CO3,6.0 g NaHCO3,1000 ml distilled water, pH 9.6
1.16 g Na2HPO4,0.1 g KCl,
0.1 g K3PO4
4.0 g NaCl (500 ml distilled water)
3. Blocking solution:
Commonly used blocking
agents are 1% BSA , serum,
non-fat dry milk, casein, gelatin
4. Wash solution:
Usually PBS or Tris-buffered saline (pH 7.4)
with detergent such as 0.05% (v/v) Tween20
5. Antibody dilution buffer:
Primary and secondary antibody should be diluted in 1x blocking solution to
reduce non-specific binding.
6. Principle of the Indirect ELISA
• Indirect ELISA is a two-step ELISA which involves two
binding process of primary antibody and labeled
secondary antibody. The primary antibody is
incubated with the antigen followed by the
incubation with the secondary antibody. However,
this may lead to nonspecific signals because of
cross-reaction that the secondary antibody may
• The four Key steps are as follows:-
1. Micro-well plates are incubated with antigens,
washed up and blocked with BSA.
2. Samples with antibodies are added and washed.
3. Enzyme linked secondary antibody are added and
4. A substrate is added, and enzymes on the
antibody elicit a chromogenic or fluorescent signal.
• 1. Dilute antigen to a final concentration of 1-20 μg/ml using PBS or Bicarbonate/carbonate
coating buffer. Coat the wells of a PVC microtiter plate with the antigen by pipeting 50μl of the
antigen dilution in the top wells of the plate. Dilute down the plate as required. Seal the plate
and incubate overnight at 4°C or 2 h at room temperature.
2. Wash plate 3 times with PBS.
3. Block the remaining protein-binding sites in the coated wells by adding 200 μl blocking
buffer, 5% non fat dry milk/PBS, per well. Alternative blocking reagents include Block ACE or
4. Cover the plate with an adhesive plastic and incubate for at least 2h at room temperature
or, if more convenient, overnight at 4°C.
5. Wash the plate 3 times with PBS.
6. Add 100 μl of diluted primary antibody to each well.
7. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.
8. Wash the plate 4 times with PBS.
9. Add 100 μl of conjugated secondary antibody, diluted at the optimal concentration
(according to the manufacturer) in blocking buffer immediately before use.
10. Cover the plate with an adhesive plastic and incubate for 1-2 h at room temperature.
11. Wash the plate 5 times with PBS.
12. Dispense 100 μl (or 50 μl) of the substrate solution per well with a multichannel pipet or a
13. After sufficient color development (if it is necessary) add 50-100μl of stop solution to the
14. Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the
8. Descriptive study of the Indirect ELISA
Different stages of the indirect ELISA:-
1. Coating ELISA Plates :-
Coating is achieved through passive adsorption of the antigen to the assay microplate. This process occurs though
hydrophobic interactions between the micro titer plate and non-polar protein residues. Although individual proteins
may require specific conditions or pretreatment for optimal binding, the most common method for coating plates
involves adding a 2-10 μg/ml solution of protein dissolved in an alkaline buffer such as phosphate-buffered saline (ph
7.4) or carbonate-bicarbonate buffer (ph 9.4).The buffer contains no other proteins that might compete with the target
antigen for attachment to the micro titer plate. Antigens ,which are protein in nature will attach passively to the micro
titer well plate during incubation in incubator at 370C.
2. Washing step:-
After incubation any excess antigen is removed by washing steps by flooding and emptying the wells with neutral
phosphate buffered saline ( PBS ) or deionized water. Washing steps are necessary to remove nonbound reagents and
decrease background, thereby increasing the signal: noise ratio. Insufficient washing will allow high background,
while excessive washing might result in decreased sensitivity caused by elution of the antigen from the well.
9. 3.Add blocking buffer :
• The binding capacity of microplate wells is typically higher than the amount of protein coated in each well and
the residual binding capacity of the plate is blocked in this step. The ideal blocking buffer will bind to all potential
sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for
• The blocking buffer is effective if it improves the sensitivity of an assay by reducing background signal and
improving the signal-to-noise ratio. Tween 20 (0.05%) by itself is more effective at blocking than any protein
tested, but because the combination of protein and Tween 20 may be more effective than Tween 20 alone in some
cases, bovine serum albumin (BSA; 0.25%) is included in the blocking buffer. Coated plates can be used
immediately or dried and stored at 4°C for later use, depending on the stability of the coated protein.
4.Add primary antibody :-
• This step involves the addition of detecting antibodies (test sample) being directed against the coated antigen.
