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123
MOLECULAR BIOLOGY TECHNIQUES
Human Immunodeficiency Virus (HIV) Structure:
• HIV belonging to the Retroviridae family and Lentivirinae subfamily is a nonsegmented, single-stranded,
diploid (two identical strands of the RNA genome), positive sense RNA (ss
RNA) virus. The following
HIV genes are important in HIV testing:
• The env gene which encodes for
• gp160 (an envelope glycoprotein which is later cleaved into gp120 and gp41)
• gp120 (binds to the CD4 receptor protein)
• gp41 (a transmembrane protein)
• The gag gene (group-specific antigen) which encodes for
• p55 (the gag precursor protein)
• p24 (capsid protein)
• p17 (matrix protein)
• p9 (nucleocapsid protein)
• p7 (nucleocapsid protein)
• The pol gene which encodes for
• p65 (a protease)
• p66 and p51 (reverse transcriptase dimer)
• p31 (integrase)
Figure 14-12B HIV ELISA (enzyme-linked immunoabsorbent assay) test. The serologic tests for HIV in-
fection are based upon detection of antibodies in the serum directed against HIV proteins. The ELISA is the
first test used clinically to detect the possibility of HIV infection. Remember that the ELISA does NOT detect
the HIV virus per se but only antibodies directed against HIV proteins. The HIV ELISA test can produce false-
positive results which means that a person’s serum may contain cross-reactive antibodies to HIV proteins
even though the person has never been exposed to HIV. Sources of false positives include multiple pregnan-
cies, multiple blood transfusions, autoimmune disorders, chronic hepatitis, chronic alcoholism, hepatitis B
vaccination, influenza vaccination, rabies vaccination, renal failure, cystic fibrosis, syphilis infection, malaria
infection (important because many Africans are exposed to malaria), infection with other human retroviruses
(e.g., HTLV 1/II), association with large animals (e.g., animal trainers, veterinarians), and hemodialysis. An
ELISA includes the following steps:
• A number of known HIV proteins are attached to a plate
• A person’s serum is applied to the plate. If the person’s serum has antibodies (Abs) that cross-react with HIV
proteins, the antibodies will bind to the HIV proteins.
• Antibody binding to HIV proteins can be detected by an HRP-labeled secondary antibody which can be react-
ed with a chromogenic substrate to produce a colormetric reaction (usually brown in color).
Figure 14-12C HIV Western blot test. If a positive ELISA result is obtained, it must be confirmed by a
Western blot. Again, remember that an HIV Western blot does NOT detect the HIV virus per se but only an-
tibodies directed against HIV proteins.
• A number of known HIV proteins are separated based on size by PAGE. The separated HIV proteins are
transferred to a nitrocellulose membrane. A person’s serum is applied to the nitrocellulose membrane. If the
person’s serum has antibodies (Abs) that cross-react with HIV proteins, the antibodies will bind to the HIV
proteins. Antibody binding to HIV proteins can be detected by an HRP-labeled secondary antibody which
can be reacted with a chromogenic substrate to produce a colormetric reaction (usually brown in color).
• Patient no. 1 shows a high positive (HP) HIV Western blot reaction.
• Patient no. 2 shows a low positive (LP) HIV Western blot reaction.
• Patient no. 3 shows an indeterminate (I) HIV Western blot reaction. Almost all HIV-infected patients with
an indeterminate result will develop a positive result within 1 month (called seroconversion).
• Patient no. 4 shows a negative (N) result.
To identify the actual HIV virus, nucleic acid-based tests (e.g., RT-PCR, Quantiplex branched DNA test)
are used to detect a 142-base target sequence in a highly conserved region of the HIV gag gene. These tests
use a patient’s white blood cells because retroviruses incorporate into host cell DNA. These tests are not used
in population screening but generally later in the disease process to assess the viral load in an AIDS patient
undergoing antiretroviral drug therapy.
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CHAPTER 14
5'
3'
3'
5'
3
4
1
2
3
4
1
2
3
4
1
5
4
3
2
1
Ligated
DNA probe
template
ss DNA
template
DNA
ligase
Ligated DNA
probes
ss DNA
ds DNA
94°
denature
55-65°
hybridize
1
2
3
4
DNA Probes
Repeat cycle
● Figure 14-13
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MOLECULAR BIOLOGY TECHNIQUES
LCR is a DNA amplification technique that detects specific
DNA sequences by amplifying the DNA probes. LCR can be used to identify tumor types, to detect
single-base mutation genetic disorders (e.g., sickle cell disease), and to detect infectious diseases
(e.g., Chlamydia trachomatis, gonorrhea). LCR involves the following steps:
• Each cycle of the LCR reaction begins with 94
C heat treatment to separate the double-stranded
DNA (dsDNA) into single-stranded DNA (ssDNA), that is, denatured.
• The DNA probes include four probes (nos. 1, 2, 3, 4) one for every side of the ssDNA. The DNA
probes hybridize to the ssDNA at 55–65
C.
• As long as there are no base mismatches at the junction of the DNA probes, DNA ligase can lig-
ate each pair of DNA probes to form ligated DNA probes.
• The cycle is repeated, whereby both ssDNA and the ligated DNA probes are used as templates.
The ability of ligated DNA probes to serve as templates in subsequent cycles leads to exponen-
tial amplification.
