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immunobiotechnology > Lecture 7 > Flashcards

Lecture 7 Flashcards

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1
Q

Why is it called ‘monoclonal’ antibody?

A

Immortalized a B cell that can become a “line” and keep on producing the same antibody indefinitely. The antibody is from a clone, and therefore a monoclonal antibody (or mAb).

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2
Q

What are the main steps in hybridoma technology?

A
  1. immunisation of animal
  2. harvest of spleen
  3. Cell fusion (with myeloma)
  4. HAT selection
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3
Q

What is used to fuse B cells with myeloma cells?

A

Polyethylene glycol (PEG)

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4
Q

What are the 3 types of cells that are preset after hybridoma fusion?

A
  1. unfused B cells (die eventually)
  2. unfused myeloma cells (no HGPRT gene)
  3. hybridomas
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5
Q

What is a drug that is used to treat leukemias and how does it work?

A

8-azaguanine

–> guanine analogue which can be incorporated into DNA by the enzyme HGPRT –> blocks purine nucleotide synthesis, with the effect of inhibiting DNA synthesis all together.

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6
Q

What is the full form of HGPRT?

A

hypoxanthine-guanine phosphoribosyl transferase

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7
Q

What kind of myeloma can become resistant to the the 8-azaguanine?

A

with mutant HGPRT

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8
Q

How is the immunisation of the mouse carried out?

A
  • adjuvant –> may contain bacterial component (doesn’t have to be pure)

-several boosters

-testing antibody timer periodically (agglutination of the immunogen)

-final booster with antigen only

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9
Q

Why is extended period of immunisation needed?

A

to drive the animals to produce IgG instead of just IgM

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10
Q

How many cells does a mouse spleen have? How many % are B cells?

A

100 million cells

40-50% are B cells

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11
Q

During fusion ,what is the ratio of B cells to myeloma cells?

A

2:1

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12
Q

What is the fusion efficiency for hybridoma technology?

A

1%

Viable hybrid in selection (HAT) medium ~1 in 10^5

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13
Q

What is H, A and T in HAT?

A

hypoxanthine
aminopterin
thymine

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14
Q

Which component of HAT blocks the de novo pathway (HGPRT gene dependent) for the synthesis of nucleotides?

A

Aminopterin

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15
Q

Which enzyme does aminopterin block?

A

dihydrofolate reductase

Dihydrofolate cannot be converted to tetrahydrofolate (THF) –> no TTP, ATP and GTP

–> targets all three types of cells

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16
Q

How is H and T from ‘HAT’ used for selection?

A

TMP can be generated from thymine (T) by thymidine kinase

IMP can be generated from hypoxanthine (H) by HGPRT

–> absence of THF (due to Aminopterin ‘A’) becomes irrelevant

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17
Q

How are fused hybridomas to be isolated as?

A

single cells

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18
Q

How are cells stored for long term usage?

A

Cells suspended in 10% dimethyl sulphoxide (DMSO) and 90% FBS

Into vials (1 ml) in isopropanol chamber

Into -80°C freezer to cool at ~ 1°C per minute

Transfer to liquid nitrogen (LN2) the next day for long-term storage (-196°C).

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19
Q

What are the main components of flow cytometry

A

Optics
-light source (lasers)
-detectors

Fluidics
-cell suspension in flow

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20
Q

For flow cytometry, does one cell in one drop = one cell per drop?

A

No.

One cell per drop: about 37% of the drops would have no cell and ~37% of the drops would have one cell. Others would have 2 or more cells.

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21
Q

If need one cell in most drops, what should be the avg?

A

< 1 drop per cell

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22
Q

What info does forward scatter provide?

A

size of cell

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23
Q

For forward scatter, what photons are collected as signal?

A

scattered photons

small scattering angle: 0.5-5 degree

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24
Q

What information does side scattering provide?

