ACTIVITY #1

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

I. Objectives

At the end of this exercise, the student should be able:

a. To familiarize oneself to the different types of ELISA
b. To identify the steps involved in the conduct of ELISA
c. To be able to know how to troubleshoot false positive results with
ELISA

II. Introduction

Enzyme-Linked Immunosorbent Assay is one the most common

immunoassay type and used to detect the presence and measure the
amount/concentration of specific analytes such as antibodies (BIO-RAD, 2021).
Developed in the early 1970s, this immunoassay utilize antibodies or antigens
covalently bound to enzymes which are selected on the basis of their ability to
catalyze the conversion of a substrate into a colored, fluorescent, or
chemiluminescent product. ELISA involves specific and non-specific interactions
via serial binding to a solid surface usually on a polystyrene mutiwell plate hence
its quantitative nature.

There are four types of ELISA: Direct ELISA, Indirect ELISA, Sandwich
ELISA and Competitive/Inhibition ELISA, where each of these types has an
advantage to one another. Their differences will be explored in the succeeding
sections.

III. Learning Platform

https://www.biointeractive.org/classroom-resources/immunology-virtual-lab
Microsoft Teams
Laboratory Manual
IV. Reagents

Coating
a. Sodium bicarbonate (NaHCO3)
b. Sodium carbonate (Na2CO2)

Blocking
c. Phosphate Buffer Saline (PBS)

Washing
d. Tween 20

Detection
e. Conjugated Antibodies (HRP)
f. p-NitroPhenyl Phosphate (pNPP)

CHECKPOINT:
Identify the functions of each reagent indicated above. Why are the reagents
segregated into coating, blocking, washing, and detection?

One of the key points in ELISA is the significance of the reagents involved in
each step of the assay. Each reagent plays a crucial role for an accurate result.
Generally, the reagents in ELISA are segregated according to their function. Each of
the functions are as follows (Thermo Fisher Scientific, 2016):

1. Coating buffer is a solution used in ELISA to immobilize or “coat” antigen or

antibody onto the microplate. The coating buffer also optimizes the attachment
of the antibody or antigen to the surface by promoting adsorption and reducing
non-specific binding. Coating buffers usually contain a pH buffer and salt
solution that stabilizes the proteins and protects it from denaturation.

a) Sodium bicarbonate (NaHCO3) - added to the coating buffer to

provide a pH around 8.0-9.6.

b) Sodium carbonate (Na2CO2) - alternative to bicarbonate and is

used for proteins with lower binding affinities and sensitive to changes
in pH.

2. Blocking buffers are solutions used in ELISA to prevent nonspecific binding

of the detection reagents such as the conjugated antibody into the plate or
 some other proteins present in the solution. Blocking buffer covers any
remaining unoccupied sites or residues to the plates that could possibly affect
the accuracy of the results. Due to this, blocking buffers reduces the possibility
of errors in the results by reducing the background noise and enhancing the
sensitivity of the procedure.

c) Phosphate Buffer Saline (PBS) commercially available reagent

usually used as a blocking buffer due to its non-toxic and non-reactive
properties.

3. Washing reagents are solutions that are used to “wash” any unbound, or non-
specifically bound molecules from the surface of the plate. Washing reagent
are usually consist of a buffer solution with a detergent or surfactant that helps
disrupt interactions that are non-specific between the detection reagents and
the surface of the microplate and eliminates any cross contamination between
added components in each step of the assay

d) Tween 20 - nonionic surfactant that is commonly used as component

of washing reagent in ELISA

4. Detection reagents are reagents used to detect and quantify the presence of
a specific analyte in focus in ELISA .

e) Conjugated Antibodies (HRP)- detection enzyme used in ELISA

that generates chemiluminescent signals that indicates presence and
quantity of a certain analyte in

f) p-NitroPhenyl Phosphate (pNPP) - widely used substrate for

detecting alkaline phosphatase in ELISA applications. It produces a
yellow water soluble color after reacting with the enzyme-linked
antibody.
Virtual Laboratory Results:

(A) (B)

Figure 1. Actual results that yielded false positive (A) and the correct plate.

Results showed that Patient A, B, C and the positive control group has
changed its color presented in Figure 1.A. While Figure 1.B indicates the correct plate
wherein Patient A is positive for SLE; Patient B might have the SLE with further testing
needed; and Patient C which is likely to be negative for SLE. Therefore, actual lab
results yielded false positives. Possible reason for having false positive results may
be because of a missed step of washing the ELISA plate that led to cross-
contamination.