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CHAPTER 14
Neutrophils
Monocytes
Lymphocytes
Cells
Fluid system
Laser
Side scatter
Forward scatter
Forward
scatter detector
Side
scatter
detector
Flourescent
detectors
Computer data
analysis
Optics
A
CD8+
27%
CD8+
CD4+
2%
CD8-
CD4-
2%
CD4+
68%
Orange
flourophore
CD4 Antibody
Red
flourophore
CD8 Antibody
Forward scatter
Side scatter
CD8 Antibody
CD3 Antibody
CD8+
1%
CD8-
CD3-
1%
CD8+
CD3+
85%
CD3+
6%
Cytotoxic CD8+ T cell
Leukemia
Labels
CD8+ cytotoxic
T lymphocytes
Labels
All T lymphocytes
CD20 Antibody
CD3 Antibody
CD20+
2%
CD20-
CD3-
96%
CD20+
CD3+
0%
CD3+
2%
Adenosine Deaminase
Deficiency (ADA; “Bubble Boy”)
Labels
All B lymphocyte
Labels
All T lymphocytes
CD20 Antibody
CD3 Antibody
CD20+ 80%
CD20-
CD3-
18%
CD20+
CD3+
0%
CD3+
2%
Di George Syndrome
Labels
All B lymphocytes
Labels
All T lymphocytes
CD20 Antibody
CD3 Antibody
CD20+
2%
CD20-
CD3-
18%
CD20+
CD3+
0%
CD3+ 80%
X-linked Infantile (Bruton)
Agammaglobulinemia
Labels
All B lymphocytes
Labels
All T lymphocytes
B
C
D
E
● Figure 14-14
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MOLECULAR BIOLOGY TECHNIQUES
Flow cytometry is a technique for the analysis of multiple parameters of cells
within a heterogenous population. Parameters include cell size, intracellular complexity (granules,
nucleus shape, etc.), and cell surface fluorescent staining.
• The fluidic system passes thousands of cells per second through a laser beam one at a time.
• As the cell passes through the laser, the cell will scatter light in the forward direction called for-
ward scatter. The forward scatter light is quantified by a detector which converts the light inten-
sity into a voltage pulse. The size of the voltage pulse recorded for each cell that passes through
the laser is proportional to cell size. Therefore, forward scatter allows the measurement of cell
size. This data is plotted on a histogram.
• As the cell passes through the laser, the cell will also scatter light in the side direction called side
scatter. The side scatter light is quantified by a detector (located 90 degrees to the laser path) which
again converts the light intensity into a voltage pulse. The size of the voltage pulse recorded for
each cell that passes through the laser is proportional to intracellular complexity. Therefore, side
scatter allows the identification of intracellular complexity. This data is plotted on a histogram.
• The histograms of the forward scatter (X axis) and side scatter (Y axis) can be combined to form
a two-dimensional dot plot. A two-dimensional dot plot of flow cytometry using peripheral
blood shows three populations: lymphocytes, monocytes, and neutrophils. Remember that each
dot on a dot plot represents a cell.
• A common method used to study cell characteristics using flow cytometry involves the use of
fluorescent-labeled antibodies that bind to the cell surface. When a fluorescent-labeled cell pass-
es through the laser, the laser will strike the fluorophore and a fluorescent signal will be emitted.
The fluorescent signal is quantified by a detector which converts the fluorescence intensity into
a voltage pulse. The size of the voltage pulse recorded for each cell that passes through the laser
is proportional to the amount of fluorescence emitted.
• A typical flow cytometry study uses two fluorescent-labeled antibodies. For example, a CD8
antibody with a red fluorophore identifies CD8
T lymphocytes and a CD4 antibody with an
orange fluorophore identifies CD4
T lymphocytes. The data for each antibody is plotted on a
histogram. The histograms can be combined to form a two-dimensional dot plot. A two-
dimensional dot plot of this example studying T lymphocytes in peripheral blood shows four pop-
ulations: a CD8
T lymphocytes making up
27% of the cells, CD8
and CD4
T lymphocytes
making up
2% of the cells, CD8
and CD4
T lymphocytes making up
2% of the cells, and
CD4
T lymphocytes making up
68% of the cells.
Figure 14-14B Two-dimensional dot plot of a cytotoxic CD8
T-cell leukemia. Peripheral blood
sample from a leukemia patient is run through flow cytometry using two fluorescent-labeled antibod-
ies: CD8 antibody which labels CD8
cytotoxic T lymphocytes and CD3 antibody which labels all T
lymphocytes. The two-dimensional dot plot shows four populations of cells with CD8
and CD3
T
lymphocytes making up 85% of the cells. This high percentage (85%) would be consistent with a cyto-
toxic CD8
T-cell leukemia where the patient has a large increase in CD8
cytotoxic T lymphocytes.
Figure 14-14C Two-dimensional dot plot of X-linked infantile (Bruton) agammaglobulinemia.
Peripheral blood sample from a patient is run through flow cytometry using two fluorescent-labeled
antibodies: CD20 antibody which labels all B lymphocytes and CD3 antibody which labels all T
lymphocytes. The two-dimensional dot plot shows four populations of cells with CD20
B lympho-
cytes making up only 2% of the cells and CD3
T lymphocytes making up 80% of the cells. These
percentages would be consistent with X-linked infantile (Bruton) agammaglobulinemia where the
patient basically has no B lymphocytes but only T lymphocytes present. Note that there can never
be any CD20
and CD3
cells because these two markers have been chosen to distinguish B lym-
phocytes from T lymphocytes; there can never be a B lymphocyte that is also CD3
, and visa versa.
Figure 14-14D Two-dimensional dot plot of DiGeorge syndrome. Peripheral blood sample
from a patient is run through flow cytometry using two fluorescent-labeled antibodies: CD20 anti-
body which labels all B lymphocytes and CD3 antibody which labels all T lymphocytes. The two-
dimensional dot plot shows four populations of cells with CD 20
B lymphocytes making up 80%
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