CELL AND MOLECULAR BIOLOGY

LABORATORY

Enzyme-linked
Immunosorbent Assay
(ELISA)
GROUP 2:
ERMINO, Andrea Alexa Joan V.

MORALES, Kaye S.

Sanchez, Shellou Grace G.

Experiment date: August 29, 2019

Submission date: September 5, 2019

Submitted to:

Mr. Paolo Antonio S. Fudalan

INTRODUCTION

In the course of advancement in science and technology, scientists introduced

more profound findings and explorations in terms of diagnosis. They have applied the
basic principles of anti-body mediated immunity to an assay for detecting infection by
specific organisms. This assay is called an ELISA. ELISA stands for Enzyme-linked
Immunosorbent Assay. It is based on the principle that antibodies produced in response
to pathogens attached to their antigen targets with great specificity to form antigen-
antibody complexes. The enzyme-linked assay is a commonly used analytical
biochemistry assay, first described by Engvall as one antibody with great particularity for
an antigen. The sample with an unknown amount of antigen is immobilized on a solid
support either non-specifically or specifically.

There are two types of ELISA tests - direct ELISA and indirect ELISA. Indirect
ELISA is used to detect infection or diseases through the testing of a patient's blood for
the presence or absence of antibodies against a particular antigen. The presence of
such antibodies indicates that the individual has been infected and that the body has
launched an immune response against the disease-causing agent.

This experiment on ELISA helps in the detection of antibodies present in the

simulated samples of random patients from A to F. This antibodies are antibodies that
aids in the diagnosis if that certain patient is diagnosed with a human immuno
deficiency virus or what is common known as HIV. If the sample, containing the
chemicals or reagents, is added with chromagen and will turn its color to violet then, it
would indicate that the patient is positive of HIV. This simply states that there are
antibodies for HIV acting upon the patient.

The objective of this activity is to have a simulation on the actual experience of

utilizing ELISA. It also helps in the distinguishing of antibodies in a given sample that
could be a way for one to detect diseases present in the body.
MATERIALS AND METHODS

The materials used in the experiment were the following: microtiter plates, where
the samples will be placed; plastic pipets to be used in pipetting, measuring and placing
the samples and reagents into their appropriate designations; the chemicals or reagents
such as the HIV antigen, secondary antibody, chromagen, and the positive and negative
control and lastly, the patient samples from A to F that were tested.

In the conduct of our experiment, we first read the profiles of the different patients
ranging from A to F then, assumed some of the possible patient samples that are likely
to contain an HIV antibody. Second, we then proceeded to the experiment proper.
Using a plastic pipet, we carefully administer a 20ml drop of Patient samples in each
well of columns A, B and C with the rows from A to F of the microtiter plate. We then
placed the positive and negative controls on the second to the last and the last rows of
the microtiter plate in which we labeled as row G and H to serve as our basis of
succession. With the use of the plastic pipets, we carefully pipetted 20ml amounts of
HIV antigen and placed in each designated wells from A to H. Next, was the pipetting of
the secondary antigen and same with the latter step, we then dispensed it on the
designated wells from A to H. The last step was the addition of the chromagen that
would indicate the presence of the antibody by turning the whole sample into color
violet. It was also dispensed to all designated wells from A to H. The amount of
chemicals or reagents added were measured by 20ml each.

GUIDE QUESTIONS

12. The basic principle of antibody-mediated immunity that is utilized in an ELISA assay
states that antibodies are produced in response to infection and that these antibodies
are designed to specifically target particular antigens and bind lightly to them.

13. Indirect ELISA test detects infection by assaying patients’ blood serum for antibody
molecules designed to target the infectious agent in question.
14. Antigen-antibody complexes formed in the initial steps of and ELISA are not visible
to the unaided eye. Therefore, a colorimetric detection system involving a secondary
antibody and chromagen is employed. The secondary antibody, which is conjugated to
an enzyme, recognizes and binds to primary antibodies of antigen-antibody complexes
if they are present. Chromagen substrates is then added; if present, the enzyme linked
to the secondary antibody changes the color of the chromogen. A color change will then
appear to signify if the sample provided was positive or negative.

15. Performing three identical trials ensure to get rid or rule out of any false negatives or
positives. Another is to justify that there are still the presence of antigens in the
samples.

17. The human immunodeficiency viruses (HIV) are two species of Lentivirus (a
subgroup of retrovirus) that causes HIV infection and over time acquired
immunodeficiency syndrome (AIDS). AIDS is a condition in humans in which
progressive failure of the immune system allows life-threatening opportunistic infections
and cancers to thrive. Without treatment, average survival time after infection with HIV
is estimated to be 9 to 11 years, depending on the HIV subtype.

In most cases, HIV is a sexually transmitted infection and occurs by contact with
or transfer of blood, pre-ejaculate, semen, and vaginal fluids. Research has shown (for
both same-sex and opposite-sex couples) that HIV is untransmissible through
condomless sexual intercourse if the HIV-positive partner has a consistently
undetectable viral load. Non-sexual transmission can occur from an infected mother to
her infant during pregnancy, during childbirth by exposure to her blood or vaginal fluid,
and through breast milk. Within these bodily fluids, HIV is present as both free virus
particles and virus within infected immune cells.