V. Procedure:

Direct ELISA (adapted from BIO-RAD Protocol with modifications)

a. Coat the microtiter plate wells with 100uL of the coating antigen at 1
ug/mL in coating buffer then incubate overnight at 4°C. Wash the plate
after incubation.
b. Add 150 uL of the blocking solution in each well, then incubate for 60
minutes at 37 °C. Wash
4 times with wash buffer
 c. Add 100 uL of biotin-conjugated detection antibody, diluted in wash
buffer, to each well. Incubate for another hour then wash 3 times using
the wash buffer.
d. Add 100 uL of enzyme-conjugated streptavidin, diluted in wash buffer,
to each well. Incubate for another hour then wash 3 times using the wash
buffer.
e. Add 100 uL of pNPP, diluted in the wash buffer, to each well. Incubate
for 30 minutes in the dark.
f. Read absorbance values at 405 nm

Indirect ELISA (adapted from BIO-RAD Protocol with modifications)

a. Coat the microtiter plate wells with 100uL of the coating antigen at 1
ug/mL in coating buffer then incubate overnight at 4°C. Wash the plate
after incubation.
b. Add 150 uL of the blocking solution in each well, then incubate for 60
minutes at 37 °C. Wash
4 times with wash buffer
c. Add 100 uL of unconjugated detection antibody, diluted in wash buffer,
to each well. Incubate for another hour then wash 3 times using the wash
buffer.
d.
Add 100 uL of enzyme-conjugated secondary antibody, diluted in wash
buffer, to each well. Incubate for another hour then wash 3 times using
the wash buffer.
e. Add 100 uL of pNPP, diluted in wash buffer, to each well. Incubate
for 30 minutes in the dark.
f. Read absorbance values at 405nm.

Sandwich ELISA (adapted from BIO-RAD Protocol with modifications)

a. Coat the microtiter plate wells with 100uL of the coating antibody at
1 ug/mL in coating buffer then incubate overnight at 4°C. Wash the plate
after incubation.
b. Add 150 uL of the blocking solution in each well, then incubate for 60
minutes at 37°C. Wash 4 times with wash buffer
c. Dilute antigen and standards in wash buffer and dispense 100 uL to
the microtiter wells in triplicate. Incubate for 90 minutes at 37°C then
perform washing.
 d. Add 100 uL of enzyme-conjugated secondary antibody, diluted in
wash buffer, to each well. Incubate for another hour then wash 3 times
using the wash buffer.
e. Add 100 uL of pNPP, diluted in wash buffer, to each well. Incubate
for 30 minutes in the dark.
f. Read absorbance values at 405nm.

Inhibition ELISA (adapted from BIO-RAD Protocol with modifications)

a. Coat the microtiter plate wells with 100uL of an antigen solution at 1
ug/mL in coating buffer then incubate overnight at 4°C. Wash the plate
after incubation.
b. Add 150 uL of the blocking solution in each well, then incubate for 60
minutes at 37°C. Wash
4 times with wash buffer
c. Prepare antigen-antibody mixture by adding 50uL of antigen to 50uL
of antibody for each well in the assay and is incubated for 1 hour.
d. After incubation, 100 uL of the mixture is added onto each well.
Incubate for 1 hour then wash 3 times.
e. Add 100 uL of enzyme-conjugated secondary antibody, diluted in
wash buffer, to each well. Incubate for another hour then wash 3 times
using the wash buffer.
f. Add 100 uL of pNPP, diluted in wash buffer, to each well. Incubate
for 30 minutes in the dark.
g. Read the absorbance at 405 nm.
 LABORATORY WORKSHEET #1
ENZYME-LINKED IMMUNOSORBENT ASSAY

Name: Bornea, Richard Christopher C. Instructor: Mr. Rainier Ulrich Velasco

Ronquillo, Kyle Kevin B.

Salva, Danielle Anne R,

Year and Section: BCM 3201L-1 Score: ___________________

Guide Questions:

1. Differentiate the various types of ELISA in terms of their procedure and applications.

As elaborated in this activity, ELISA has various types which differ in terms of their
procedure and applications. Each of the types, their differences and specific applications
are as follows:

1. Direct ELISA - In this type of ELISA, the antigen of interest is directly immobilized
onto the surface of the microplate or other solid support used in the assay. The
primary antibody specific to the antigen is then added, followed by a labeled
secondary antibody that binds to the primary antibody. This type of ELISA is
relatively simple and straightforward, but it is less sensitive than other types. Direct
ELISA is commonly used when analyzing immune response to an antigen.

2. Indirect ELISA- In this type of ELISA, the primary antibody specific to the antigen
is immobilized onto the surface of the microplate or other solid support used in the
assay. A labeled secondary antibody that binds to the primary antibody is then
added. This type of ELISA is more sensitive than direct ELISA because multiple
secondary antibodies can bind to each primary antibody, resulting in signal
amplification. This type of ELISA is used best in quantifying levels of antibody in a
sample.
 3. Sandwich ELISA - In this type of ELISA, two antibodies specific to the antigen are
used. One antibody is immobilized onto the surface of the microplate or other solid
support, and the other antibody is labeled and added to the sample. The labeled
antibody binds to the antigen, and the unbound labeled antibody is removed by
washing. This type of ELISA is highly sensitive and specific because it uses two
antibodies to detect the antigen hence can be used in analyzing complex samples.