The antibody is usually diluted in blocking buffer to prevent non specific attachment of protein in the antiserum on
the solid phase. The antibody present in the serum which are specific to the antigen, binds the coated antigen on
5.Washing step :-
• Excess antibody or unbound antibodies are removed by washing step and is followed by addition of blocking
10. 6.Add secondary antibody (antibody enzyme conjugate):
The next step is the addition of secondary antibody, diluted in blocking buffer directed against the primary antibody.
Followed by incubation to the achieve the binding of the enzyme-conjugated secondary antibody. The choice of
antibody enzyme conjugate is determined by the goals of the assay. If it is necessary to detect all antibodies that bind
to antigen, conjugates prepared with antibodies specific for Ig κ and λ light chains should be used. Alternatively,
protein A or protein G–enzyme conjugates may be preferable when screening monoclonal antibodies. Such
antibodies are produced against immunoglobulins (Ig’s) of species in which the detecting antibodies are produced and
are termed anti-species conjugates. Thus, if detecting antibodies are produced in rabbits, the enzyme-labeled
antibodies would have to be anti-rabbit Ig’s in nature. This allow greater flexibility in use of anti-species conjugates in
that different specificities of conjugate can be used to detect particular Ig’s binding in the assay. For example, the anti-
species conjugate could be anti- IgM, igg1, igg2 and so on. The enzyme can be linked to a protein such as
streptavidin if the primary antibody is biotin labeled. The most commonly used enzyme labels horseradish
peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained
widespread acceptance because of limited substrate options. These include β-galactosidase, acetylcholinesterase and
Unbound antibody enzyme conjugate is washed away after incubation phase.
11. 8.Adding substrate :
Substrates are critical for the detection and visualization steps of an ELISA. The step involves the addition of suitable
substrate solution for the particular enzyme conjugated to the antibodies. The objective is to allow development of
color reaction through enzyme catalysis. A large selection of substrates is available for performing the ELISA with an
HRP or AP conjugate. TMB (3, 3’, 5, 5’-tetramethyl benzidine) is the most commonly used substrate for the enzyme
horseradish peroxidase (HRP).The substrates of alkaline phosphatase (AP) , 4-methylumbelliferyl
phosphate (MUP) and PNPP (p-Nitro phenyl-phosphate) are nontoxic and relatively stable. Solutions of p-nitro-
phenyl phosphate (NPP) are stable for months at 4°C, while solutions of 4-methylumbelliferyl
phosphate (MUP) can be kept for months at room temperature without any significant spontaneous hydrolysis.
The biggest disadvantage if NPP is used as a substrate is that, the yellow color of the nitro
phenyl product is relatively difficult to detect visually. Using the substrate MUP instead of NPP can greatly
enhance the sensitivity of the assay. The fluorogenic system using MUP is 10 to 100 times faster than the
chromogenic system using NPP, and appears to be as sensitive as an enhanced chromogenic assay in
which alkaline phosphatase generates NAD+ from NADP. The disadvantage of using fluorogenic substrates is that
they require a microplate fluorometer costing twice as much as a high quality micro titer plate spectrophotometer.
The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-
detection (spectrophotometer, fluorometer or luminometer).
12. 9.Stop solution :
The reaction is allowed to progress for a defined period after which the reaction is stopped by altering the ph of the system. Stop
Solution is a used to terminate the enzyme substrate reaction for ELISA applications after attaining the desired color intensity
which is an indication of analyte level. For e.g. The TMB substrate reacts with immobilized horseradish peroxidase (HRP)
conjugated secondary antibodies to produce a blue solution. Reaction may be stopped by 0.2 M sulphuric acid which offers a
yellow end product read at 450 nm. AP stop solution (0.5M NaOH) does not change the yellow color or the absorbance of the
chromogen, and so the absorbance is read at 405 nm to 420 nm.
Specially designed spectrophotometers are available which reads through the micro titer wells either singly or in rows. Several
ELISA plate readers are available, with increasing levels of sophistication. Some of these provide a measurement of optical density
while some tabulate data and apply statistical analysis. Compatibility with a small computer, and availability of a suitable program
to process the results and transform the optical density readings into concentrations of protein are important additional things to
look for when selecting an instrument. Most ELISA readers can be set to measure the absorbance of the colors produced by the
action of antibody- conjugated enzymes on their respective substrates the microplate reader works by shining a particular type
of light at each of the samples in the micro well plate.
• Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved
fluorescence and fluorescence polarization. A light source illuminates the sample using a specific wavelength (selected by an
optical filter, or a monochromator), and a light detector located on the other side of the well measures how much of the initial
(100%) light is transmitted through the sample, the amount of transmitted light will typically be related to the concentration of
the molecule of interest. This is called absorption detection. The range of application of fluorescence intensity detection is
much broader than when using absorbance detection, but instrumentation is usually more expensive. Microplate readers feed
the absorbance or fluorescence measures into a computer program that analyses the particular information being collected.