18. According to the Department of Health and Human Services, to reduce the risk of
HIV infection is to use condoms correctly, don’t inject drugs and if so, use only sterile
injection equipment and water and never share your equipment with others. If not an
HIV positive but are at high risk of becoming infected with HIV, talk to your health care
provider about pre-exposure prophylaxis (PrEP). PrEP involves taking a specific HIV
medicine every day to reduce the risk of HIV infection. For those who are not yet
infected, scholars and experts of the study highly encourage the people to follow the
ABCD steps. First, abstain from casual sex, Second, Be Mutually-Faithful to your
partner. Third, Condom Usage and Fourth, Detect Early. People who are infected
already are undergoing treatment and taking medicines as prescribed by the doctors.
They should keep their body healthy as they are prone to different diseases. Moreover,
they should also stay in a place where they are safe from any harm or transmission of
diseases either due to place or people.

Results

Before we started the experiment, we, the analysts inferred that Patients A, D
and F are positive patients while Patients B, C, and E are negative for the infection. And
here are the results of the experiments.

SAMPLE COLOR TEST RESULT

Postive Control Purple Positive
Negative Control No Color Negative
Patient A Purple Positive
Patient B No Color Negative
Patient C No Color Negative
Patient D Purple Positive
Patient E No Color Negative
Patient F Purple Positive
DISCUSSION

With the given situations, Patients A, D and F are more likely to be infected. For
Patient A, she is sexually active with his boyfriend who's positive with the infection. So
there's a high tendency that she will be infected though they were protected. Patient B
for us is negative, deep kissing cannot transmit the virus though saliva is a body fluid
and even if we say the partner has cut while kissing, it is not likely to transmit the virus
from one person to another. Patient C is also negative. It is not true at all time that
attending to a patient with HIV will directly infect you. Though he is dealing with blood
and he has cut, the contact between the two is not directly to the bloodstream of blood.
Patient D is positive. A polygamous partner will likely get the virus. The more partners
you have, the more likely you are to have a partner with poorly controlled HIV or to have
a partner with a sexually transmitted disease (STD). Both of these factors can increase
the risk of HIV transmission. Patient E is negative, he maybe once a partner of the
victim, big tendency that his partner is infected, most unlikely to happen prior to their
break up. Patient F is positive. Sharing injection drug equipment (works), such as
needles, with someone who has HIV will mainly spread the virus.

There's no cure for HIV/AIDS, but many different drugs are available to control
the virus. Such treatment is called antiretroviral therapy, or ART. Each class of drug
blocks the virus in different ways. ART is now recommended for everyone, regardless of
CD4 T cell counts. It's recommended to combine three drugs from two classes to avoid
creating drug-resistant strains of HIV. Doctors will monitor the viral load and CD4 T cell
counts to determine the response to HIV treatment. CD4 T cell counts should be
checked every three to six months. Viral load should be tested at the start of treatment
and then every three to four months during therapy. Treatment should lower your viral
load so that it's undetectable. That doesn't mean your HIV is gone. It just means that the
test isn't sensitive enough to detect it.

The enzyme-linked assay uses a solid-phase enzyme immunoassay (EIA) to

detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies
directed against the protein to be measured. ELISA has been used as a diagnostic tool
in medicine, plant pathology, and biotechnology, as well as a quality control check in
various industries.

In the simplest form of an ELISA, antigens from the sample are attached to a
surface. Then, a matching antibody is applied over the surface so it can bind to the
antigen. This antibody is linked to an enzyme, and in the final step, a substance
containing the enzyme's substrate is added. The subsequent reaction produces a
detectable signal, most commonly a color change.

GENERALIZATION

ELISA (Enzyme-linked assay) is a helpful tool especially in the field of medicine.

Since it can detect infectious diseases. With that, scientists and doctors will be given an
idea on how to improve their works and studies. But the test has limitations, it can be
true positive, false negative and false positive results. Commonly, the results it
produces are seconded by other tests to confirm its validity. ELISA is often used as a
screening tool before more in-depth tests are ordered.

HIV is a virus that targets and alters the immune system, increasing the risk and
impact of other infections and diseases. Without treatment, the infection might progress
to an advanced disease stage called AIDS. However, modern advances in treatment
mean that people living with HIV in countries with good access to healthcare very rarely
develop AIDS once they are receiving treatment.

AIDS can open the door to a range of infections known as opportunistic

infections that pose a severe risk to health. Some are extreme or prolonged
presentations of infections that would normally resolve quickly in a person with healthy
immune function. And here's ELISA that helps a lot in determining this infectious
disease that can harm many people.
REFERENCES

2016. Sinco, A. Cell and Molecular Biology Lab Manual

ELISA. 2019 Sep 4.

Overview of ELISA.

Prevent HIV with ABCD.

The Basics of HIV Prevention Understanding HIV/AIDS. 2019 Apr 29.

APPENDIX

Scenarios

 Patient A has just learned that her ex boyfriend was diagnosed with the
infection. Although they often used protection when sexually active, she is
concerned that she could have contracted the virus from him.

 Patient B is the current boyfriend of Patient A. Although they are not sexually
active, they have engaged in deep kissing. Patient B is concerned for his
girlfriend and for himself, and decides to get tested along with her.

 Patient C is an EMT who treated a bleeding car accident victim's injuries, he

realized that his glove was torn and his hand cut.

 Patient D just broke up with his boyfriend after learning about several lies he
told her. She worries that he has not been monogamous and decides to be
tested for the infection

 Patient E is simply married with young children when he gets a call from an
old college girlfriend who is positive for the infection. She doesn't know when
she contracted the virus and is contacting all her old partners so that they can
be tested as well.

 Patient F experimented with drugs during collegr anf used some intravenous
substances. Although he has been clean and sober for years, he remembers
sharing a needle with a friend at a social function and decides to be tested for
the infection.
 Picture 1. A Untested samples provided

Picture 1.B Results of the experiment proper