4. Competitive ELISA - In this type of ELISA, a labeled antigen competes with the
antigen of interest for binding to a limited amount of immobilized antibody. The
amount of labeled antigen that binds to the immobilized antibody is inversely
proportional to the amount of antigen in the sample. This type of ELISA is useful
for quantifying the amount of antigen in a sample. This method is typically utilized
when there is only one antibody accessible for the targeted antigen. Additionally,
it is appropriate for identifying minor antigens that are unable to attach to two
different antibodies, as in the case of the sandwich ELISA approach.

2. Identify the specific functions/concepts behind the steps in coating, blocking, washing
and detection. Why are the said stages performed in a stepwise manner? What will
happen if the steps are mixed up?

COATING - Coating refers to the first step of ELISA, which involves the incubation and
adsorption of antibodies or antigens to the surface of the well (Thermo Fischer Scientific,
2023). This process occurs passively due to the hydrophobic interactions between the
amino acid side chains of the antibody or antigen used for coating and the plastic surface.
Various factors affect this process, including time, temperature, antibody/antigen
concentration, diffusion coefficient, surface area ratio, and coating buffer pH, determining
the rate and extent of coating (Creative Diagnostics, 2023). The coating buffers stabilize
the antibody or antigen coating in the microplate wells, optimizing its adsorption and
interaction with the target antibodies or antigens (Bio-Rad Laboratories, 2023).
.
BLOCKING - Blocking refers to the addition of irrelevant molecules that do not react with
any of the antibodies used in the detection step to cover the unsaturated surface-binding
sites of the microplate wells (Creative Diagnostics, 2023). Blocking these sites is
necessary to prevent the nonspecific binding of the following reactants, resulting in lower
specificity and sensitivity and subsequently causing a high background signal (Bio-Rad
Laboratories, 2023).

WASHING - Washing removes nonspecifically unbound molecules in the microwell plate

after incubation procedures (Thermo Fischer Scientific, 2023). Incubation allows high
affinity and specific interactions to form among the reactants. By washing 3-5 times, the
excess reactants are diluted and removed to an undetectable background level. This
process uses washing reactants, usually a buffer solution consisting of detergent or
surfactant that disrupts the nonspecific interactions between reagents and the microwell
plate surface (Bio-Rad Laboratories, 2023). In addition, this process avoids cross-
contamination between added reagents during ELISA, which can lead to false results
(Creative Diagnostics, 2023).

DETECTION - Detection involves the interpretation of signals generated by the

interaction of enzyme-linked antibodies with the substrate (ThermoFischer Scientific,
2023. This process determines the absence or presence of the target substance,
including its concentration. The analysis of the ELISA assay during detection can be
qualitative, quantitative, or semi-quantitative. The qualitative manner involves comparing
the ELISA assay to a blank well to determine the absence or presence of the target
antigen or antibody. The quantitative method aims to calculate the concentrations of the
target antigen or antibody by comparing it with a standard curve, which is a serial dilution
of the identified, purified antigen or antibody. Lastly, the semi-quantitative methods
involve comparing the relative levels of antigens or antibodies in assay samples,
reflecting their relative concentrations (Creative Diagnostics, 2023).

The correct order of steps in ELISA is necessary to obtain a reliable and accurate
result. These sequentially performed steps generally involve plate coating, plate blocking,
washing, and detection. It is critical to perform these steps accordingly since their
functions are related. For instance, we cannot accomplish the detection process correctly
without ensuring that there are no nonspecific bindings in the assay, done by performing
the coating, blocking, and washing steps. Without the preceding steps, the successive
steps will be deemed worthless. Failure to perform these steps accordingly will create a
false result.

3. Why do we need to set a blank for each ELISA plate? Identify its specific function in a
plate.

Blank plate samples refer to the ELISA plates that serve as an assay control to
validate the results. During ELISA, 3-8 wells are typically assigned as plate blanks, each
consisting of a liquid, usually a buffer or water, not having the target analyte (Mabtech,
2023). These plates function as a negative control that checks for the nonspecific binding
during assay that can result in a false positive result. It allows the subtraction of
background absorbance from the rest of the other wells to ensure an accurate optical
density reading, which measures the presence and concentration of the target analyte
(Thermo Fisher Scientific, 2023). A background interference with these plates can
indicate that the assay is detecting another substance other than the target analyte. Thus,
a false positive result (Science Squared, 2018).

4. Troubleshooting:

Problem Proposed Solution

No enzyme signal detected (low -> review the protocol; retrace steps,
absorption)
-> ensure that all reagents used are fresh
and correctly prepared and stored.