14. Principle of Direct ELISA
• Initially in a direct ELISA test which is considered to be the simplest type of ELISA the antigen is
adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin) is added
to block all the other binding sites.
• While an enzyme is linked to an antibody in a separate reaction, the enzyme-antibody complex is
applied to adsorb to the antigen.
• After excess enzyme-antibody complex is washed off, enzyme-antibody bound to antigen is left. By
adding in the enzyme's substrate, the enzyme is detected illustrating the signal of the antigen.
• Direct ELISA, when compared to other forms of ELISA testing, is performed faster because only one
antibody is being used and fewer steps are required. This can be used to test specific antibody-to-antigen
reactions, and helps to eliminate cross-reactivity between other antibodies.
15. Direct ELISA Protocol
1. Coat ELISA plate (96 well plate) with testing antigen (10 μg/ml to 0.01 ng/ml in 50 mM Na2C03, pH 9.6, adjust based on
the reactivity of antibody, 100 μl/well. Seal the plate and incubate overnight at 4°C.
2. Wash plate 3 times with PBS-T (0.05 % Tween-20 in PBS).
3. Block plate with 0.2% non-fat dry milk in PBS at room temperature for 1 hour at 4°C
Note: Milk should be thoroughly dissolved. It's recommended to dissolve 0.5 g milk in 50 ml PBS (1%) for at least 30
minutes at room temperature with rotation or stirring, then dilute to 0.2 %, and keep rotating or stirring for another 10-15
minutes at room temperature. If high background is experienced, 1% milk in PBS could be applied for both blocking and
4. Wash plate 3 times with PBS-T.
5. (a) Incubate with biotinylated, affinity-purified rabbit IgG (0.1-0.5 μg/ml in PBS, 100 μl/well) at room temperature for 1
hour, followed by washing 6 times with PBS-T, then go to 8a.
(b) Alternatively, incubate with affinity purified rabbit IgG (0.2-1 μg/ml in PBS, 100 μl/well) at room temperature for 1
hour, followed by washing 6 times with PBS-T, then go to 8b.
6. (a) Incubate with HRP-Streptavidin (1:4000-10,000 dilutions) in 0.2 % milk-PBS, 100 μl/well, at room temperature for 1
(b) Incubate with HRP-anti-rabbit IgG (1:3,000-10,000 dilutions of 1 mg/ml or 0.25 μg/ml) in PBS, 100 μl/well, at room
temperature for 1 hour.
7. Wash plate 8 times with PBS-T.
8. Develop color using TMB as a substrate (100 μl/well) and incubate at room temperature for 15-30 minutes without
9. Stop reaction by addition of 2N H2S04 (100 μl/well). Record the absorbance at 450 nm on a plate reader within 30
minutes of stopping the reaction.
16. Different stages of the Direct ELISA
1.Coating ELISA Plates:-
This stage is same as indirect ELISA.
Same as indirect ELISA
3. Adding blocking buffer:-
Same as indirect ELISA
4. Washing step:
Same as indirect ELISA
Same as indirect ELISA
Same as indirect ELISA
In this method sample which contain the antibody is treated with the enzyme to link them and they result into enzyme
linked antibody which will then mixed in the blocking buffer and then react with the coated antigen which will gives
the color indication when treated with the substrate.
Note:- This method is much faster then the indirect ELISA because it does not required the secondary antibody treatment.
18. Principle of Sandwich ELISA
• Sandwich ELISA is a less common variant of ELISA, but is highly efficient in sample antigen detection.
Moreover, many commercial ELISA pair sets are built on this sanwich ELISA.
• The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and detection antibody). The
antigen to be measured must contain at least two antigenic epitope capable of binding to antibody, since at least
two antibodies act in the sandwich. Either monoclonal or polyclonal antibodies can be used as the capture and
detection antibodies in Sandwich ELISA systems. Monoclonal antibodies recognize a single epitope that allows
fine detection and quantification of small differences in antigen. A polyclonal is often used as the capture antibody
to pull down as much of the antigen as possible. The advantage of Sandwich ELISA is that the sample does not
have to be purified before analysis, and the assay can be very sensitive (up to 2 to 5 times more sensitive than
direct or indirect ELISA), but lower than ELISpot.
• Sandwich ELISA procedures can be difficult to optimize and tested match pair antibodies should be used. This
ensures the antibodies are detecting different epitopes on the target protein so they do not interfere with the other
• The principle is as follows: (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen
present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked
secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme
to detectable form