Antibodies are highly specific, sensitive proteins that are created by
specialized leukocytes called plasma cells. Every antibody is a Y-shaped
molecule composed of four polypeptide chains linked by disulfide bonds; two
heavy and light chains each.
Theoretically, there are about 1030 antibodies in the human
body, however, there are only about 20000 genes in the body (Nee,2017). This is
possible due to antibody diversity. This diversity arises as a result of
rearrangement of the V region in the genome of an immature B cell prior to
Antibodies have very strong
affinity for antigens, thus when the body encounters an unknown antigen, it
binds to a portion of the antigen’s surface called the epitope. In a typical
immune response, when an antibody binds to an antigen, plasma cells are
stimulated to produce antibodies specific to that antigen. Most antigens have
various epitopes, the immune system therefore uses different plasma cells to
produce antibodies that bind to different epitopes on the same antigen. These
antibodies are known as polyclonal antibodies (pAb). Monoclonal antibodies
(mAb) on the other hand, are antibodies that all come from the same type of
plasma cells all bind to the same epitope on the antigen (Janeway et al.,
mAbs have various applications in
therapy as they can recognize specific structures in targets such as viruses,
bacteria and also cancer cells (Zola, Thomas and Lopez, 2013). In order to
study mAbs, we have to isolate the specific plasma cell that produces that
antibody. However, even if one were to isolate the specific plasma cell, and
place it in a culture, it would die out soon following the removal from the
Nonetheless, mAb can be produced using hybridoma technology. This
technology aims to provide immortality to the specific plasma cells by
hybridizing them with myeloma cells. This results in a hybridoma.
of a successful fusion is extremely small, so very few hybridoma cells are
achieved. In order to select successful
hybridoma cells, the cells are grown in hypoxanthine-aminipterin-thymidine (HAT)
medium. HAT selection relies
on the principle that cells can synthesize purines and pyrimidines by two pathways:
the de novo and the salvage pathway.
Aminopterin blocks the de novo pathway. This causes the cell to use the
salvage pathway, which bypasses the aminopterin block by converting the
nucleotides into DNA directly. However, the enzymes hypoxanthine-guanthine
phosphoribosyl transferase (HGPRT) and thymine kinase (TK) are fundamental in
the salvage pathway. If a cell is deficient in either one of these enzymes, the
salvage pathway will be blocked (Luttmann et al., 2006).
The HAT medium has aminopterin, which blocks the de novo pathway, and
hypoxanthine and thymidine which allows growth via salvage pathway. When a cell
with a mutated TK and another cell with a mutated HGPRT are fused, only hybrid
cells will contain the necessary enzymes for growth on HAT medium via the
salvage pathway. Thus, only hybridomas will grow in HAT medium (Luttmann et
Since mAbs are highly specific, they are often used to target specific
antigen, which in this case is human immunodeficiency virus (HIV). HIV is a
virus that attacks specific immune system cell, CD4+ helper
lymphocyte. This damages the immune system, making it tough for your body to
fight even minor infections. HIV mainly affects young people, being transmitted
by sexual intercourse, sharing or needles and from mother to fetus. This is why
so much emphasis has been placed on research regarding this virus (Nye and
The common methods used in STD clinics to screen HIV include, ELISA,
rapid HIV test and western blot, which has been used in this experiment as well
In this experiment, hybridoma technology will be used to produce mAbs
against HIV. HIV will be further discussed in the discussion section.
Methods: Throughout this experiment, mAbs were produced, screened and mapped to antigens.
In order for mAbs to be produced, equal amounts of B cells and myeloma
cells are fused with the help of polyethylene glycol (PEG). The B cells are
obtained from the spleen of a mice that has been immunized with HIV.
After the cell fusion, they are placed in HAT medium. As mentioned in
the introduction, only hybridoma cells will survive in HAT medium. The cells in
HAT medium are cultured in microtiter wells and incubated.
After that, screening of hybridoma cells is carried out using the Dot
Blot technique. A miniscule amount of supernatant from each well was placed on
a nitrocellulose paper, including positive and negative controls. It is then
blocked with blocking solution and eventually washed with PBS/tween.
Substrate solution is then added to help visualize which hybridoma cells
are producing antibodies.
