Enzyme Linked Immunoabsorbant Assay: Detection of Antibodies and Its Applications

This presentation provides an overview of Enzyme Linked Immunoabsorbant Assay (ELISA) and its applications in detecting antibodies or antigens in clinical practices. It explains the steps and materials required for performing an ELISA test. The presentation also includes the results and discussion of an experiment to detect mock HIV antibodies.

The + and – control in the result are the determine what the + and – result looks like. The +ve
control row changed its color to brown and the and – result looks like. The +ve control row
changed its colour to brown and the -ve control row remained the colorless in results.
Moreover the coloru of the wells of DS1(Row 3) matched with the –ve control wells
indicating wells of DS1(Row 3) had –ve result for this ELISA test. This leads to the
conclusion that the donor serum 1 did not had the protein that we were detecting our ELISA
test which is the mock HIV Ab. Due to this, the 20 Ab had nothing that it could bind with and
change the colour of the substrate from colorless to brown. On the other hand, the wells of
donor serum 2(Row4) was brown in color which match with colour of the wells in the +ve
control row. This result in the conclusion that my DS2 show the result for the ELISA test that
means the DS2 (Row4) wells has mock HIV Ab that we were detecting in our experiment
which binded to the epitope of the immobilized HIV Ag. The Anti IgG enzyme conjugate 20 Ab
that added to the wells binded with specific sites on these present mock HIV 10 Ab in the
well of DS2 row which resulted the reaction of the enzyme attached to the 20 and
substrate added and thus colour changes.
Enzyme Linked Immunoabsorbant Assay
Author list and affiliation
Materials and Methods
Results and Discussion
Subsection Title
Conclusion
Summary
- Showing the interaction between enzyme and substrate.
- Based on binding of antibody and antigen.
- Detecting the mock HIV antibody
References Cited
Enzyme-linked immunosorbent assay (elisa). (2018). Retrieved from
https://www.ukessays.com/essays/sciences/enzyme-linked-immunosorbent-assay-elisa.php
Hosseini, S., Vázquez-Villegas, P., Rito-Palomares, M., & O. Martinez-Chapa, S. (2018).
General Overviews on Applications of ELISA. In Enzyme-linked Immunosorbent Assay
(ELISA). Singapore: Publisher Name Springer.
Problems and Solutions in ELISA Experiments-CUSABIO. (2018). Retrieved from
https://www.cusabio.com/c-15109.html
Introduction
The ELISA- Enzyme Linked Immunoabsorbant Assay is used for the detection of antibody in the blood. It is based on
he specificity of antibody-antigen interaction. It works on the principle enzyme substrate interaction. It helps in
diagnosis like cancer infectious diseases and transplantation drugs. It is very sensitive test. ("Enzyme-linked
mmunosorbent assay (elisa)", 2018)
This test has many application like in food industry, pregnancy test, cancer detection, toxicology and transplantation.
Hosseini, Vázquez-Villegas, Rito-Palomares & O. Martinez-Chapa, 2018)
Here, we are going to perform the ELISA test to separate the antibodies (10 20) for the detections and ELISA test that
here are performing have 10Ab and 20 Ab in which 10Ab is the one we are finding.
The question being investigated with this experiment is whether the patients' serum contains the mock HIV antibodies in
t or not?
Predicted results
The donor serum does contain a mock HIV antibody in it, then there will be coloured final product(substrate) because
he mock HIV antibody binding with the adsorbed antigen (HIVAg). If, there are no HIV antibody (Ab) in the donor
erum, then there will be no colour in the final product(substrate) because there were no mock HIV antibody bonded
with the antigen(Ag ).
Objective
The aim of this experiment is to determine/detect which donor serum contains the mock HIV antibody and understand
how the immunodetection technique – ELISA works to detect antibodies or antigen in the clinical practices
Following methods used for the experiment:
Step 1: Adsorption of HIV Antigen on the well
Step 2: then add human serum and binding of human serum
Step 3: addition of Anti-IgG (Rabbit) and horseradish peroxidase enzyme, binding of 20
Antibodies.
Step 4: get colourless substrate and coloured product
Procedure
We have used following step for performing the experiment
Step1: Incubate a protein mixture containing antigen in the well of a microlite plate.
Step2: the 10Ab which is specific to the antigen is incubated in the wells of the plate.
Step3: the 20 Ab is covalently linked to the enzyme so that it can
detectable.
Step 4: A colourless substrate of the enzyme is added to the wells.
Four types of
ELISA
•
• Figure: Types of ELISA ("Problems and
Solutions in ELISA Experiments-CUSABIO",
2018)
Steps for
ELISA
Test
•
Material
required
• Microlite plate Mock HIV antigen(HIV Ag)
• Primary HIV Antibody(10AB ; Positive Control IgG)
• Anti -IgG – Peroxidase – Conjugate Secondary Antibody(20 AB)
• Peroxidase – Enzyme Substrate(sub)
• Transfer Pipets P-1000 and P-200 Micropipettes
• Phosphate buffer saline(PB5), Donor serum 1 and 2(DS1 and DS2)
• 370c Incubator ,Student waste beaker
A) B)
Figure A: Showing ELISA Test results Figure B: Diagram for ELISA test results
.
As result of this experiment suggest that DS2 serum contains the antibody for HIV antigen and
therefore contain HIV itself. The DS2 wells were all brown in colour and matching with the
positive control wells. This result in the determination that DS2 had HIV antibodies in it which
binded with the HIV antigen and leads to change of colour of the substrate. On the other hand,
DS1 did not change the colour and was colourless. It was matched with the negative control
indicating that DS1 did not have HIV antibody for attaching with the HIV antigen.
It also indicates that when antigen binded to the antibody in the reaction of enzyme- substrate
than it changes the colour from colourless to brown. When there are no HIV antibodies to
attach with the HIV antigen and the therefore, no reaction between enzyme- substrate. That is
why colour of the substrate remained as it was earlier – colourless.