MCB 150L - Unit 1 Questions With Complete Solutions
3H thymidine incorporation assay Correct Answers -
radioactive nucleotide (3H-thymidine) incorporated into
DNA of dividing cells
- cells are incubated with 3H thymidine for 12-48 hours and
then lysed to harvest DNA onto fiber filters
- relative amount fo radioactivity absorbed onto the filter
paper is measured as counts per minute (cpms) on a
scintillation counter
- relative amount of cpms indicates the relative amount of
3H-thymidine incorporated into cells
ex: can test human peripheral blood mononuclear cells
(PBMCs) for reactivity to SARS-CoV-2 and control
51 Cell release assay Correct Answers target cells are
"labeled" with chromium and then effector cells are added
in different ratios to get a % lysis (% of target cells lysed by
effector cells)
effector cells: CTLs or NK cells
target cells: viral infected cells, tumor cells
challenges:
1) high material costs
2) handling and disposal of radioactive 51Cr
3) difficult to standardize
,ACE2 receptor binding domain (RBD) Correct Answers -
homotrimers of the S protein make up the distinctive spike
structure on the surface of the virus and is the main target of
neutralizing antibodies
after precipitation the proteins can be used... Correct
Answers 1) in conjunction with Western blots to determine
the presence of antigen
2) for enzymatic studies
3) for ligand binding studies
aggregate precipitation Correct Answers method of
purification of immune complexes
when enough antigen-antibody complexes are present, large
aggregates may form. these aggregates may be pelleted by
centrifugation at high speeds
analysis parameters Correct Answers 1) detection with
visible light
- forward scatter = size
- side scatter = relative measurement of granularity (relative
because there is no fixed number for a certain amount of
granularity corresponding with a particular cell
2) detection of fluorescent light: when the lasers shine
through the droplet containing the cell, the fluorochromes
reflect the laser light and give excitation beams that can then
be measured in wavelength ranges by the various sensors
,3) compensation for detection of fluorescent light: overlap of
emission spectra from different fluorochromes makes
compensation necessary to differentiate the signals from
different fluorescent labels
analytical measurements for DNA quantitation Correct
Answers cells are fixed so that dyes can easily enter cells
relative DNA quantitation is given based on the amount of
dye staining = amount of DNA can be used to determine
stage in cell cycle (G0/G1, S, G2) or the induction of
apoptosis
antibodies binding by Fab or Fc Correct Answers can bind
using both Fab or Fc
many IgG monoclonal antibodies bind to allow 75% of Fab
to bind antigen
antibodies Correct Answers = immunoglobulins
membrane or surface Ig is the B cell receptor
secreted antibodies are a soluble form of the B cell receptor
that seek to bind to antigen
each B cell makes only one Ab with a unique antigen
specificity
therefore, each antibody binds to only one epitope or
antigenic determinant
, antibodies/immunoglobulins Correct Answers can bind to
literally anything
each B cell makes only one Ab with a specific antigen
specificity
therefore, each antibody binds to only one epitope or
antigenic determinant
antibody conjugated beads Correct Answers method of
purification of immune complexes
agarose or sepharose beads can be used as a solid phase
method to separate the ag/ab complexes from the cell lysates
by simple centrifugation. we will be using agarose beads that
have goat anti-mouse IgG antibodies attached --> coupled
antibody-bead complex permits the use of far less antibody
for the direct precipitation of the antigen-antibody
complexes from solution
antibody conjugated beads with primary antibodies or
secondary antibodies (monoclonal or polyclonal) can be used
to purify ag/ab immune complexes
antigen coat ELISA steps Correct Answers 1) coat
polystyrene plates with known antigen
2) block all non-specific sites with irrelevant protein
3) add test sample with unknown number of antibodies
4) add secondary ab conjugated with enzyme that binds to
primary antibody
3H thymidine incorporation assay Correct Answers -
radioactive nucleotide (3H-thymidine) incorporated into
DNA of dividing cells
- cells are incubated with 3H thymidine for 12-48 hours and
then lysed to harvest DNA onto fiber filters
- relative amount fo radioactivity absorbed onto the filter
paper is measured as counts per minute (cpms) on a
scintillation counter
- relative amount of cpms indicates the relative amount of
3H-thymidine incorporated into cells
ex: can test human peripheral blood mononuclear cells
(PBMCs) for reactivity to SARS-CoV-2 and control
51 Cell release assay Correct Answers target cells are
"labeled" with chromium and then effector cells are added
in different ratios to get a % lysis (% of target cells lysed by
effector cells)
effector cells: CTLs or NK cells
target cells: viral infected cells, tumor cells
challenges:
1) high material costs
2) handling and disposal of radioactive 51Cr
3) difficult to standardize
,ACE2 receptor binding domain (RBD) Correct Answers -
homotrimers of the S protein make up the distinctive spike
structure on the surface of the virus and is the main target of
neutralizing antibodies
after precipitation the proteins can be used... Correct
Answers 1) in conjunction with Western blots to determine
the presence of antigen
2) for enzymatic studies
3) for ligand binding studies
aggregate precipitation Correct Answers method of
purification of immune complexes
when enough antigen-antibody complexes are present, large
aggregates may form. these aggregates may be pelleted by
centrifugation at high speeds
analysis parameters Correct Answers 1) detection with
visible light
- forward scatter = size
- side scatter = relative measurement of granularity (relative
because there is no fixed number for a certain amount of
granularity corresponding with a particular cell
2) detection of fluorescent light: when the lasers shine
through the droplet containing the cell, the fluorochromes
reflect the laser light and give excitation beams that can then
be measured in wavelength ranges by the various sensors
,3) compensation for detection of fluorescent light: overlap of
emission spectra from different fluorochromes makes
compensation necessary to differentiate the signals from
different fluorescent labels
analytical measurements for DNA quantitation Correct
Answers cells are fixed so that dyes can easily enter cells
relative DNA quantitation is given based on the amount of
dye staining = amount of DNA can be used to determine
stage in cell cycle (G0/G1, S, G2) or the induction of
apoptosis
antibodies binding by Fab or Fc Correct Answers can bind
using both Fab or Fc
many IgG monoclonal antibodies bind to allow 75% of Fab
to bind antigen
antibodies Correct Answers = immunoglobulins
membrane or surface Ig is the B cell receptor
secreted antibodies are a soluble form of the B cell receptor
that seek to bind to antigen
each B cell makes only one Ab with a unique antigen
specificity
therefore, each antibody binds to only one epitope or
antigenic determinant
, antibodies/immunoglobulins Correct Answers can bind to
literally anything
each B cell makes only one Ab with a specific antigen
specificity
therefore, each antibody binds to only one epitope or
antigenic determinant
antibody conjugated beads Correct Answers method of
purification of immune complexes
agarose or sepharose beads can be used as a solid phase
method to separate the ag/ab complexes from the cell lysates
by simple centrifugation. we will be using agarose beads that
have goat anti-mouse IgG antibodies attached --> coupled
antibody-bead complex permits the use of far less antibody
for the direct precipitation of the antigen-antibody
complexes from solution
antibody conjugated beads with primary antibodies or
secondary antibodies (monoclonal or polyclonal) can be used
to purify ag/ab immune complexes
antigen coat ELISA steps Correct Answers 1) coat
polystyrene plates with known antigen
2) block all non-specific sites with irrelevant protein
3) add test sample with unknown number of antibodies
4) add secondary ab conjugated with enzyme that binds to
primary antibody
