Biology Department

College of Arts & Sciences Date submitted:

BIOL 117L – Immunology Group No.:
Author: MRB Dacar & NM Odchimar
Members:

EXERCISE 1: IMMUNOLOGICAL TECHNIQUES

Immunology is the study of the immune system that deals with how our immune system protects
us from infection through various lines of defense, the basic notions of immunity,
immunoglobulins, and antibody production. Immunological techniques remain an important and
widespread technology in detection, identification, measuring and characterizing immune
responses. It includes both experimental methods to study the immune system and methods to
generate or use immunological reagents as experimental tools.

Enzyme Linked Immunosorbent Assay (ELISA), a sensitive method to quantify or test for the
presence of antigen or ligand. ELISA works on the principle that specific antibodies bind the target
antigen and detect the presence and quantity of antigens binding. Flow Cytometry, a technique
used to detect and measure physical and chemical characteristics of a population of cells or
particles. Its main principle is based on scattering of light and emission of fluorescence.
Immunoblotting, an in vitro technique for detecting specific proteins using antibodies and based
on the principle of immunochromatography where proteins are separated into polyacrylamide gel
according to their molecular weight.

TASK 1: Watch the following video links provided and answer the corresponding questions at the
end of this activity.
Enzyme Linked Immunosorbent Assay
The Enzyme Linked Immunosorbent https://www.youtube.com/watch?v=zR_xlV5v_f4
Assay
The Principle of Immunoassays/ ELISA https://www.youtube.com/watch?v=Svoipyl6IRc
Quantitative ELISA https://www.youtube.com/watch?v=849HN1ueUhs
Flow Cytometry
Intro to Flow Cytometry https://www.youtube.com/watch?v=mcnFTjcmykE
Flow Cytometry Animation https://www.youtube.com/watch?v=EQXPJ7eeesQ
Immunoblotting
Western Blot Protocol https://www.youtube.com/watch?v=3LXrOEdwPlA
Western Blot (WB) Visual Protocol https://www.youtube.com/watch?v=uFu8aie4QFI

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TASK 2: Perform ELISA simulation via The Virtual Immunology Lab (separate paper for the image outputs)
In this activity, do this task twice with both results recorded (PRINTABLE SUMMARY
PAGE)

1. Copy and paste this link: https://media.hhmi.org/biointeractive/vlabs/immunology/index.html to

your own browser and provide a screenshot (Label as Image 001<FamilyName>).
2. Follow the instructions on the screen.
3. On a separate sheet of paper (for taking notes) write down all the steps in completing this activity.
4. Provide a screenshot image of your ELISA Plate after step 3&4 (Label as Image
002<FamilyName>).
5. Provide a screenshot image of your ELISA Plate after step 11 (Label as Image 003<FamilyName>).
6. Provide a screenshot image of last feedback provided by The Virtual Immunology Lab (Label as
Image 004<FamilyName>).

TASK 3: Perform Flow Cytometry simulation via Learn.Genetics (separate paper for the image outputs)

1. Copy and paste this link: https://learn.genetics.utah.edu/content/labs/flowcytometry/ to your own

browser and provide a screenshot (Label as Image 004<FamilyName>).
2. Click COMPLETE BLOOD COUNT and follow the instructions provided.
3. On a separate sheet of paper (for taking notes) write down all the steps in completing this activity.
4. Provide a screenshot image of the flow cytometer result (Label as Image 005<FamilyName>).

This material is owned by the Biology Department – Xavier University and it is for exclusive use. No part of this page
shall be reproduced in any form or by any electronic or mechanical means (including photocopying, recording, CD
reproduction or any information storage and retrieval system) without written permission from the Biology
Department of Xavier University
QUESTIONS:

1. Why is Phosphate Buffer Saline used as a negative control?

A negative control should never produce a positive response. The PBS contains table salt and
the amount of salt in it is about the same amount in our blood. In short, they keep the solution
from becoming too acidic and basic.

2. What is the positive control used and its purpose on the experiment?

The positive control is used in the titer of anti-DNA primary antibody. Its purpose is to produce
a positive response if the reagents and conditions are correct.

3. How step 6 and 9 affects the result of the experiment?

Steps 6 and 9 remove any antibody that did not react with SLE antigen in the wells. IF not
removed properly, the anti-human antibody added in the next step will recognize and react with
any remaining antibody in the well, whether they are specific for SLE antigen or not, and this
may lead to a false positive result.

4. What is the purpose of Horseradish peroxidase?

The Horseradish peroxidase are enzymes that can be attached to antibody. This enzyme together
with hydrogen peroxide acts on a chemical called ABTS to produce a yellow solution that is
estimated by eye or measured in a spectrometer at 414 nanometers.

5. Enumerate the factors that affects false positive result in the virtual experiment.

Factors that affect false positive result in the virtual experiment are small amounts of antigen,
reaction with non-SLE antibody, reaction of a non-SLE antibody with second antibody.

6. What are the properties that a flow cytometer can measure?

The properties that a flow cytometer can measure include the particle’s relative size, relative
internal complexity, and relative fluorescence intensity.

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shall be reproduced in any form or by any electronic or mechanical means (including photocopying, recording, CD
reproduction or any information storage and retrieval system) without written permission from the Biology
Department of Xavier University
7. What is the use of fluorescent tags on flow cytometry analysis?
Flow cytometry utilizes fluorescent antibodies to tag proteins on the outside or inside of a cell.
Flow cytometry can also utilize fluorescent dyes to stain nucleic acids whether it be DNA or
RNA.

8. In simulated activity (task 3), what is the difference on the scatter plot results of the cbc
count (our patient vs. healthy patient)?

In Task 3 Flow Cytometry, an unhealthy amount of immature white blood cells was detected
from our patient’s blood sample. In comparison to the healthy patient’s sample, immature white
blood cells are fewer and even untraceable form the test performed. Our patient’s blood sample
result suggests acute leukemia.

9. The importance of western blot analysis?

The importance of western blot is researchers are able to identify specific proteins from a
complex mixture of proteins extracted from cells.

10. What is the purpose of the protein ladder in western blot?

Western blot protein ladders are a mixture of pre-stained proteins. It enables fluorescent
visualization of protein gels and chemiluminescent or colorimetric immunodetection of western
blots.

Reflection/Generalization
It was the first exercise for this topic, and the students were requested to become familiar with the
many methods used in various scientific disciplines, particularly medicine, in order to study
immunological phenomena. The complex process of Enzyme-linked immunosorbent assay
(ELISA) tests measures the quantity of antibodies, antigens, etc. in order to identify certain
diseases that may be present in the body. On the other hand, a method called flow cytometry is
used to count numerous cells at once. Through their simulations, us students had learned that
immunology offers a different perspective to those who wish to explore the outside world,
particularly the irregularities in human health brought on by infections, bacteria, etc.

END

This material is owned by the Biology Department – Xavier University and it is for exclusive use. No part of this page
shall be reproduced in any form or by any electronic or mechanical means (including photocopying, recording, CD
reproduction or any information storage and retrieval system) without written permission from the Biology
Department of Xavier University