ELISA Activity

NAME: Mel Florante P. Avenido

Text SECTION: 2MT-K

DATE:29/4/2022

1.What is the role of the substrate? Is it really necessary to add substrate in ELISA? Explain why, or why not.
- The enzyme-linked immunoassay (ELISA) is a kind of ELISA. It is an essential approach for detecting antibodies
in blood that is commonly used in laboratories . It is necessary to add substrate because it relies heavily on
the substrate. The substrate must be very sensitive in order to achieve excellent detection. Colorimetric ELISA
enzyme-substrate reactions produce soluble products with a spectrophotometer-measurable absorbance
(optical density).

2.What is the label used in ELISA? Can we use different label? Justify your answer.

- Horseradish peroxidase (HRP) and alkaline phosphatase are the most often utilized enzyme labels (AP). Other
enzymes such as -galactosidase, acetylcholinesterase, and catalase have also been employed. Commercially
available substrates for performing ELISA with an HRP or AP conjugate are plentiful. The sensitivity of the test
and the instrumentation available for signal detection (spectrophotometer, fluorometer, or luminometer)
influence the substrate selection.
Yes, we can use different label because it is versatile many primary antibodies can be made in one species and
the same labeled secondary antibody can be used for detection.

3.Explain elaborately the principle involved in ELISA.

- In an ELISA test, different antigen-antibody combinations are employed, always with an enzyme-labeled
antigen or antibody, and enzyme activity is colorimetrically assessed. The enzyme activity is determined by
changing the color of a substrate when it is modified by the enzyme. The product's light absorption after
adding the substrate is measured and translated to numerical values. The assay is referred to as a direct
ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, and so on, depending on the antigen-antibody
combination.

4. What are the different applications of ELISA?

The ELISA can be applied in laboratory processes such as

- In a viral test, to assess the concentration of serum antibodies.

- Rapid testing kits are used to assess the presence of antibodies in blood samples to analyze the spread of the
disease, such as during the current COVID-19 epidemic.

5.Explain the positive and negative results of ELISA.

- An ELISA test works by attaching specialized enzymes to antibodies in your blood. A sample of your blood is
mixed with a recognized substance on specific absorbent plates for the test. The test can use a variety of
enzymes and discover a variety of antibodies, depending on your doctor's diagnosis. The enzymes on the plate
will bind to the antibody your doctor is seeking for if your blood contains it. The plates change color in
positive tests, but not in negative tests. The lab can determine whether you have a specific condition based
on the change. They can even evaluate the severity of the illness in some circumstances.

6.Why do you think it is called indirect ELISA?

- Indirect ELISA gets its name from the fact that the secondary antibody, which carries the enzyme that
produces color when it combines with the chromogenic substrate, binds to the primary antibody that is
coupled to the antigen rather than the antigen itself. The antigens that are exposed to primary antibodies are
coated in the wells in indirect ELISA. After that, secondary antibodies that will bind to the primary antibodies
are introduced. After that, the secondary antibodies are exposed to a color-producing chromogenic substrate.

As a result, the "detection antibody" detects the antigen through the interaction of the main antibody and is
considered to be "indirectly" linked to it. Because different colors absorb light at different wavelengths, we
may compare the color's absorption to a standard curve made with known analytes to determine the
 quantity of the analyte.

7.What are the different solid phases that antibody or antigen can bind to, in ELISA?

- Fusion-tag binding plates its coating is Glutathione (GST tag binding) or nickel or copper coated (His tag
binding) and its application protein-protein interactions or genetically engineered fusion proteins.

- Antibody binding plates its coating is Protein A, G, L or A / G and its application is it binds to the FC region (VL
for protein L) of capture antibodies to properly orient while leaving antigen binding capability

- Modified polymer surfaces its coating is its various modifications to increase hydrophobicity or hydrophilicity
of the plate and its application is it increase passive binding of molecules based on their physical and chemical
features

- Biotin-binding plates its coating is Streptavidin or neutravidin and its application is it binds small molecules
(sometimes biotinylated) that are difficult to bind by passive adsorption

8.What are the different materials needed in the experiment?

- Primary antibodies, secondary antibodies (in case of indirect or sandwich ELISA), blocking buffers, wash
buffers, and equipment to assess the color parameter are all required (chromogenic a standard absorbance
plate reader; fluorescence: fluorometer; chemiluminescence: luminometer plate reader)

9.What is the importance of washing? When does washing is performed?

- Washing is critical because it prevents unbound antigens from attaching to unbound antibodies, causing a
false positive result. This step is performed after adding any antibody (primary or secondary) or sample to the
well to avoid free floating antibodies or antigens from binding to one another and producing a false positive
test result. Following these methods, each well is usually cleansed four times.

10. Assuming that these are the 12 microplates. What is wrong with the result of the test? What do you think
thecause of this occurrence? What should the medical technologist do?
- The GC patient's sample should be redone by the medical technologist. In ELISA, each sample is normally
tested three times to ensure accuracy and precision. The positive controls are found in the first three wells,
whereas the negative controls are found in the next three wells. Both are acceptable since we detect color in
the positive controls (indicating that the ELISA assay is functioning) and no color in the negative controls
(indicating that the ELISA assay is not functioning) (meaning that there was no signs of contamination).

In this example, we have two patients: GC and AA. We can observe that the three wells assigned to AA's
sample all return negative findings, indicating that the chemical was not found in this patient's sample.
However, one of GC's samples does not appear to be consistent. Two of the wells produced from the same
sample were colorless, indicating that the analyte was most likely not identified in GC's sample. One of them,
however, is positive, which contradicts the results of the other two samples. The medical technologist most
likely did not thoroughly wash the samples, causing free-floating antigen to bind to free-floating antibodies,
resulting in a false-positive result. The medical technologist should retest GC's sample since the quality
control (negative and positive controls) were satisfactory.

References:

Overview of ELISA Thermo Fisher Scientific – PH April 29, 2022

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#:~:text=Versatile%20because%20many%20primary%20antibodies,because%20it%20is%20not%20labeled.

What is ELISA? - Types, Procedure, Principle, and Applications BYJUS December 28, 2021
https://byjus.com/biology/elisa-
technique/#:~:text=Applications%20of%20ELISA&text=To%20determine%20the%20concentration%20of,antibodies%20in%20the%
20blood%20sample.
The ELISA Test: What It's For, When It's Necessary, and More WebMD

https://www.webmd.com/a-to-z-guides/what-is-elisa-test

The principle and method of ELISA, The principle and method of ELISA | MBL Life Science -JAPAN-

https://ruo.mbl.co.jp/bio/e/support/method/elisa.html#:~:text=In%20ELISA%2C%20various%20antigen%2Dantibody,when%
20modif ied%20by%20the%20enzyme

ELISA Substrates
https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/enzymes-substrates/elisa-substrates#:~:text=The%20substrate
%20is%20a%20crucial,be%20measured%20in%20a%20spectrophotometer.