A

granularity of the cell

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25
Name two fluorochromes?
1. Fluorescein isothiocyanate --> thiocyanate (FITC) is a reactive group for easy coupling to other compounds such as an antibody. 2. Phycoerythrin (PE)
26
What is the function of dichroic, monochromatic mirrors and bandpass filters?
dichroic: pass light of certain wavelengths while reflecting others monochromatic: transmit a narrow range of wavelengths, Bandpass: transmit a specific range or "band" of wavelengths while blocking others outside this range
27
What is used to stain DNA?
propidium iodide
28
What is the cell cycle of eukaryotes?
1. Interphase -G1 -S -G2 2. Mitosis 3. Cytokinesis
29
What happens in interphase?
G1 -cell growth S - DNA synthesis G2 - Replication of mitochondria and organelle and preperation for genomic separation
30
What is cytokinesis?
division of cytoplasm
31
How does the number of chromosomes change during the S phase of cell division?
from 46 to 92
32
Why is indirect assay used in hybridoma mAB analysis instead of a direct assay?
If use direct assay, need to label ALL mAbs INDIVIDUALLY --> use second antibody e.g. a goat anti-mouse antibody with a label
33
In flow cytometry, what is used for the fixation of cells?
1% formaldehyde in PBS
34
What is CD3?
T cell antigen associated with T cell receptor
35
What is CD4?
Helper T cell marker, also on macrophages
36
What is CD8?
cytotoxic T cell marker
37
What is CD16 and where is it found?
Fc Receptor NK cells, Macrophages, Neutrophils sometimes on monocytes
38
What is CD56?
Neural Cell Adhesion Molecule-1, also on NK
39
What is CD19?
B cell marker
40
What is the B cell marker?
CD19
41
Now you have a number of hybridomas, what do you do?
-Confirm the antibody is against the antigen intended -Characterize the antibody, IgM, IgG1, etc. -Its functional effect? Inhibiting, activating, neutral? -Is the epitope always expressed?
42
How to 'make' GFP encoded protein?
tagging the GFP domain to a protein cDNA GFP
43
During cell sorting, cells are separated based on their __________.
net charge
44
For the production of monoclonal antibodies from the hybridomas in tissue culture media, what is the media supplemented with?
10% FBC (bovine Ig ~ 50ug/mL) --> serious problem of contamination
45
How to solve the problem of Ig contamination from FBS during the production of mAbs from hybridomas in tissue culture media>
-batch-test FBS -change to lower FBS concentration media as time goes on i.e. from 10% to 1 % to serum-free media --> not all hybridomas can adapt -use ultra low IgG FBS --> other "goodies" may also be removed
46
Apart from Ig contamination upon usage of BSA in producing mAbs from hybridomas, what else could be another challenge? How to prevent it?
Bovine spongiform encephalopathy (BSE) i.e. Mad cow disease test of FBS from non BSE animals
47
What is the mechanism of mad cow disease?
Chain reaction caused prions to form plagues (in the brain) leading to neurodegeneration and death.
48
What are the symptoms of mad cow disease?
loss of muscle control, hence “mad cow” behaviour with unsteady gait.
49
Apart from using tissue culture to produce antibodies from hybridomas, what other methods could be used?
Mouse Ascites
50
Describe the 'mouse ascites' method to produce antibodies from hybridoma?
-prime with 0.5ml pristine (10days), Freunds incomplete adjuvant (no bacteria) -inject hybridoma into peritoneal cavity of mouse (1 mil cells) -harvest 7-21 days -mAb conc ~ 10mg/mL -
51
What is Ascites?
abnormal accumulation fluid in the abdominal (peritoneal) cavity. Introducing a tumor (hybridoma) to a mouse is creating an artificial disease state.
52
What are the advantages of using tissue culture to produce mAbs from hybridomas?
no need of animals --> animal facilities can be expensive + require trained personnel + need IACUC approval --> issue of animals rights sentiment avoided
53
What is the disadvantage of using tissue culture to produce mAbs from hybridomas?
-low yield (100ug/ml) -contamination from FBS
54
What are the advantages of using mouse ascites over tissue culture for producing mAbs from hybridomas?
-Some hybridomas may not grow well in tissue culture -No contamination of bovine immunoglobulins -High yield (10mg/mL)
55
