1.1.Objective myeloma cells were fused using centrifugation

1.1.Objective

To
produce and understand the usage of monoclonal antibodies in identification of Human
Immunodeficiency Virus

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1.2. Background
information

Although
government agencies and health organization (Stefano
Bertozzi, 2006) intervened, HIV has continually spread to all regions of
the world. Of the accumulated 38.6 million people infected with HIV worldwide up
till 2006, 25 million of those affected have died (Viviana Simon, 2006). The causative agent of death is due to the
development of AIDs in HIV infected people. This fatal disease leads to immune
system break down and infected personals becomes susceptible to wide spectrum
of infections (Boyd JE, 1992).

Infections
are able to proliferate in vivo due to the ability of HIV to infect T helper
lymphocyte, monocytes, macrophage and white blood cells progenitors in bone
marrow (Boyd JE, 1992). This leads to the
defect in cellular function mediation of lymphocytes, hence, immune system of
infected host is unable to respond to new and old pathogens.    

2.     
Methods (213 words)

2.1. Production of
hybridoma cells through cellular fusion

Mouse B cells and myeloma cells were fused
using centrifugation technique and pellet was suspended in polyethylene glycol.
After suspension of pellet, RPMI was added slowly and tube was rocked gently.
Tube was stood at 37? followed by centrifugation. Pellet was extracted and
suspended in HAT medium. Supernatant was plated out and incubated at 37? in 5%
CO2 for 8 days.

2.2. Screening for
antibody producing hybridomas

After incubation, supernatants were
spotted across nitrocellulose paper. Plate containing supernatants was kept and
stored at -20? for further use. Nitrocellulose paper was blocked in
PBS/tween/milk power and washed in PBS/tween. After washing, nitrocellulose
paper was conjugated with horse radish peroxidase. An additional wash was performed
followed by an addition of substrate solution (diaminobenzidine
tetrahydrochloride, hydrogen peroxide in PBS). Wells containing antibody
producing hyridomas were recorded.

2.3. Identification of
specific HIV antigen on Western strip

Pre-blotted western strips (HIV antigens)
were blocked with PBS/milk powder/tween and antibody producing hyridomas
(3wells) were pipetted onto respective western strips. After blocking was
performed, western strips were washed with PBS/tween. Following the wash,
diluted secondary antibody conjugate was added and incubated. Another wash was
performed using PBS/tween and strips were rinsed in tap water. Substrate
solution was added to western strips and reaction was allowed to take place.

3.     
Results

3.1. Growth check of
fusion culture under inverted microscopy (205 words)

To provide evidence of successful
generation of hybridomas, microtiter plate containing supernatants was placed
under inverted microscopy.

Figure.1 View
of well A4 under inverted microscope. Presence of Hybridomas could be seen in
HAT solution.

 

Microtiter
plate (Figure.1) showed abundance growth of hybridomas in HAT solution. Successful
production of hybridoma cells were cultured from fusion of mouse B cells and
myeloma cells.

3.2. Dot blot analysis

Figure.2 Dot blot assay of
nitrocellulose paper dotted with sera from microtiter hydridoma culture. Wells
containing antibody producing hybridoma were labelled. Positive and negative
control were performed at cell 63 and 64 respectively.

 

Hybridomas
were tested for antibody production by dot blot assay. Figure.2 shows the
result yielded from dot blot assay of hybridoma spotted onto nitrocellulose
paper. Positive “brownish dots” were observed on nitrocellulose paper which
were spotted with supernatants from wells A4, A11, B10, C10, D3 and D12. Out of
the 56 wells prepared in microtiter plate, 6 wells contained antibody producing
hybridmas, the fusion efficiency is 10%. 3 wells were selected for
identification of HIV antigen on 3 pre-blotted western strip.

3.3.Characterization
of antibody specificity by western blot strips

Figure 3. Pre-blotted western
strips are shown along with positive and negative control. Western strips are
incubated with sera from well B10, A4 and A11 respectively. Band development
and their respective HIV antigens are labeled above.

 

4ul of sera taken from
wells B10, A4, A11 was sufficient to produce result as shown in Fig.3. HIV
gp120, HIV p66-pol and HIV gp41 proteins were identified by antibody producing
hybridomas from wells B10, A4 and A11 respectively. Positive control strip showed
all 4 HIV antigen proteins (gp120, p66-pol, gp41 and p24) while negative
control strip showed no antigenic identification.

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