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Group 5 - Activity 1 - BCM 3201L-1
Group 5 - Activity 1 - BCM 3201L-1
I. Objectives
II. Introduction
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.
https://www.biointeractive.org/classroom-resources/immunology-virtual-lab
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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):
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
4. Detection reagents are reagents used to detect and quantify the presence
of a specific analyte in focus in ELISA .
(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:
LABORATORY WORKSHEET #1
ENZYME-LINKED IMMUNOSORBENT ASSAY
Name: Bornea, Richard Christopher C. Instructor: Mr. Rainier Ulrich Velasco
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:
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.
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).
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:
No enzyme signal detected (low -> review the protocol; retrace steps,
absorption)
-> ensure that all reagents used are fresh
and correctly prepared and stored.
Blank sample exhibited coloration -> increase the number of washing cycles
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.
Conclusion:
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