What is the characteristic of CELLine 2-compartment bioreactor?
-10kDa semipermeable membrane (top) -Silicone membrane Polydimethylsiloxane (PDMS), perameable to gases but not liquids (bottom)
56
What are some nutrients required and waste produced during the production of mAbs from hybridomas in tissue cultures?
Nutrients: glucose, glutamine Waste: lactate and ammonium
57
What are the advanatges of using monoclonal antibodies from rat over mouse?
-Generation of monoclonal antibodies against mouse antigens. -Rat, a larger animal, more lymphocytes can be obtained from its organs, namely spleen. -Rat myeloma cells support more effective fusion than mouse. -More ascites fluid from rat (again being a larger animal) for antibody production. -Rat hybridoma more stable than mouse hybridomas
58
What are the advantages of using monoclonal antibodies from rabbit over rat/mouse?
-able to generate a larger range of high affinity antibodies. -Able to generate antibodies against small epitopes, useful for peptide antigens -Rat and mouse evolutionary very close, thus many proteins are similar and are therefore ineffective in elicit an immune response from each other
59
What are the disadvantages of using monoclonal antibodies from rabbit over rat/mouse?
-Slow in establishing rabbit mAb because of lack of myeloma/plastocytoma lines.
60
How are mAbs purified from ascits fluid?
ppt by 20% Na2SO4 at RT. then, chromatography
61
Immunoprecipitation would not have worked for mAbs against monomeric soluble antigens, unless __________________________ and unless_____________________________.
1.the antigen is with multiple subunits 2. the antigen is with multiple epitopes each recognized by a different mAb
62
What is direct Coombs test?
-wash fetal red blood cell coated with maternal antibody -add rabbit anti-human antibody --> agglutination?
63
What is indirect Coombs test?
- Add Rh+ red cells to maternal serum and wash out unbound antibody -Add rabbit anti-human antibody --> agglutinations
64
What is the Rh incompatibility?
-Mother Rh- -first fetus Rh+ --> mother make antibody for Rh+ after birth of child -when pregnant with 2nd fetus also Rh+, antibody cross the placenta and fetus RBC destroyed by phagocytosis --> anemia of fetus and newborn
65
What are southern blots, northern blots and western blots for?
Southern blots - DNA Northern blots -RNA Western blots - proteins
66
What gel is used for southern and northern blotting?
Agarose gel (agarobiose)
67
Why is agarose gel used over agar gel in southern and northern blotting?
Agar made of agaropectin and agarobiose agaropectin --> anionic groups such as sulphate, pyruvate and glycuronate --> interfere with mobility of DNA/RNA
68
What is used to stain DNA to visualise it under UV?
ethidium bromide
69
DNA of 3000bp would have MW of how much?
2 million da
70
How is DNA separated in electrophoresis?
DNA are uniformly charged (phosphates). Electrophoresis in agarose would separate DNA by weight/charge.
71
What is the purpose of SDS in western blotting?
1 SDS per 2 AA --> uniformly negatively charge --> electrophoresis from cathode (-) to anode (+)
72
What gel is used in western blotting/ SDS-PAGE?
Polyacrylamide gel -use electric current to drive the transfer (smaller pores than in agarose gel)
73
After SDS-PAGE to separate proteins, what happen next?
Transfer to nitrocellulose membrane Bind with primary antibody followed by secondary antibody
74
Why is the detection of maternal anti-Rh antibody difficult?
Rh antigen on the RBC is sparse and does not cause agglutination.
75
What is the purpose of 'priming with pristane' in mouse for the production of mAbs using ascites?
Pristane --> mineral oil to induce inflammation and stimulate antibody production in animal
76
How to purify mAbs from tissue cultures?
use specific Ig binding proteins from bacteria e.g. Protein A, protein G and protein L
77
What are the properties of gels?
- highly cross-linked system -no flow when in steady state -solid supports for holding liquid
78
What 2 components in polyacrylamide gel?
acrylamide and bis-acrylamide
79
Explain the difference in the electrophoresis of DNA and proteins?
DNA: cathode (-) to anode (+) Protein: anode (+) to cathode (-)

immunobiotechnology (8 decks)

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