Then, the specificity of the mAbs are tested using strips of western
blot containing HIV proteins that the B cells are producing antibodies against.
Supernatant is taken from three wells which are identified to be producing
mAbs, and added to three pre-blotted strips on three separate weighing boats
along with blocking solution. It is then washed and substrate solution is
Substrate solution is removed when reaction has occurred and nitrocellulose
paper is dried.
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Figure 1 depicts the screening of mAb producing hybridoma cells via a
dot blot assay on the right. As shown in the picture, wells A3, C5, E2, E6, G4
and H1 are producing mAb, including the positive control (PC) on D7
and negative control (NC) on E8.
Supernatant from all 50 wells, including positive and negative control,
was placed on the nitrocellulose paper. PC indicated a successful experiment
and ideal result while negative indicated a fail experiment. With comparison to
the PC, the 5 wells mentioned above were proven to be producing mAb as their
supernatant was visualized as a brown spot on the nitrocellulose paper
identically to the PC. These brown spots were caused by the substrate solution.
The spots of supernatant from the other wells remained colorless, similar to
NC. This shows that the other wells were not producing mAbs.
Supernatant was taken from wells
E2, G4 and H1 to test for antigen specificity. It was placed on the western
strips that were pre-blotted with recombinant proteins of HIV antigens like;
p24, gp41, gp120.
Supernatant from well E2 is
specific to p24
Supernatant from well G4 is
specific to gp41
Supernatant from well H1 is
specific to gp120
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Discussion: Figure 1 shows the image of a dot blot assay. Dot blot assays are a
class of rapid tests that are highly specific and sensitive for detecting
antibodies to HIV. As discussed in the results section, the presence of mAb
producing hybridoma cells is depicted through the brown spots. These brown
spots are seen on the nitrocellulose paper where the supernatant was taken from
well; A3, C5, E2, E6, G4, H1. This simply means that the sera in those wells
contained cells producing mAb against HIV.
Figure 2 shows
the identification of antigen specificity of mAbs by western blot. Western blot
assay is a technique where proteins from HIV are loaded onto polyacrylamide gel
electrophoresis. This denatures the proteins and then separates them according
to size. Larger proteins migrate slowly and smaller proteins migrate quicker.
The viral proteins are then transferred onto nitrocellulose paper and then
reacted with mAb producing hybridoma cells from the wells as discovered through
the previous dot blot assay.
Any HIV antibody
detected will result in a colored band after being treated by the substrate
solution. Based on the results, supernatant
from every well was specific to different proteins.
In a western blot assay, controls are very crucial. A positive control
as shown in figure 2 will ensure that the antibody recognizes target protein. A
negative control, on the other hand, will not have any antibody, thus no
binding will occur.
It can be seen from the results and discussion that mAb and pAb are very
diverse. They differ in cross-reactivity, epitope specificity, life span(?)
This is mainly due to the technique by which mAbs are formed. While pAb are
produced by plasma cells upon antigenic stimulation, mAb are produced by hybridoma
technology. In this experiment, hybridoma technology was used to produce mAb
against key proteins of HIV.
As mentioned in the introduction, HIV is a virus that targets the T-lymphocytes.
This results in impaired organs and cells in the immune system, and basically, increasing
immunodeficiency. As the virus progresses, it can lead to diseases like acquired
immune deficiency syndrome (AIDS). AIDS is a severe disease that up till today
has no cure despite rapid scientific advancements (Whiteside, 2008).
This is why the ability to immune screen HIV is important in biomedical
sciences. Screening will allow the detection of HIV antigens or antibodies
produced against HIV in the blood. This is important as HIV might be
asymptomatic initially, and the only way to detect it will be through screening.
(Bayer and Oppenheimer, 2013).
Therefore, many measures have been taken to enhance HIV screening.
Singapore can be taken as an example; HIV testing can be done conveniently in
hospitals and clinics all over Singapore. Conventionally, HIV is tested by
taking a blood sample from the patient and sending it for an enzyme immunoassay
(EIA). If the sample indicates positive, then it has to be further screened through
western blot. This is time consuming.
Which is why, clinics now allow
HIV to be tested via rapid HIV test kits. They produce results fast, only
taking around twenty minutes.