-> identify incorrect steps and incorrect

reagents

-> repeat assay following protocol and

using correct reagents.

Blank sample exhibited coloration -> increase the number of washing cycles

-> ensure that all reagents used are fresh

and stored at optimal temperature.

-> ensure that protocols are thoroughly

followed and aseptic techniques should
be observed.

-> ensure that all equipment used were

properly calibrated

-> perform substrate incubation in the

dark

-> ensure that the plates are not

contaminated.
 Strong enzyme signal detected (high -> review the protocol; retrace steps
absorption)
-> reduce the concentration of primary
and secondary antibody/antigen.

Slow color development -> ensure that plates and reagents are
stored at room temperature.

-> ensure that all reagents and samples

are freshly prepared.

-> ensure that all reagents and samples

are at optimal pH.

Conclusion:

Enzyme-linked immunosorbent assay is a laboratory test that detects substances

including antibodies, antigens, proteins and hormones. It is commonly used to determine
if a patient has an infectious or an autoimmune disease wherein a specific antibody
connected to the illness is found in the blood. Results of this test are shown by the
measure of color that appears in the wells. There are four types of ELISA; (a) Direct ELISA
that uses a single primary antibody to detect an antigen; (b) Indirect ELISA that has the
same steps as direct but has secondary antibody; (c) Sandwich ELISA that uses matched
antibody pairs; and lastly, (d) Competitive ELISA which measures target substance by
detecting signal interference. In this activity, a virtual laboratory of indirect ELISA was
done.

The case done was testing 3 different patients to detect an antibody whether the
patients may or may not have Systemic Lupus Erythematosus. The results yielded false
positives compared to the correct plates where Patient A and B likely to have the SLE.
The result that yielded false positives may be because of the step/s that were not done
properly including washing the ELISA plates. The missed step can produce cross-
contamination resulting in a false positive. Therefore, since this test has delicate
procedures, it is important to follow it thoroughly to get the best result possible.
References:

• Alhajj, M., & Farhana, A. (2022, February 2). Enzyme Linked Immunosorbent
Assay. Nih.gov; StatPearls Publishing.
https://www.ncbi.nlm.nih.gov/books/NBK555922/
• Bio-Rad Laboratories. (2023). ELISA: Helpful Hints. Retrieved February 26, 2023,
from Bio-Rad Laboratories: https://www.bio-rad-antibodies.com/helpful-elisa-
hints.html
• Bio-Rad. (2015). ELISA Troubleshooting & Advice | Bio-Rad. Bio-Rad.
https://www.bio-rad-antibodies.com/elisa-troubleshooting.html#high
• Bio-Rad. (2015). Types of ELISA | Bio-Rad. Bio-Rad. https://www.bio-rad-
antibodies.com/elisa-types-direct-indirect-sandwich-competition-elisa-
formats.html
• Creative Diagnostics. (2023). Practical Tips of ELISA. Retrieved February 26,
2023, from Creative Diagnostics: https://www.creative-diagnostics.com/Practical-
Tips-of-ELISA.html
• Direct and Indirect ELISA. (2016). Thermofisher.com.
https://www.thermofisher.com/ph/en/home/life-science/protein-biology/protein-
biology-learning-center/protein-biology-resource-library/pierce-protein-
methods/overview-elisa.html#10
• ELISA Blocking Buffers and Reagents | Thermo Fisher Scientific - PH. (2023).
Thermofisher.com. https://www.thermofisher.com/ph/en/home/life-
science/antibodies/immunoassays/elisa-kits/elisa-blocking-buffers-reagents.html

• Mabtech. (2023, January 17). Step-by-step Guide to ELISA. Retrieved March 6,

2023, from Mabtech: https://www.mabtech.com/knowledge-hub/step-step-guide-
elisa
• Science Squared. (2018, February 14). Troubleshooting a Faulty ELISA. Retrieved
March 6, 2023, from Bite Size Bio: https://bitesizebio.com/37935/troubleshooting-
faulty-
 elisa/#:~:text=The%20blank%20wells%20on%20the,analyte%20you%20want%2
0to%20assay.
• Thermo Fischer Scientific. (2023). Overview of ELISA. Retrieved February 26,
2023, from Thermo Fischer: https://www.thermofisher.com/ph/en/home/life-
science/protein-biology/protein-biology-learning-center/protein-biology-resource-
library/pierce-protein-methods/overview-elisa.html
• Thermo Fisher Scientific. (2023). ELISA Data Analysis. Retrieved March 6, 2023,
from Thermo Fisher: https://www.thermofisher.com/ph/en/home/life-
science/protein-biology/protein-biology-learning-center/protein-biology-resource-
library/pierce-protein-methods/overview-elisa/elisa-data-analysis.html